Method of mass production of multimeasured mannan-binding lectine

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.

EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.

6 cl, 11 dwg, 9 ex

 

The technical field

The present invention relates to a developing method by which the mass production of multimeric recombinant minnesotabased lectin person (rhMBL).

The prior art inventions

Minnesotabuy lectin (hereinafter referred to as "MBL") is a whey protein, a component of innate immunity (genetically predetermining immunity). Molecular weight MBL is 32 kDa. MBL consists of a C-terminal uglevodorodnogo domain (CRD), a collagen domain and rich in cysteine region on the N-end. Three MBL molecules form a complex through the formation of a triple helix, using collagen domains, and then up to 6 complexes can be combined together via intermolecular disulfide bond (-s-s-) between residues cysteine at N-end, leading to the formation of a multimeric molecule MBL, consisting of up to 18 molecules MBL.

The oligomeric form of MBL may form a complex with other proteins, such as associated with MBL serine protease (MBSP-1, MASP-2 or MASP -3) or associated with MBL protein (Map19). Although the overall molecular structure and function MBL involved in the activation of complement, similar to C1q, the first component of the complement system, in contrast to C1q MBL system activates the complement him, splitting C4 and C2. This process is the activation of complement, called latisevicius way, using associated with MBL serine protease, which is activated by the binding of MBL with the microorganism. Activation of the complement system by MBL is one of the functions of MBL in the primary protection against microbial infection, before you receive the adaptive immune response. Binding of MBL with the microorganism through uglevodorodnaya domain (CRD)that recognize the unique patterns of glycosylation on the surface protein of the microorganism, is a necessary condition for the activation of complement. Binding of MBL bacterium activates associated with MBL predecessors serine proteases, MASP-1 and MASP-2, leading to the cleavage of C4 and C2 of the complement system, leading to the formation of C4b2a and C3b. Some of the proteins, resulting in activation of complement, directly attached to the surface of the microorganism as opsonin, and MBL can also function as opsonin to more effectively carry out phagocytosis associated with MBL microorganisms by phagocytes. Some activated complement proteins are lysed directly penetrating microorganisms, forming membranaceous complex (MAC), and other protein fragments of complement, obtained during the activation process, contribute highlighted the th cytokine, causes inflammation.

In previous reports described the MBL gene expression in various cells, such as CHO cells, hepatoma cells HLF or cells HEK293-EBNA. In particular, the level of expression in CHO cells was very high, but produced MBL mainly consisted of monomers or dimers (Katsuki Ohtani et al., J. Immunol. Methods, 222: 135-144, 1999). In cells HEK293-EBNA were products of oligomeric forms of MBL, but overall productivity was less than 1 µg/ml (T. Vorup-Jensen et al., International Immunopharmacology, 1: 677-687, 2001). Products oligomeric forms of MBL extremely important as monomers and dimers, consisting of 3 subunits and 6 subunits, respectively, have little, if any, activities. Also an important issue in the development of pharmaceutical product from MBL - get cell line-superproducer, as it required several milligrams.

MBL can also play an important role in adaptive immunity. Cells involved in phagocytosis, such as neutrophils and macrophage play an important role in the removal of penetrating foreign microorganisms by phagocytosis. In addition, the macrophage is an antigen presenting cell (APC), which activates T-cells, leading to induction of acquired immune response. Therefore, a multimeric MBL involved in the activation of the complement system is AK opsonin, plays an important role not only in the primary protection as a component of the innate immune system, but also in the induction of acquired immune responses.

The amount of MBL in serum varies with individuals, being in the range from 50 ng/ml up to several µg/ml due to genetic variability of the MBL gene. For example, if there was a point mutation in codon 52, 54 or 57 of exon 1 of the gene MBL, MBL molecules do not form oligomeric forms, resulting in a nonfunctional smaller complexes, consisting of not more than 3 molecules MBL. If the mutation occurs in the promoter regions of MBL gene, the expression level of MBL is reduced, leading to a significant change of MBL level in the blood among the affected people. Basically, those infants or patients with neutropenia who have an MBL level below a certain level, have a weak immunitet, becoming highly susceptible to microbial infections (Sumiya, M. et al., Lancet, 337: 1569-1570, 1991; Summerfield, J. A. et al., Lancet, 345: 886, 1995; Garred, P. et al., Lancet, 346: 941, 1995; Summerfield, J. et al., Br. Med. J., 314: 1229, 1997; Mullighan, C. G. et al., Scand. J. Immunol, 51: 111-122, 2000; Nefh, O. et al., Lancet, 358: 614-618, 2001; Peterslund, N. A. et al., Lancet, 358: 637-638, 2001; C. G. Mullighan et al., Blood, 99: 3524-3529, 2002).

According to a study of patients with chronic hepatitis b virus MBL level in the blood is an important factor in the prediction of disease development (Hakozaki Y. et al., Liver 22, 2-34, 2002). In particular, among patients who developed fulminant liver failure in chronic infection by hepatitis B virus, the mortality of patients in whom the level of MBL in the blood was more than 3 μg/ml, was 0%, whereas patients in whom the level of MBL in the blood was 1.5 μg/ml and less than 0.5 ág/ml, the mortality rate was 58% and more than 80%, respectively. This study shows a significant correlation between the level of MBL in blood and mortality of patients with infectious disease. This study also suggests that the addition of MBL patients with low levels or absence of MBL can be beneficial.

