Method of human recombinant lactoferrin obtaining

FIELD: medicine; biotechnologies.

SUBSTANCE: adenoviral vector carrying in the genome structure a human lactoferrin gene, administer into an allantois of the 9-10 day chicken embryoses. The subsequent planting is performed by egg incubation at temperature of 37°C within 70-75 hours. Then allocate the recombinant protein from the allantoic liquid of a chicken embryos.

EFFECT: depression of expenses and obtaining simplification of the recombinant human lactoferrin.


The invention relates to biotechnology, in particular to the use of eukaryotes as hosts for adenoviral vectors, and can be used to produce biologically active recombinant lactoferrin (hereafter LF) of a person.

LF - stranded metallovedeniye glycoprotein. A large number of LF is expressed by epithelial cells of the mammary gland, lacrimal, salivary glands. LF is also included in the composition of bile and pancreatic fluid. It is well known that this natural protein has a number of medicinal properties, including bactericidal and bacteriostatic activity, participates in the regulation of humoral and cellular immunological responses, anti-inflammatory and other processes. In this regard, the main task is to obtain LF as the main ingredient for the industrial manufacture of the product.

There are two main directions in obtaining LF.

The first selection from natural sources. Human LF obtained from donor breast milk (n-t of the Russian Federation No. 1709606). However, the disadvantages of the known method of obtaining LF, including the use of donor human milk as a source of LF are the limited availability of raw materials and the necessity of strict control on the presence of infectious agents.

The second is the synthesis of recom is anantnag proteins in prokaryotic and eukaryotic systems. For this purpose, widely used cells of bacteria, yeast, plants, mammals and insects. Recombinant LF Express in various prokaryotic and eukaryotic organisms, including aspergillus (US Pat. No 6.080.559), horned cattle (US Pat. No 5.919.913), rice, corn, yeast Sacharomcyes (US Pat. No 6.228.614) and Pichia pastoris (US Pat. No 6.455.687, 6.277.817, 6.066.469). Each of these methods of synthesis of recombinant proteins has a number of disadvantages, namely:

in prokaryotes, as well as in the cells of many eukaryotes (yeast, insects, plants), except vertebrates, the correct post-translational modification and installation (folding) of the synthesized protein of human rights does not occur at all or is performed incorrectly, which significantly reduces its biological activity;

- receiving recombinant human protein in the culture of cells of vertebrate animals or through their transgenes is a high-cost technology.

Among bioreactors cell-based vertebrates can be classified into three types: cell culture, somatic transgenes and genetic transgenes. As genetic transgenes use different classes of vertebrates, mainly birds and mammals. Bird cages unlike mammalian cells have the advantage that they do not contain viruses, pathoge the data for a person, and prions which could serve as a safe source of recombinant proteins for medical purposes. However, the production of proteins in cell cultures of birds currently in use is limited, due to problems with the delivery of genetic material.

A method of obtaining recombinant human proteins from protein chicken eggs obtained from transgenic chicken, in which the gene is by lentiviral vector was incorporated gene corresponding recombinant protein (U.S. Pat. No 6730822). The use of transgenic birds is related to the duration of the implementation period (up to 24 weeks), high costs, not only for obtaining transgenic herds, but also its contents.

Closest to the claimed invention by a combination of traits is a method for LF, including the transformation of eukaryotic cells with a vector containing a nucleic acid sequence encoding a variant of LF, the cultivation of transformed eukaryotic cells in a suitable nutrient medium before the formation of LF and its secretion (U.S. Pat. No 6111081). However, the known method does not allow to achieve technical results to be obtained from use of the claimed invention, for the following reasons:

1. Requires additional cleaning and testing the resulting p is eparate recombinant LF in the presence of impurities and toxic substances, in particular mycotoxins, which are products of fungi. Allocated originally the Aspergillus aflatoxin potential carcinogens - have a toxic effect on the liver of mammals, birds, fish.

2. The impossibility of using growth medium containing recombinant LF for any purpose without the cleanup phase, whereas allatoona liquid chicken embryo containing recombinant LF, can be used as a dietary Supplement without the cleanup phase.

