Recombinant plasmid dna pul32hcmv ensuring the expression of the fragment of the ul32 gene of human cytomegalovirus, encoding the hydrophilic part of the basic matrix protein pp150, in the cells of the bacteria escherichia coli

 

(57) Abstract:

The invention relates to biotechnology, particularly genetic engineering. Recombinant plasmid DNA contains a fragment of the UL32 gene of human cytomegalovirus (HCMV), encoding the entire hydrophilic part (3 antigenic epitope) main matrix phosphoprotein RR and located on the 3'end of the DNA fragment encoding polyhistidine tract for affinity purification the protein. Design provides efficient biosynthesis polypeptide. The obtained recombinant polypeptide has antigenic properties of human cytomegalovirus. Use in immunoassay analysis of this antigen to detect specific for cytomegalovirus immunoglobulin in the blood of patients shows 98-99% sensitivity and specificity for IgG-HCMV and 80-90% for IgM HCMV. 4 Il.

The invention relates to biotechnology, in particular genetic engineering, and is designed in vitro recombinant plasmid DNA obtained by the modification of the plasmid pUR290, containing a fragment of the UL32 gene of human cytomegalovirus (HCMV) encoding the entire hydrophilic part (3 antigenic epitope) main matrix phosphoprotein pp150, and also the Oh purification the protein. The design provides for the cells of E. coli efficient biosynthesis of the polypeptide as a fusion with framenum-galactosidase and (6*His) at the C-end fragment RR HCMV. The obtained recombinant polypeptide has antigenic properties of human cytomegalovirus. Use in immunoassay analysis of this antigen to detect specific for cytomegalovirus immunoglobulin in the blood of patients shows 98-99% sensitivity and specificity for IgG-HCMV and 80-90% for IgM HCMV and can be used in clinical practice for serological diagnosis of HCMV.

The main matrix phosphoprotein pp150 human cytomegalovirus is a phosphoprotein with a molecular weight of 150,000 daltons and is a major component of the viral matrix". This hydrophobic viral protein possessing the properties of antigen cytomegalovirus [1] with high specificity against HCMV-specific antibodies, but have little serological affinity with other herpesvirus proteins [1,2].

Known methods for producing this protein in pure form [2] of the purified virus. The disadvantage of this method is the use of cytomegalovirus, human pathogens, and use the serum, other expensive materials and process techniques, such as ultracentrifugation. However, even the obtained high-purity matrix protein pp150 not guarantee against nonspecific cross-reactions with other viruses when using this antigen in the enzyme-linked immunosorbent assay (ELISA). This is due to the presence of homologous with other viruses and cellular proteins antigenic epitopes in the composition of the protein.

Known methods for producing this protein microbiological synthesis [2,3,4], showing its immunochemical activity. However, the expressed part of the gene encodes not only the unique antigenic determinants, but also the region of homology to other viral proteins. In addition, the low level of expression and the lack of affinity of the target for the subsequent chromatographic purification of the synthesized protein is not allowed to use the resulting enzyme-linked immunosorbent assay.

Closest to the claimed technical solution (prototype) is the method described in [3] . Recombinant plasmid DNA pUR(ppl50) contains a fragment of the UL32 gene, encoding the entire hydrophilic part of the protein pp150, in the composition of the IPTG-inducible gene LacZ expressing plasmids pUR290. OPI is and. The level of expression in the strain - producing E. coli reaches 1 - 3% of the amount of cellular proteins.

The disadvantage of the prototype method is the relatively low level of synthesis of the encoded polypeptide pp150 (1 - 3% of the total cellular protein), the presence of full-galactosidase as a nonspecific antigen in subsequent ELISA and low chromatographic purification (no more than 50 - 60% of a - galactosidase and total cellular protein).

An object of the invention is the preparation of recombinant plasmid DNA, providing a higher level of expression in cells of E. coli fragment UL32 gene of human cytomegalovirus, encoding the entire hydrophilic part of the basic matrix phosphoprotein pp150, and 95-98% level of affinity purification the recombinant protein.