In order to recombinant MBL can be used as a therapeutic agent, a recombinant protein MBL must be made in large quantities at a reasonable price in the form of active oligomeric molecules. Unlike cytokines he needed several milligrams for the patient at each visit. For example, the volume of blood in an average of approximately 5 liters, which requires 25 mg for the 5 μg/ml MBL. Given the size of other body fluids, you may need a much larger quantity at each visit. So was a very important issue in developing a commercial product MBL that is created n the only recombinant cell line superproducer, but also cell line, which produces reactive polymer MBL.

Thus, the authors of the present invention have made extensive efforts to create recombinant cell line, which could produce MBL on an industrial scale and, at the same time, which could produce functionally active oligomeric form of MBL, which is able to activate the complement system, specifically binding to glycoproteins that bind MBL, in the presence of MASP. As a result of these efforts, the authors of the present invention have prepared this invention, arguing that active rhMBL, suitable for therapeutic applications, it is possible to produce on an industrial scale in the form of a multimeric polymer in Cho cell line, transtitional cloning vector containing polynucleotide encoding sequence rhMBL.

Description of the drawings

Figure 1 is a schematic illustration showing the genetic map of plasmid vector pMSG-MBL.

Figa and Figv represent the image MBL produced by a recombinant cell line CHO CHO MBL/D1-3, which transformed pMSG-MBL. Figa is a type of analysis SDS-PAGE and figv shows the analysis of Western bottom, using specific MBL monoclonal antibodies.

3, not only is em a picture with the electron microscope, showing oligemia forms of MBL. Clearly oligemia MBL molecules consisting of 2, 3, 4, 5 and 6 of the trimeric subunits MBL.

Figure 4 is a photograph of scanning electron micrographs showing the complex recombinant protein MBL and gold nanoparticles, coated, pre-S, in the presence of calcium ion.

On figa and figv shown that rhMBL specifically binds to pre-S, glycoprotein or mannan activity, quantitatively similar to natural MBL.

Figure 6 shows the relative binding capacity of large polymeric forms of MBL and smaller forms, consisting mainly of monomers and dimers of three-dimensional subunits. Smaller forms have little, if any, binding ability.

7 shows a comparison of the ability to activate C4 large shapes and smaller form rhMBL.

On Fig shown that rhMBL specifically binds with a variety of microorganisms.

Figure 9 shows that rhMBL inhibits infection of embryonic cells, monkey kidney (FRhK-4) SARS-CoV.

Figure 10 is a series of images of phase-contrast microscope, showing cells FRhk-4 infected with SARS-CoV. Without treatment rhMBL cells look unhealthy, whereas MBL present in healthy cells.

11 is a schematic illustration showing the diagnostic kit received the first using recombinant MBL person.

Description

The purpose of the invention

The purpose of the present invention consists in the construction of Cho cell line, which transliterowany a vector containing a polynucleotide encoding the sequence MBL person, with the aim of producing functionally active oligomeric forms of MBL in large quantities that will be used for commercial purposes.

Additionally, the purpose of the present invention is to provide a method of cleaning rhMBL from the environment of the cultivation of recombinant Cho cell line.

The invention

In the present invention serves expressing vector pMSG-MBL containing a sequence coding region rhMBL presented on the map plasmids in figure 1.

The present invention also offers a line of master cells, transfusiona expressing vector.

The present invention further provides a method of mass production of multimeric recombinant MBL in accordance with the following stages:

(1) obtaining host cells, expressing transfected with the vector containing the sequence coding region MBL person;

(2) the production of recombinant MBL human cell line transformed by expressing the vector sequence, the coding region MBL brow of the ESA, in the system of cultivation; and

(3) purification of recombinant MBL person obtained at stage (2).

The present invention also features a kit for determination of recombinant MBL person, including : (i) solid-phase substrate with the test line and control line, where mouse antibodies against minnesotabased lectin person is fixed in the test line and antibodies against mouse IgG or antibodies against pre-S is fixed in the control line; (ii) a substrate for the dye adsorbed conjugate with dye and connected with the lower part of the solid-phase substrate, where, if the control line is fixed antibodies against mouse IgG, conjugated with the dye is conjugated mouse antibodies against minnesotabased lectin person and gold and if the control line fixed antibodies against pre-S conjugate with dye represents a conjugate pre-S and gold; and (iii) substrate sample connected with the lower part of the substrate with the dye, which loads the sample.

Detailed description of the invention

In the present invention serves expressing vector pMSG-MBL containing polynucleotide encoding rhMBL presented on the map plasmids in figure 1, which is sufficient for the expression of oligomeric recombinant MBL person who can activate the system complemen is a, specifically by binding to MBL binding glycoprotein in the presence associated with MBL serine proteases.

In a preferred implementation of the present invention MBL cDNA man (Gene Bank NM_000242) embedded in the vector pMSG (No. of access: KCCM (Korean center of microorganisms) 10202)containing MAR-element (element of the binding site with the nuclear matrix) beta-globin gene, a poly-A SV40 virus and complex transcription terminator gene gastrin, which led to the creation of a design unique vector denoted by pMSG - MBL.

The present invention also provides cell-owners, transfetsirovannyh expressing vector containing polynucleotide encoding rhMBL, which is sufficient for the expression of recombinant MBL man who is able to activate the complement system, specifically binding to a glycoprotein on the surface of microorganisms in the presence associated with MBL serine proteases.

