Recombinant plasmid dna pul 44hcmv ensuring the expression of the fragment of the ul44 gene of human cytomegalovirus, encoding the immunodominant part of the dna-binding protein p 52(icp36), in cells of the bacterium escherichia coli

 

(57) Abstract:

The invention relates to biotechnology, particularly genetic engineering. Recombinant plasmid DNA contains a fragment of the UL44 gene of human cytomegalovirus (HCMV), encoding the immunodominant part (2 antigenic epitope) DNA-binding protein p52 (ICP36) virus. The design provides in E. coli cells, the biosynthesis of the polypeptide in the form of a fragment P52 HCMV with polyhistidine tract for affinity purification, possessing antigenic properties of human cytomegalovirus. The use of antigen to detect specific for cytomegalovirus immunoglobulin M(IgM HCMV) in the blood of patients shows 98-99% sensitivity and specificity in enzyme-linked immunosorbent assay. Described plasmid construction allows to increase the synthesis of the target polypeptide in 40-50 times in comparison with the prototype. 4 Il.

The invention relates to biotechnology, in particular genetic engineering, and is designed in vitro recombinant plasmid DNA containing a fragment of the UL44 gene of human cytomegalovirus (HCMV), encoding the immunodominant part (2 antigenic epitope) DNA-binding protein p52 (ICP36) virus with 200 431.to. Design is His) tract for affinity purification, possessing antigenic properties of human cytomegalovirus. This purified using affinity chromatography recombinant protein can be used as a HCMV antigen for serological testing of cytomegalovirus in clinical practice. The use of this antigen to detect specific for cytomegalovirus immunoglobulin M (IgM HCMV) shows 98-99% sensitivity and specificity in enzyme-linked immunosorbent assay.

The main late DNA-binding protein p52(ICP36) of human cytomegalovirus encoded UL44 gene, is a protein with a molecular weight of 52000 daltons, and is one of the proteins associated with HCMV polymerase. This hydrophobic viral protein possessing the properties of antigen cytomegalovirus containing 3 antigenic epitope [1], two of which have a high specificity towards HCMV-specific antibodies and not have serological affinity with other herpesvirus proteins [2].

Unknown ways to get that protein in its pure form [1] of the purified virus. There is a possibility of use in immunological analysis of this protein antigen only in the composition of the viral suspensions or in the form of a lysate of cells infected with qi the ESA, and the use of expensive developments viral material, requiring large expenditures of human embryonic serum, other expensive materials and expensive techniques, such as ultracentrifugation. Even obtained highly purified virus does not guarantee against nonspecific cross-reactions with other viruses when using this antigen in the enzyme-linked immunosorbent assay (ELISA).

Known methods for producing viral protein p52 (ICP36) microbiological synthesis [3], showing its immunochemical activity. However, according to the proposed methods, expressed part of the gene encodes not only the unique antigenic determinants, but also the region of homology with other viral proteins, and there is no affine target for subsequent chromatographic purification of the synthesized protein.

Closest to the claimed technical solution (prototype) is the method described in [1]. Recombinant plasmid DNA contains a gene fragment UL44. Described plasmid construction allows to Express the recombinant protein in the form of a slit with a full-galactosidase protein. The level of expression in the strain, the expression of only parts of the sequence, encoding the immunodominant region; the relatively low level of synthesis of the encoded polypeptide p52 (1 - 3% of the total cellular protein) and the presence of full-galactosidase as a nonspecific antigen in subsequent ELISA and low chromatographic purification of not more than 50-60% of the - galactosidase and total cellular protein.

An object of the invention is the preparation of recombinant plasmid DNA, providing the expression of the fragment of the UL44 gene of human cytomegalovirus, coding all of the immunodominant part p52HCMV with 200 431.K. in the composition of the bacterial plasmid vector encoding a 6*His target for affinity purification of the protein. The design should provide a higher level of biosynthesis of recombinant p52HCMV in E. coli cells and the level of affinity purification using the 6*His target is not less than 95-98% of the recombinant protein.