The problem is solved by introducing the restriction sites BamHI and HindIII recombinant plasmid DNA pUL32HCMV with a plot, encoding polyhistidine tract, plasmid pUR290, and delete parts of the sequence, the encoding-galactosidase on sites restrictio EcoRV and BamHI. Received expressing design provides IPTG-induced biosynthesis polypeptide, obladaushih the fragment of HCMV pp150, with (6*His) at the C'- end for affinity purification.

Recombinant plasmid DNA pUL32HCMV encoding the immunodominant part of the main matrix phosphoprotein pp150 human cytomegalovirus, characterized by the following features:

- has a molecular weight of 2.09 in megadalton (3,165 etc., O.);

- encodes the amino acid sequence (398 A. K.) the immunodominant part of the main matrix phosphoprotein RR HCMV [3];

- consists of:

EcoRV/HindIII - fragment DNA (1949 p. O.) vector plasmids pUR290 [3] , which contains a fragment of the LacZ gene, expressing only 1/3 part-galactosidase (660 A. K.);

- BamHI/HindIII fragment of plasmid pUR (ppl50) [3], including the UL32 gene encoding all of the immunodominant protein HCMV pp150 (1193 p. O.) [3];

- contains:

as a genetic marker gene bla-lactamase, which determines the stability of the transformed plasmid pUL32HCMV cells to ampicillin;

- nucleotide sequence (23 p. O.), encoding polyhistidine tract in the frame read UL32 gene, consisting of 6 molecules of the amino acid histidine;

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

Cloned - 2082; StyI - 1741; HindIII - 2558; NruI - 1361; SacII - 2290;

Pv is considerably reduced portion of the amino acid sequence of-galactosidase in fused protein pp150 and C'- terminal region polyhistidine tract, all of which gives a higher level of synthesis of the target protein to 10 - 15% with the yield of the pure product after affinity chromatography to 98 - 99% of total cellular proteins.

The thus obtained plasmid construction provides IPTG-induced biosynthesis of recombinant antigen of human cytomegalovirus in bacteria E. coli. 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 shows 98-99% sensitivity and specificity for IgG-HCMV and 80-90% for IgM HCMV.

The list of graphical materials

Fig. 1. Physical map of recombinant plasmid pUL32HCMV.

Fig. 2. The nucleotide sequence UL32 gene encoding a hydrophilic immunodominant protein pp150 with the adjacent C'- terminal region polyhistidine tract and the N'- terminal region fragment-galactosidase.

Fig. 3. Amino acid sequence of the immunodominant fragment of the protein ppl50HCMV encoded by the recombinant plasmid pUL32HCMV.

Fig. 4. Elektroforeticheskiy 150HCMV (lane 3); the recipient strain, the (track 4); recombinant protein ppl50HCMV, purified affinity chromatography on Ni-NTA-resin (lane 2). The arrow indicates the recombinant ppl50HCMV.

The invention is illustrated by the following examples:

Example 1. Construction of intermediate recombinant plasmid DNA pUR(ppl50) with a sequence that encodes polyhistidine tract.

10 μg of plasmid DNA pUR290 [1] is treated with restrictase BamHI and HindIII in accordance with the methodology described in [2], and from the resulting hydrolysate is isolated in a 4% polyacrylamide gel vector fragment length 3,291 T. p. O.

10 μg of DNA from the reaction mixture after the polymerase chain reaction with matrix DNA plasmids pUR(ppl50) in accordance with the methodology [2], modified so that the reverse primer was introduced nucleotide sequence encoding polyhistidine tract, treated with restrictase BamHI and HindIII and from the resulting hydrolysate is isolated in a 4% polyacrylamide gel fragment length 1,216 T. p. O.

The obtained fragment and the vector portion of the plasmid pUR290 sew using a ligase reaction in 30 μl of buffer for ligation [1]. 10 - 20 µl reaction mixture is used for transformation to the clones secrete plasmid DNA and analyze DNA restriction analysis. At the same time the obtained clones after IPTG induction analyzed by SDS-electrophoresis in the presence of recombinant protein 160 kilodaltons, as described previously [2]. DNA clones are selected based on the presence of theoretically predicted fragments and induced recombinant protein secreted in pure form by affinity chromatography on Ni-NTA-resin.

Example 2. Construction of recombinant plasmid DNA pUL32HCMV.