Cell owners used in the present invention are, in General, the cells of animals and, preferably, selected from the group consisting of CHO cells (Chinese hamster ovary), hepatocytes, cells HEK (human embryonic kidney) and so on

In a preferred embodiment of the present invention, the transformant was obtained by embedding the cloning vector pMSG-MBL containing nucleotide, encoding rhMBL, which is sufficient for the expression of recombinant MBL person, able to activate the complement system, specifically binding to a glycoprotein on the surface of microorganisms in the presence associated with MBL serine proteases, in line host cells, such as CHO cells, and then adapting transformants to terms with MTX (methotrexate) leading to the selection of transformant, which is able to Express in a large number of protein rhMBL in multimeric form of the polymer. The selected transformant identified MBL D1-3 (cell line CHO) and deposited in the Korean collection of type culture, Daejeon, Korea, may 16, 2003 (No. of access: KCTC 10472BP).

Recombinant MBL man, produced by transformants described in the present invention, is mainly in the form of a multimeric form, which is similar to natural MBL from human blood. Cell line obtained in the present invention, was produced mainly functionally active multimeric form of MBL and showed a high expression level, providing the opportunity to use the transformed cell line CHO to obtain large quantities of functionally active protein rhMBL.

The present invention further provides a method of obtaining a recombinant MBL person, comprising the following stages:

(1) How is Holocene host cells, transfected design DNA, including the cloning vector containing polynucleotide encoding rhMBL, which is sufficient for the expression of recombinant MBL person, able to activate the complement system, specifically binding to a glycoprotein on the surface of microorganisms in the presence associated with MBL serine proteases.

(2) a method of obtaining a recombinant MBL man by the expression of polynucleotide encoding MBL human, animal cell and from the cell, applying massive system cell culture.

(3) Method of purification of recombinant MBL obtained at stage (2).

In particular, the use of characters of MBL binding to glycoprotein at the stage (1) and MBL binding glycoprotein, examples of which are glycosylated envelope protein of a virus containing the pre-S hepatitis b glycosylated bacterial protein, glycosylated fungal protein and synthetic glycoprotein.

In a preferred embodiment of the present invention stage production of recombinant MBL at the stage (2) is characterized by stages of suspension culture cells in serum-free/protein-free medium and pereselenia such cells are well adapted to serum-free/protein-free medium in order to gradually increase production, resulting in receiving the Oia in a large number of protein rhMBL in the form of a multimeric polymer.

At stage (3) recombinant MBL protein was purified from the environment of the cultivation of the transformants MBL gene, using anion-exchange chromatography and affinity MBL relative to the pre-S hepatitis B. Method of purification MBL consists of the following stages: (a) separation of samples containing multimeric recombinant MBL person using anion exchange chromatography; (b) prepare column by filling its substrate, on which the immobilized pre-S hepatitis B virus; (c) specific capture of recombinant MBL immobilized on pre-S hepatitis B virus during passage through the column sample containing recombinant proteins in MBL the presence of calcium ions, after equilibration of the column with buffer containing calcium ions; and (d) elution of the recombinant protein MBL by adding a buffer solution with the addition of EDTA or EGTA to the column, which was grasped by recombinant MBL at the stage (c).

At stage (a) used a substance which is usually used for anion exchange chromatography and Q-sepharose was one of such substances for the column. Sample containing MBL proteins as a monomer or dimer, were separated in the presence of 150 - 200 mm NaCl, and another portion containing high molecular weight protein MBL in the form of a copolymer, suirable in the presence of 350 - 400 mm NaCl.

At the stage (b) used sub is spending, which is usually used for affinity chromatography, and sepharose represented one of the commonly used substrates.

At the stage (c) the equilibration of the column was performed by adding a buffer solution or the same solution as used for affinity binding of recombinant MBL and pre-S hepatitis C. the Binding of recombinant MBL with pre-S hepatitis b virus is preferably conducted in the presence of calcium ions and then the calcium concentration was preferably 2 to 20 mm. The sample containing the recombinant protein may be a supernatant obtained from the environment culturing cells-transformant producing MBL or MBL solution, suirvey with column fractionation MBL mentioned above.

At stage (d) elution of the protein rhMBL was performed using a buffer solution of EDTA or EGTA in the absence of calcium ions. The buffer solution may be a solution containing EDTA or EGTA at a concentration of 2 to 10 mm, for example distilled water or buffer Tris-Cl, pH 7,4.

Eluent obtained in stage (d), liofilizirovanny or deliberately before lyophilization, resulting in obtaining a purified recombinant protein MBL.

Method of purification protein rhMBL of the present invention is also applicable to clean MBL derived from natural biological samples, in addition to the recombinant protein MBL, the scientists from transformant. The biological sample can be a as an example, blood, plasma or serum.

The present invention also features a kit for determination of recombinant MBL person, including: (i) solid-phase substrate containing a test line and control line, where mouse antibodies against minnesotabased lectin person is fixed in the test line and antibodies against mouse IgG or antibodies against pre-S is fixed in the control line; (ii) the substrate with adsorbed dye conjugate with dye and connected with the lower part of the solid-phase substrate, where, if the control line is fixed antibodies against mouse IgG, conjugated with the dye is conjugated mouse antibodies against minnesotabased lectin person and gold and if the control line is fixed antibodies against pre-S conjugate with dye represents a conjugate pre-S and gold; and (iii) a substrate for sample connected with the lower part of the substrate of the dye, which loads the sample.

In the present invention nitrocellulose membrane, polyvinylidenedifluoride (PVDF) and nylon membrane can be used as a solid-phase substrate. The substrate with the dye can be used, but not limited to, polyester, fiberglass, etc.