3. The resulting recombinant LF biological properties is not identical to a protein derived from a natural source.

The claimed invention is directed to solving the problem of preparative production of biologically active recombinant human LF.

Use in clinical practice, the proposed method allows to achieve several technical and economic results:

- the identity of the protein obtained in accordance with the inventive method, biological properties, including biological activity, protein derived from a natural source, through proper post-transcriptional modification and folding of proteins;

- the security of the resulting product due to the fact that it does not contain human pathogens, infectious agents and other dangerous is x impurities;

- short term organization of production up to 10 weeks.

- low cost.

These technical and economic results by carrying out the invention are achieved due to the fact that in the same way as in the known method of recombinant human LF is obtained by injection of viral vector, carrying in their genome a gene human LF, in eukaryotic cell system, cultivation and subsequent isolation of recombinant protein.

The feature of the proposed method lies in the fact that, as a viral vector using recombinant adenovirus birds or mammals, as eukaryotic cellular systems use a chicken egg with the embryo, the cultivation is carried out by incubation at 37 °C for 60-90 hours.

The essence of the invention consists in the introduction in allantoin cavity chicken eggs with embryo recombinant adenoviral vector carrying the genome expressing the cassette with the gene LF cheloveka subsequent expression of the gene of this protein and its accumulation preparative quantities allantoine fluid of chicken embryos. Chicken egg with embryo infected with recombinant adenovirus, plays the role of a natural bioreactor for production of recombinant LF.

Compared to known you upomyanutyi systems in the expression of the preparation of recombinant proteins in allantoine liquid eggs with embryo has a number of features and advantages. Found that in the cells of birds occurring glycosylation and folding of proteins, similar to those in mammalian cells. As a consequence, the synthesized recombinant human protein has all the properties of natural protein derived from milk.

On the other hand, in the case of use in the pharmaceutical industry of the claimed invention, it is possible to achieve high economic effect. On the one hand, the cost of extraction and purification of the target protein from allantoine fluid of chicken embryos is comparable to the cost of extraction and purification from other systems. On the other hand, the content of the embryos does not require expensive equipment, and agricultural production scale source products guarantee the possibility of industrial production of recombinant drug. The result is greatly reduced cost of production raw material.

The method is as follows.

Gene human LF is obtained by amplification for DNA synthesized by RT-PCR (Reverse Tpanscrip'tion System "Invitrogene" No. 12236-014, USA), matrix RNA isolated from the genome of human cells using TRIZOL ("Invitrogene" No. 15596-018, USA). The DNA fragment carrying the gene for human LF, clone into plasmid vector pGEM-T Easy (Promega No. A). Cloning is carried out according to the Protocol attached to Kinabalu for cloning PCR products pGEM-T Easy. Received legirovannoi mixture transform cells of E. coli DH5α ClRb method. Transformed cells are selected on agar LB medium with antibiotic ampicillin (40 g/ml). Plasmid DNA secrete the method of alkaline lysis, analyze using restricted EcoRI, Ncol (No. ER0271, ER0571 "Fermentas", Lithuania) and selected clones carrying the plasmid of the expected size (5316 BP). The primary structure of the cloned fragment in the resulting plasmid pGEM-Lf confirm restriction mapping by restriction endonucleases Bgll (No. ER0071 "Fermentas", Lithuania) and sequencing by the method of Sanger.

For the preparation of recombinant human adenovirus 5-th serotype Ad5-Lf, containing the gene for human LF, produce the cloning of human LF (Lf) from the plasmid pGEM-Lf, hydrolyzed according to the NotI site in the commercial vector pShuttle-CMV, which is set to "AdEasy Adenoviral vector system", on the website for restrictase NotI under the control of the CMV-promoter and polyadenylation signal. The presence of LF gene in the resulting Shuttle vector pShuttle-CMV/Lf confirm restriction fragments length polymorphism analysis using endonuclease EcoRI, EcoRV and PCR. The preparation of recombinant adenovirus Ad5-Lf carried out according to the methodology "AdEasy Adenoviral vector system" (Stratagene Cat. No 240009). The presence of the gene in human LF in the genome of the recombinant adenovirus Ad5-Lf confirmed by PCR.