The problem is solved by constructing recombinant plasmid DNA pUL44HCMV, coding IPTG - induced biosynthesis of a polypeptide having antigenic properties of human cytomegalovirus in E. coli cells in the form of a fragment p52HCMV(c 200 431. K.), 6*His at the N-end for affinity purification.its protein p52 human cytomegalovirus, characterized by the following features:

- has a molecular weight 2,73 megadalton (4,14, etc., O.);

- encodes the amino acid sequence of the immunodominant part of the protein p52HCMV(c 200 431.K.);

- consists of:

- BamHI/ > PST fragment DNA plasmid pQE30 (3,424 etc., O.) [4];

- BamHI/ > PST fragment UL44 gene (716 p. O.), encoding all of the immunodominant protein p52HCMV (200 431.K.);

- contains:

as a genetic marker gene lba-lactamase, which determines the stability of the transformed plasmid pUL44HCMV cells to ampicillin;

- the nucleotide sequence encoding polyhistidine tract in the frame read UL44 gene, consisting of 6 molecules of the amino acid histidine, for further purification using affinity chromatography on Ni-NTA-resin;

unique recognition sites of restriction endonucleases, with the following coordinates:

SmaI - 498; NaeI - 475; NcoI - 530;

BamHI - 146; > PST - 862.

Significant advantages of the proposed plasmid constructions unlike the prototype is the presence of the expressed fragment UL44 gene encoding only the immunodominant protein p52HCMV without - galactosidase and N - terminal region polyhistidine tract that owls is matography 98 - 99% of the total cellular proteins.

The list of graphical materials

Fig. 1. Physical map of recombinant plasmid pUL44HCMV.

Fig. 2. The nucleotide sequence of UL44 gene encoding a hydrophilic immunodominant protein p52.

Fig. 3. Amino acid sequence of the immunodominant fragment of the protein p52HCMV encoded by the recombinant plasmid pUL32HCMV.

Fig. 4. Electrophoregram lysates of E. coli cells (strain TG-1), transformed with plasmid pUL44HCMV synthesizing p52HCMV (lane 3); the recipient strain, the (track 2); recombinant protein p52HCMV, purified by affinity chromatography on Ni-NTA-resin (lane 4). The arrow indicates the recombinant protein p52HCMV.

The invention is illustrated by the following examples:

Example 1. Construction of intermediate recombinant plasmid DNA pUC(UL44).

10 μg of plasmid DNA pUC19 [5] treated with restrictase BamHI > PST and in accordance with the methodology described in [6], and from the resulting hydrolysate is isolated in a 4% polyacrylamide gel vector fragment length 2,664 T. p. O.

10 μg of DNA from the reaction mixture after the polymerase chain reaction with genomic DNA of cytomegalovirus strain AD169 in soo is isolated in 4% polyacrylamide gel fragment length 0,716 T. p. O.

The obtained fragment and the vector portion of the plasmid pUC19 sew using a ligase reaction in 30 μl of buffer for ligation. 10 - 20 ál of the reaction mixture used to transform competent cells of E. coli TG-1[5]. Transformants plated on LB-agar containing 100 μg/ml ampicillin. From grown clones secrete plasmid DNA and analyze DNA restriction analysis. Selected DNA clones for the presence of theoretically predicted fragments.

Example 2. Construction of recombinant plasmid DNA pUL44HCMV.

10 μg of plasmid DNA pUC(UL44) is treated sequentially with restriction endonucleases > PST and BamHI in accordance with the methodology described in [5] , and from the resulting reaction mixture is isolated in a 4% polyacrylamide gel DNA fragment length 0,716 T. p. O.

10 μg of plasmid DNA pQE30 treated with restrictase BamHI > PST and in accordance with the methodology described in [4], and from the resulting hydrolysate is isolated in a 4% polyacrylamide gel vector fragment length 3,424 T. p. O.