The present invention also affords the fast way to get set for the determination of recombinant MBL person, includes the following stages:

(1) preparation of conjugates with dyes by binding of antibodies against minnesotabased lectin person or protein pre-S with gold particle;

(2) sorbirovaniya conjugates with dye on a substrate for a dye;

(3) binding of antibodies against minnesotabased lectin person with a solid-phase substrate in the test line and antibodies against pre-'s man with a solid-phase substrate in the control line, when compared with the dye are gold particles with antibodies against minnesotabased lectin person, or binding of antibodies against pre-S with a solid-phase substrate as the control line, when compared with the dye are gold particles with a murine antibody against MBL person; and

(4) attaching a substrate to pattern the substrate with the dye to the bottom of the solid-phase substrate.

In the present invention the proteins pre-S antibody against MBL person can be used, but not limited to, as a conjugate with dye.

The present invention also proposes a method of determining the MBL using a set of definitions for MBL, which includes the following stages:

(1) applying a sample containing MBL, on a substrate with a dye that is connected with the lower part of the solid phase substrate; and

(2) you the statement, the antibodies bind to the test line and control line solid-phase substrate.

Recombinant MBL man of the present invention similar to the natural form, purified from human blood. He is an oligomeric form, and it is active against binding to the microorganism, thus activating the complement system. And a high level of production of the product (50 µg/million cells/day) provides commercial production of functionally active MBL for new product development. Thus, the obtained rhMBL can be used effectively for the treatment of the individual, infetsirovanna virus, bacteria or fungi. It can also be used for the manufacture of a diagnostic kit for determination of recombinant MBL person.

Examples

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following examples.

However, it will be appreciated that the experts in this field can make modifications and improvements within the essence and scope of the present invention.

Example 1: Getting transformant MBL gene and expression of recombinant MBL person.

<1-1> Design expressing vector

pEZ-MBL 2-5 designed, kloner what I MBL cDNA, obtained by PCR from a cDNA library of human hepatocytes, in vector pEZ, and then confirmed that the nucleotide sequence of cDNA MBL authentic known nucleotide sequence of the MBL Gene Bank NM_000242). PCR was performed using pEZ-MBL 2-5 as template and using a primer set (forward primer 1 and reverse primer 2) for amplification of cDNA MBL approximately 750 tysp Each primer contained a website recognition restricts and Kozak sequence, so that amplified the entire coding region. Then pMSG-MBL received, cloning MBL cDNA in the vector pMSG (No. of access: KCCM 10202), and then confirmed built nucleotide sequence MBL (Fig. 1).

Direct primer 1 (SEQ. ID. No. 1)

ctagctagcc accatgtccc tgtttccatc actc (34-Mer)

Reverse primer 2 (SEQ. ID. No. 2)

gaagatctca gatagggaac tcacagacg (29-Mer)

<l-2> Transformation of host cells pMSG-MBL

<l-2-l> Obtaining expressing plasmid DNA pMSG-MBL

E.coli was transfusional pMSG-MBL and the obtained transformant was cultured in 100 ml of LB medium with the addition of ampicillin at a concentration of 100 µg/ml (Sigma, USA). DNA pMSG-MBL were separated using the kit for purification of plasmids (QUIAPREP Plasmid Midi Kit, Quiagen, USA). Purified DNA in order to make it linear, were digested with restriction enzymes Sea I, which was separated by means of a set of purification of PCR product (QIAQuick PCR product purification kit, Quiagen, USA).

<l-2-2> Obtaining host cells

After culturing host cells CHO DG44 (dhfr-/dhfr-) in the environment α-MEM with the addition of 10% cFBS number of cells was counted in hemocytometer. The cells were distributed (2 × 105cells/well) in medium α-MEM with the addition of 10% cFBS, which were cultured in the incubator CO2within 24 hours.

<l-2-3> Transformation

2 μg of vector pMSG-MBL was mixed with 5.3 ál reagent Dosper™ and 16 ng DNA vector pDCHlP (a plasmid containing the gene for DHFR, Venolia, L. et al., 1987, Somat. Cell MoI. Genet. 13, 491-501)to induce the reaction at room temperature for 45 minutes. The resulting product was added to cells-owners. Cells were cultured at 37°C for 6 hours, then the medium was replaced with freshly prepared medium α-MEM with the addition of 10% cFBS (3 µl/well), followed by further cultivation. After 2-3 days, when the transformed cells were grown sufficiently, added trypsin was added to the cells 2 ál α-MEM (wt./vol.), containing 10% dFBS (4×105cells/well), after which again was followed by further cultivation. Every 2-3 days, the medium was replaced with freshly prepared and the condition of the cells and formation of single colonies was observed in the microscope. After 10 days has been adapted early cells.

<l-3> Selection expressing MBL cell Lin and and gene amplification MBL

After receiving adapted early cells gradually increased the concentration of MTX (methotrexate) in the environment, in order to induce the MBL gene amplification, built-in transformed cells. The cells were placed in 4×105cells/well and then cultured in medium (α-MEM+10% dFBS) with addition of 10 nm MTX until the merger, during which the medium was replaced with freshly prepared every 2-3 days. Cells were again distributed in the amount of 4×105cells/well and have adapted to the environment with the addition of 100 nm MTX, followed by the same procedure as described above, and then adapted to the conditions of 1 μm MTX. During amplification MBL gene was performed Western blot analysis on each stage in order to observe changes in the level of expression of MBL. Here the expression of MBL increased with increase in the content of MTX in the environment.