To obtain recombin nnogo adenovirus birds CELO-Lf, containing the gene for human LF, produce the cloning of human LF (Lf) from the plasmid pGEM-Lf, hydrolyzed according to the NotI site in the Shuttle plasmid vector according to the website for restrictase ESO under the control of the CMV-promoter and polyadenylation signal. This Shuttle plasmid vector is part of a system for the preparation of recombinant adenovirus birds CELO according to the method described by M. Cotten (Michou, A.I., Lehrmann H., Saltik, M., Cotten, M., Mutational Analysis of the Avian Adenovirus CELO, Which Provides a Basis for Gene Delivery Vectors, Journal of Virology, February, 1999, p.1399-1410, Vol.73, No. 2), according to which then retrieves recombinant adenovirus birds CELO-Lf. The presence of the gene in human LF in the genome of the recombinant adenovirus CELO-Lf confirmed by PCR.

Next, determine the titer of recombinant adenoviruses Ad5-Lf and CELO-Lf method belascoaran on the culture of 293 cells (cells of embryonic human kidney) and LMH (Leghorn male hepatoma cells hepatoma cock the Livorno).

The CELO virus-Lf or Ad5-Lf infect 9-10-day SPF chicken eggs with embryos. As a result of earlier experiments found that the infection of the embryo at this stage of development is the most effective accumulation of recombinant protein in allantoine fluid. The infection produced through a hole with a diameter of 1-2 mm in the eggshell from the side of the air chamber, injecting a needle of the syringe 1.5-2.0 SMOD shell to injection of infectious material under chorioallantoic membrane in the allantois of the embryo.

Chicken embryos infect recombinant adenovirus CELO-Lf or Ad5-Lf in doses of 1×106-1×107The BATTLE/the embryo and 1×107-1×109The FIGHT/embryo, respectively. The optimality of this dose was established in previously performed experiments where it was shown that the dose reduction leads to lower product yield (recombinant protein)and increasing the dose to the premature death of a chicken embryo, so that the product yield is also reduced.

Infected chicken embryos incubated at a temperature of 37°C for 60-90 hours. It is established,that during this period of incubation in allantoine fluid accumulate the maximum amount of recombinant human LF.

Control the level of expression of recombinant human LF is carried out by sampling aliquot (0.5 - 1 ml) allantoine fluid through a hole in the eggshell, a small percentage of infected embryos during the whole period of incubation. The level of expression of recombinant human LF in the selected aliquot allantoine fluid produced by ELISA. After completion of the incubation period in allantoine fluid levels of recombinant protein human LF account for 0.9±0.07 mg/embryo during infection with a virus CELO-Lf and 0.2±0.08 mg/embryo during infection with a virus Ad5-Lf.

On abuses stage produces the selection and purification of recombinant human LF of allantoine fluid by the method of affinity chromatography sorbent antibodies to human lactoferrin (At) - sepharose 4 C. the Yield is 64±4% and 60±3% if using recombinant adenovirus birds CELO-Lf and human Ad5-Lf, respectively.

Of the selected recombinant human LF examined for its biological (antioxidant) activity in vitro and in vivo. These methods are based on the ability of recombinant LF to inhibit lipid peroxidation (LPO) in the liver of mice. The analysis of the biological activity (in vitro and in vivo) recombinant protein obtained in accordance with the inventive method, showed his identity natural human LF isolated from donor breast milk.

The inventive method has significant advantages and meets the criteria of patentability.

A method of obtaining a recombinant human lactoferrin, by introducing a viral vector, carrying in their genome a gene of human lactoferrin in eukaryotic cells, culturing and subsequent secretion of the recombinant protein, characterized in that as a viral vector using recombinant adenovirus birds or mammals, as eukaryotic cells use 9-10-day egg with an embryo that infect recombinant adenovirus CELO-Lf or Ad5-Lf by injection into the embryo allantois in doses of 1×106-1×107 The BATTLE/the embryo or 11×07-1×109The FIGHT/embryo, respectively, the cultivation is carried out by incubation at 37 °for 70-75 hours


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