The ends of the received fragment and the vector connecting using a ligase reaction in 30 μl of buffer for ligation. 5 - 10 ál of the reaction mixture used to transform competent cells of TG-1 [5]. Transformlist her by processing a set of restriction endonucleases SmaI, NaeI, NcoI, BamHI and > PST and subsequent electrophoretic analysis of the lengths of restriction fragments in a 4% polyacrylamide gel. Of the 10 analyzed clones 10 showed the desired set of restriction fragments. The target plasmid pUL44HCMV contains a unique recognition sites of restriction endonucleases, with the following coordinates:

SmaI - 498; NaeI - 475; NcoI - 530; BamHI - 146; > PST - 862.

The final structure of recombinant DNA pUL44HCMV confirmed by determining the nucleotide sequence in the region of the embedded fragment containing the gene fragment UL44HCMV (Fig. 2).

The expression of the target gene UL44HCMV check on the presence of the recombinant protein of 28 kilodaltons allocated using affinity chromatography on Ni-NTA-resin, after IPTG induction of the transformed target plasmid pUL44HCMV cells of E. coli TG-1 (Fig.4).

Thus, the claimed technical solution allows you to get expressing plasmid DNA pUL44HCMV encoding a fragment of the gene UL44HCMV. Transformed by this plasmid cell culture of E. coli TG-1 after induction of IPTG provides biosynthesis polypeptide size 28 kilodaltons, consisting of a fragment of the protein p52 (200 431.K.) human cytomegalovirus containing 2 antigenic epitope, and RAS is Holocene high-purity up to 98 - 99% recombinant antigen p52HCMV, and increase the synthesis of the target polypeptide in the 40 - 50 times. This recombinant protein after affinity chromatography can be used as a HCMV antigen for serological analysis of cytomegalovirus in clinical practice. The level of detection specific for cytomegalovirus immunoglobulin in the blood of patients using this protein shows 98 - 99% sensitivity and specificity for IgM HCMV in enzyme-linked immunosorbent assay.

The list of references.

1. Landini M. R., Guan, M. H., Jahn G., W. Lindenmeier, M. Mach, Ripalti A. , A. Necker, T. Lazzarotto, B. Plachter Large scale screening of human sera with recombinant cytomegalovirus antigens //J. Clin. Microbiology. 1990. V. 28. P. 1375-1379.

2. Landini, M. R., M. Mach are seaching for antibodies specific for HCMV: it Is diagnostically useful? When and How // Scand. J. of Infect. Dis. 1995. V. 99. P. 18-23.

3. European patent N 0252531, CL 4 C 12 N 15/00, 1988

4. The QIAexpressionist. Hilden: QIAGEN, Summer 1992. P. 70.

5.Maniatis T., Fritsch, Sambuc J. (1984) Molecular cloning. TRANS. from English., M. The World.

6. M. A. Susloparov, P. A. Belavin, A. Century of Krendelyova, A. I. Beketov, M. M. Bakhtin, Century Century Gutorov, I. C. Babkin, A. A. the town Chepurnov (1996) Cloning and primary structure of protein coding genes A and pp150 of human cytomegalovirus (HCMV) //Molecular genetics, microbial gene fragment UL 44 human cytomegalovirus, encoding the immunodominant part of the DNA-binding protein p 52(ICP36), in cells of Escherichia coli bacteria, characterized by the following features:

has a molecular weight 2,73 megadalton (4,14, etc., O.);

encodes the amino acid sequence of the immunodominant part p52 HCMV (200 431.K.);

consists of

BamHI/ > PST fragment DNA plasmid pQE30(3,424 etc., O.);

BamHI/ > PST fragment of the gene UL 44 (716 p. O.), encoding all of the immunodominant protein p52 HCMV (200 431.K.);

contains:

as a genetic marker gene bla-lactamase, which determines the stability of the transformed plasmid pUL 44HCMV cells to ampicillin;

the nucleotide sequence encoding polyhistidine tract in the reading frame of the gene UL 44, consisting of 6 molecules of the amino acid histidine, for further purification using affinity chromatography on Ni-NTA-resin;

unique recognition sites of restriction endonucleases, with the following coordinates: SmaI-498; NaeI-475; NcoI-530; BamHI-146; > PST -862.

 

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