<l-4> Selection one cell line

In order to separate one cell line showing a high degree of MBL expression, cells cultured in medium (α-MEM+10% dFBS)containing 1 μm MTX, were distributed in 96-well tablet to get 0.5 cells/well, and then cultured in medium containing 1 μm MTX. After 2 weeks, when he confirmed the formation of single colonies of cells was transferred into a 24-well plate, which was then cultured until then, the cells sufficiently proliferated. Some of them were frozen and stored, and some of them used to compare expression by Western blot analysis. And transformant D1-3 in the form of a single cell, which, as confirmed, has produced high-molecular form of MBL in large quantities, was chosen as a cell line of the present invention. The selected transformant identified MBL D1-3 (cell line CHO) and deposited in the Korean collection of type culture, Daejeon, Korea, may 16, 2003 (No. of access: KCTC 10472BP). Analysis PAGE recombinant MBL protein, expressed by transformants, was carried out at adenocarinoma conditions and it was found that the protein was a multimeric form of MBL, which is very similar in characteristics with MBL natural origin.

<l-5> Quantitative analysis of recombinant MBL expressed by transformants

Based on a standard amount of purified MBL obtained from human serum, assessed the level of expression of recombinant protein MBL of transformed CHO cells MBL/D1-3. Cells were placed in a T25 flask at a concentration of 4×105and then cultured in medium (α-MEM(wt./vol.)+10% dFBS) to 90% confluence. Then added 3 ml of medium (α-MEM(wt./vol.)+5% dFBS) and then were cultured for 4 days. The cultural medium was diluted 10 times, followed by Western-blot the analysis. The result was compared with the standard amount of natural MBL. The level of expression of recombinant protein MBL, as confirmed, was approximately 50 μg/106cells/day as cultivated in the flask for cell culture.

Example 2: the Way of mass production of protein rhMBL

<2-l> Obtaining cell lines adapted for suspension culture in serum-free medium

Dependent on the culture substrate of the cell line expressing the MBL, were cultured in medium (α-MEM(wt./about.) with the addition of 10% dFBS and 1 μm MTX. After recovery of viable cells were inoculable roller in a flask with a volume of 250 ml containing 100 ml containing protein environment HyQ SFM4 Cho (Hyclone, USA) with addition of 0.15% sodium bicarbonate in the amount of inoculum 4×105cells/ml Cultivation roller in the flask was carried out at 37°C incubator with 5% CO2, which stirred all the time at about 40./minutes When the number of cells reached 1,0-2,0×105cells/ml, cells were again inoculable on Wednesday HyQ SFM4 SNO with the addition of 0.15% sodium bicarbonate in the amount of inoculum 5×105cells/ml cell Viability was determined by way of exception Trypanosoma blue. In particular, the viability of over 90% was confirmed at a later stage adaptation.

Cell hell is adapted to serum-free protein-free environment, cultivated up until the density of the cells is not reached 2.5×106cells/µl. Then the cells were recovered and resuspendable in freezing environment. The cells were placed in cryoprobes to obtain the concentration of 2.8×107cells/μl, which was then stored in liquid nitrogen.

<2-2> create a culture in the bioreactor, using a cell line that is adapted to serum-free suspension cultivation

Cells for suspension cultivation was inoculable roller in a flask with a volume of 250 ml containing 100 ml of serum-free/protein-free medium (medium HyQ SFM4 SNO) with the addition of 0.15% sodium bicarbonate in the amount of inoculum 5×105cells/µl. When the number of cells was increased to 1,0-2,0×106cells/ml, cells were inoculable in roller flask 500 ml, containing 200 ml of medium HyQ SFM4 SNO when the value of inoculum 5×105cells/µl. Then, when the number of cells was again increased to 2.0 × 106cells/ml, cells were inoculable in roller flask 1000 ml containing 400 ml of medium HyQ SFM4 SNO when the value of inoculum 5×105cells/µl. Thus, cells were cultured with a gradual increase, leading to obtaining 1 liter for sowing culture with a concentration of 2.5×106cells/µl.

Cultures the cells for sowing, obtained above, was inoculable in the bioreactor with a volume of 7.5 l (working volume of 5 l) and cultured at 34°C for 5 days (50 rpm, DO 50, pH 7,2-7,4).

Example 3: Method of purification of recombinant multimeric protein MBL person

Expressing MBL CHO cells were cultured in medium HyQ SFM4 CHO and then the cultural medium was centrifuged and passed through a membrane filter with pore size of 0.45 μm to remove cells. Anion exchange chromatography was performed for purification of recombinant proteins MBL. The number of purified proteins was determined using a set of MBL ELISA.

<3-l> the Division of the sample containing multimeric polymer, using columns with Q-separate

After optimization of the pH-value and coefficient of filtration of supernatant smaller forms of MBL, such as monomer and dimer were removed using a column Packed with Q-separate, and collected only MBL large forms. Sample containing smaller forms of MBL, were separated by passing through 150-200 mm NaCl buffer solution and the sample containing large forms of MBL was suirable using 350-400 mm NaCl buffer solution. These results show that more than 80% expressionalism in the form of a high molecular weight copolymer forms of MBL cell line, which is proposed in this invention (Fig. 2A).

<3-2> Getting speakers with pre-S-separate

Recombinantly the protein pre-S, used in the present invention, received, expressive gene pre-S, which is a part of the surface protein of hepatitis b virus in Sacharomyces cerevisiae, and then cleaning it as glycosylated in a high degree molecules (international patent publication number WO 02/094866).

One gram of powder sepharose 4B CNBr activated, was dissolved in 1 mm HCl, and then several times followed by washing. In order to use recombinant protein pre-S as a ligand, 6.4 mg of protein pre-S was dissolved in consolidating buffer (0,2 M NaHCO3and 0.5 M NaCl and pH 8,3), obtaining a concentration of 0.5 - 10 mg/ml. Activated CNBr resin sepharose 4B was mixed with a solution of pre-S and subjected to interaction at room temperature for 2 hours. The mixture is then transferred to blocking buffer (0.1 M Tris-Cl, pH 8.0), followed by further interaction at room temperature for 2 hours. After washing immobilized on sepharose pre-S was confirmed by analysis of the Western blot and, as discovered almost all recombinant proteins pre-S immobilizates on column sepharose, which was used for purification of recombinant protein MBL. In conclusion, a column pre-S-separate for purification of recombinant protein MBL of man has been filling the column with the resin pre-S-sepharose 4B.

<3-3> Purification of recombinant MBL person

A column Packed with pe-S-separate, balanced buffer for binding (20 mm Tris, 150 mm NaCl and 10 mm CaCl2pH of 7.6). The fraction of columns with Q-sepharose containing recombinant proteins MBL, or the cultural medium containing MBL, were loaded on the column for adsorption MSL. Then the column was washed several times in binding buffer. Eluting buffer (20 mm Tris, 150 mm NaCl and 5 mm EDTA, pH 7,6) without Ca2+was passed through the column to be eluted recombinant MBL person. The obtained eluent was analyzed by using SDS-PAGE. The result has been purified recombinant MBL person with 99.9% purity (Fig. 2A and Fig. 2B).

Example 4: Verification of a multimeric recombinant MBL through EM and activity of MBL binding to the surface of the organism.

<4-1> evidence of the formation of multimeric recombinant MBL person

To confirm forms whether or not the purified recombinant MBL person multimeric polymer in the form of a bouquet of flowers, purified recombinant MBL person was treated amorphous carbon and then form of the protein was examined under transmission electron microscope (Tecnai 12, FEI, Netherlands) and found that purified recombinant MBL man was a multimeric polymers in the form of a bouquet (Fig. 3).

<4-2> the Binding of recombinant MBL man with gold particles coated with pre-S

<> The ability of recombinant MBL person to contact the surface of the microorganism was observed using pre-S surface protein of hepatitis B. Proteins Pre-S attached to the surface of the gold particles, which have a size of 20-40 nm, which, therefore, made as if the microorganism. Recombinant MBL person was added thereto in the presence of calcium. Then under transmission electron microscope (Tecnai 12 watched joined whether recombinant MBL person to proteins pre-S, leading to confirmation of complex formation. As shown in figure 4, squirrels pre-S printed on gold particles were surrounded by recombinant MBL person in the presence of calcium. It demonstrates how similar binding rhMBL with the surface of the microorganism. In fact, MBL binding to a microorganism, could mechanically block the infectivity of specific organisms, such as viruses to cells, in addition to activation of the complement system and act as opsonin.

Example 5: the Confirmation of binding activity of recombinant MBL person

In order to investigate the biological activity of rhMBL, the activity of binding to the pre-S and mannan investigated in the presence of calcium ions.

Mannan or protein pre-S hepatitis B virus was dissolved in 50 mm carbonate-bicarbonate buffer solution was placed in titration the first microplate (Nunc Maxisorp Immunoplate), to get 1 ug per well, then incubated overnight at 4°C. the plate is coated with a pre-S or mannan four times washed with wash buffer (20 mm Tris, 150 mm NaCl, 10 mm CaCl2, 0,05% Tween-20, pH 7,6), followed by blocking with the use of 0.2% BSA at room temperature for one hour. After washing with wash buffer three times a sufficient number of MBL was added to each well to obtain the amount of MBL in each hole, as indicated in the figure. To do this 1 µg of recombinant MBL person in 1 ml binding buffer (20 mm Tris, 1 M NaCl, 10 mm CaCl2, 0,1% BSA, 0.05% of Tween - 20, pH 7,6) serially diluted to get the required number of MBL in 100 μl, which was then added to each well and incubated at room temperature for 2 hours. The tablets are then again washed with wash buffer 6 times. Then mouse monoclonal antibody against MBL person (MBL8F6, Dobeel Corp., Korea) was diluted 10,000 times and add 100 ál to each well, followed by incubation at 37°C for 1 hour. IgG-HRP against mouse was diluted 10,000 times and add 100 ál to each well. Tablet incubated at 37°C for one hour and then staining showed adding 150 μl of TMB solution. After 20 minutes the reaction was stopped by adding 50 μl of 2M H2SO4. OD450change the Yali, using a spectrophotometer to read the tablets ELISA (Multiskan Ex, Labsystems, USA). In Fig. 5A shows the binding of natural MBL purified from human serum with mannan and protein pre-S, and in Fig. 5B shows the binding of recombinant MBL person with pre-S and mannan. In conclusion, recombinant MBL human and natural MBL person had similar binding activity in respect of pre-S and mannan, and both of them were associated with bovine serum albumin (Fig. 5A and Fig. 5B).

Example 6: Comparison of the biological activity of high molecular weight multimeric recombinant MBL person and low molecular weight oligomeric recombinant MBL person

The biological activity of greater and lesser forms of recombinant MBL person were studied in order to show which of the recombinant protein MBL of man, more forms or lesser forms, including monomer and dimer, binds more effectively with pre-S and mannan in the presence of calcium ions.

<6-l> MBL Binding to glycosylated protein pre-S

Squirrels Pre-S was applied to the tablet (Nunc Maxisorp Immunoplate) under the same conditions as described in example 5. Then a large shape or a smaller form was dissolved in binding buffer (20 mm Tris, 1 M NaCl, 10 mm CaCl2, 0,1% BSA, 0.05% of Tween-20, pH 7.6) and was added to the coated pre-S tablet in various concentrations to obtain the necessary if estvo in each well. Further analysis of binding were performed as in example 5. As described previously, the activity of MBL binding protein pre-S was much more multimeric recombinant MBL, compared with a smaller form, which includes, mainly, the monomers and dimers (Fig. 6).

<6-2> Activation of C4 in the complement system

On immunoplate Nunc Maxisorp Immunoplate was applied to 500 ng protein pre-S in the hole, to which were added various concentrations of recombinant MBL person, allowing MBL contact pre-S at room temperature for 2 hours. As a source of protein MASP 100 μl containing MBL serum diluted 100 times, was added to each well and 90 minutes were incubated at room temperature. The tablet is then washed with wash buffer 6 times, then added to 500 ng C4, followed by further interaction at room temperature for 2 hours. Antibody-HRP against C4 was diluted 2000 times and 100 μl was added to each well. Then let the reaction be carried out at room temperature for one hour. Added a hundred and fifty μl of OPD solution and staining was shown in the course of 20 minutes. Then the precipitate C4b was measured after adding 50 µl of 3 M HCl and read OD at OD492using the spectrophotometer to read the tablets ELISA (7). For experiments and multimeric recombinant MBL more f RMI, proposed in the present invention, and recombinant MBL person lesser forms such as monomers or dimers, used respectively for comparison of their activity. As shown in Fig. 7, C4 was significantly activated multimeric recombinant MBL more forms, but barely activated recombinant proteins MBL person lesser forms. Therefore, it is confirmed that recombinant MBL person in the form of a multimeric polymer offered by the present invention, have superior activity against activation of C4.

Example 7: the Binding of recombinant MBL person with different microorganisms

Eleven strains including gram-positive bacteria and gram-negative bacteria and fungi were cultivated in a liquid medium suitable for each of them. In order to mobilitat cultured bacterial cells, 100 μl of the cultured cells were placed on the tablet (Maxisorp Immunoplate, Nunc)to get 1×106cells/well, and incubated at 4°C during the night. Blocking was carried out using 0.2% BSA at 37°C for one hour. Five hundred ng of recombinant MBL man was dissolved in 100 MK binding buffer (20 mm Tris, 1 M NaCl, 10 mm CaCl2, 0,1% BSA, 0.05% of Tween-20, pH 7,6), was added to each well, followed by the interaction of p and 37° C for one hour. Mouse monoclonal antibody against MBL person MB1B5 (Dobeel Corp., Korea) was diluted 10,000 times and add 100 ál to each well, followed by interaction at 37°C for one hour. IgG-HRP against mouse was diluted 10,000 times and add 100 ál to each well, followed by interaction at 37°C for one hour. Then there was added 100 μl of TMB solution and staining was shown in the course of 30 minutes. The reaction was stopped by adding 50 μl of stop solution (H2SO4) and then measured OD450using the spectrophotometer to read the tablets ELISA (Multiskan Ex, Labsystems, USA).

The microorganisms used in the present invention, showed different activity binding to recombinant MBL person and, as shown in Fig. 8, C. albicans, H. influenzae ATCC 51907, S. aureus CCARM 3197 and S. aureus ATCC 29213 showed very high activity binding to recombinant MBL person, but S. pyrogenes ATCC 8668, S. aureus CCARM 3114 and E. faecalis ATCC 29212 showed an average level of activity linking. K. pneumoniae ATCC 10031, S. epidermis ATCC 12228, S. epidermis CCARM 35048 and E. faecium CCARM 5028 had a low activity of binding to MBL. In conclusion, it is proved that the recombinant MBL person like natural MBL is associated with microorganisms, recognizing the pattern of glycosylated surface protein, and the activity of binding varies mi is reorganiza target.

Example 8: Inhibition of infection with SARS-CoV recombinant MBL person

Cells FRhk-4 cells, monkey kidney, were cultured in MEM. To a solution of cultivation was added recombinant MBL man, who then infected SARS-CoV. Recombinant MBL man bred by the environment of the cultivation of cells four times consecutively, starting from a concentration of 2.5 μg/ml, and used the SARS-CoV, a primary isolate from a patient (Ksiazek TG, et al. N. Eng. J. Med 348, 1953-1966, 2003; Peiris et al., Lancet 361, 1319-1325, 2003).

In order to confirm whether the host cells were infected with SARS-CoV and multiply, quantitative real-time PCR (GeneAmp PCR system, Applied Biosystems, USA) was performed using primer sets specific for SARS-CoV. Cells FRhk-4 infected with SARS-CoV and not treated with recombinant MBL person, used as a control group.

As shown in Fig. 9, infection with SARS-CoV inhibited during the processing of recombinant MBL person dependent on dosage. For example, when cells were cultured in the presence of recombinant MBL person at a concentration of 2.5 µg/ml, the proliferation of the virus is inhibited to a significant extent (less than 15% proliferation) compared with the control group. Data 15% most likely originate from viral supernatant.

Fig. 10 is a photograph of a phase-contrast is on microscope showing that cells FRhk-4 infected with SARS-CoV. In Fig. 10 (1) healthy cells were not observed in the control group that was not treated with recombinant MBL man, and Fig. 10 (2-6) healthy cells observed in the test groups treated with recombinant MBL person. In particular, the number of healthy cells increased with increasing concentrations of recombinant MBL person.

Example 9: Determine number of MBL contained in clinical samples

The authors of the present invention have developed a method of determining the number of MBL, part of the clinical samples, such as blood, serum, saliva, etc. that are designed to use the ability of the protein pre-S hepatitis B virus to contact MBL.

<9-l> Immobilization of pre-S on the surface of the gold particles

Preparing particles of colloidal gold the size of 20-80 nm and protein pre-S (approximately 100 μg/ml) was added thereto under conditions of a pH of 6.0-8.0, which was stirred at room temperature for holding. After completion of the reaction, a sufficient amount of 10% BSA was added to obtain a final concentration of 0.3% (weight/volume), which was stirred at room temperature for 15 minutes to further interaction, leading to the blocking surface. Centrifugation was carried out at 12000 rpm./minutes for 15 minutes to DL the separation of the supernatant liquid. Sediment suspended in the buffer solution with addition of 2% BSA. Because proteins Pre-s uniformly contact the surface of colloidal gold particles, created conjugates with dyes that can be used in the preparation of the test set MBL. Such conjugates with dyes are red or purple coloration due to colloidal gold particles.

<9-2> preparation of diagnostic strips

Using an inkjet printer or injection printer, the test line and control line were applied to nitrocellulose membrane (S&S, USA)to obtain a test strip MBL. To get the plank line, used a mouse monoclonal antibody against MBL person, MB1B5 (Dobeel Corp., Korea) and is specific to pre-S monoclonal antibody used for the control line. Compared with the dye prepared above, barbirolli on the substrate for the dye, which is made of polyester or glass fiber, fixed to the lower part of the solid-phase substrate diagnostic strips.

When a drop of the clinical specimen containing MBL person, dripped on the substrate to the sample, which is attached to the substrate for dye diagnostic strips, the sample migrated to nitrocellulose membrane by capillary phenomena and diffusion. When the sample has reached the test line, the complex of the MBL-pre-S-gold particles were captured mouse monoclonal antibody against MBL person on the test line, so the test line was red. And when the sample has reached the control line, and complexes MBL-pre-S-gold particles, and pre-S-gold particles were captured monoclonal antibodies against pre-S, so that the control line was red. Therefore, when the sample contained the MBL, and the test line and control line showed a positive reaction, becoming red. On the other hand, when the sample did not contain MBL, only the control line was red, showing a negative reaction (Fig. 11).

Applicability in industry

As illustrated above, the methods for obtaining recombinant multimeric minnesotabased lectin person (rhMBL) in bulk quantity as proposed in the present invention represent a significant advancement in the development of MBL as applicable commercial product. In a specific multimeric MBL effectively communicates with microorganisms, thus he could effectively inhibit the infection by virus, bacteria or fungi, so it could be used to develop drugs for the prevention and/or treatment of infection by microorganisms such as viruses, bacteria or fungi, in particular, SARS-CoV, and manufacture of a diagnostic kit for determination of MBL person.

Special designed for the professionals in this field will appreciate, what concepts and specific implementation shown in the foregoing description, it can easily be used as a basis for modifying or creating other implementations to accomplish the same purposes of the present invention. Specialists in this field will also appreciate that such equivalent embodiments do not depart from the essence and scope of the invention as defined in the attached claims.

1. Line of master cells of the Chinese hamster ovary (Cho), transfusiona containing polynucleotide encoding minnesotabuy lectin (MBL) of the person expressing vector pMSG-MBL shown on the plasmid map in figure 1, producing MBL.

2. Line host cells according to claim 1, where the cell line is a cell line Cho MBL D1-3, deposited in the Korean collection of type culture under access number XTS VR.

3. The method of mass production of multimeric recombinant MBL person using a cell line according to claim 1, comprising the following stages:

(1) culturing the cell line of claim 1 with the accumulation of recombinant MBL person as a result of expression of the sequence coding region MBL person in cultivated cell, and

(2) purification of recombinant MBL person obtained in stage (1).

4. The method according to claim 3, in which stage (1) characterizes the following stages:

(a) suspension culture cells in serum-free/protein-free medium, and

(b) passage of these cells adapted to serum-free/protein-free environment for gradual scaling.

5. The method according to claim 3, in which stage (2) is characterized by the following stages:

(a) separation of samples containing multimeric recombinant MBL person, using anion-exchange chromatography;

(b) prepare column by filling its substrate, on which the immobilized MBL binding protein;

(c) specific binding of recombinant MBL person with MBL binding protein by adding a column of a sample containing recombinant MBL person, in the presence of calcium ions, after equilibration of the column, and

(d) elution of the recombinant protein MBL of a man with a column when adding a buffer solution with addition of EDTA or EGTA.

6. The method according to claim 5, in which MBL binding protein selected from the group consisting of glycosylated envelope protein of the virus pre-S, glycosylated bacterial protein, glycosylated fungal protein and synthetic glycoprotein.



 

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