10 μg of plasmid DNA pUR(pp150), coding polyhistidine tract, treated sequentially with restriction endonucleases EcoRV and BamHI, 5'-ends of the complete DNA polymerase I (fragment maple) in accordance with the methodology described in [1], and from the resulting reaction mixture is isolated in a 4% polyacrylamide gel DNA fragment length 3,165 etc., of O. the Ends of the obtained fragment connect using a ligase reaction in 30 μl of buffer for ligation [1]. 5 - 10 ál of the reaction mixture used to transform competent cells of TG-1 [1]. Transformants plated on LB-agar containing 100 μg/ml ampicillin. From grown clones secrete plasmid DNA pUL32HCMV and analyze it by processing a set of restriction endonucleases kpni restriction sites StyI, HindIII, NruI, SacII, PvuII and EcoRI, followed by electrophoretic analysis of the lengths restructurizing fragments. The target plasmid pUL32HCMV contains a unique recognition sites of restriction endonucleases, with the following coordinates:

Cloned - 2082; StyI - 1741; HindIII - 2558; NruI - 1361; SacII - 2290;

PvuII - 53, 1413; EcoRI - 2589 (Fig. 1).

The final structure of recombinant DNA pUL32HCMV confirmed by determining the nucleotide sequence in the region of the embedded fragment containing the gene fragment UL32HCMV and the nucleotide sequence encoding polyhistidine tract (Fig. 2).

The expression of the target gene UL32HCMV check for the presence of recombinant protein 112 kilodaltons allocated using affinity chromatography on Ni - NTA-resin, after IPTG induction of the transformed target plasmid pUL32HCMV the cells of E. coli TG-1 (Fig.4).

Thus, the claimed technical solution allows you to get expressing plasmid DNA pUL32HCMV encoding a fragment of the gene UL32HCMV. Transformed by this plasmid culture of cells of E. coli TG-1 after induction of IPTG provides biosynthesis polypeptide size 112 kilodaltons, consisting of the fused fragment-galactosidase (660 amino acids and 68.5 kilodaltons) containing 3 antigenic epitope hydrophilic segment (397 amino acids - 43,5 kilodaltons) main matrix phosphoprotein pp1 what, s with the prototype to simplify the process of obtaining high-purity up to 98-99% of the recombinant antigen ppl50HCMV due to the introduction of the C'- terminal part of the protein polyhistidine tract, and also due to decrease galactosidases part to 1/3 to increase the synthesis of the target polypeptide 5 to 10 times.

References

1. The Carajás N. C. Cytomegalovirus infection - modern diagnosis/Clinical laboratory diagnostics. 1998. So 2. S. 16-17.

2. Van-Zanten J. , Harmsen, M. C. at al. Humoral immune response against human cytomegalovirus (HCMV)- specific proteins after HCMV infection in lung transplantation as detected with recombinant and naturally occuring proteins //Clin. Diagn. Lab. Immunol. 1995. V. 2. N 2. P. 214-218.

3. 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 proteins gene IE2 and pp150 of human cytomegalovirus (HCMV) //Molecular genetics, Microbiology and Virology, 1996, N1, PP 32-35.

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

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

Recombinant plasmid DNA pUL 32HCMV ensuring the expression of the gene fragment UL 32 human cytomegalovirus encoding the hydrophilic part of the basic matrix protein HCMV pp150, in the cells of the bacteria Escherichia coli; mol. m 2.09 in megadalton size 3,165 etc., of O., containing EcoR V/Hind III DNA fragment (1949 p. O.) vector plasmids pUR 290, with pUR (pp150), including gene UL 32, encoding all of the immunodominant protein HCMV pp150 (1193 p. O.); as a genetic marker gene bla-lactamase, which determines the stability of the transformed plasmid pUL 32HCMV cells to ampicillin; the nucleotide sequence of (23 p. O.), encoding polyhistidine tract in the reading frame of the gene UL 32, consisting of 6 molecules of the amino acid histidine; unique recognition sites of restriction endonucleases, with the following coordinates: Cloned-2082; StyI-1741; HindIII-2558; NruI-1361; SacII-2290; PvuII-53,1413; EcoRI-2589.

 

Same patents:

The invention relates to propertytaxsession systems that require cleavage product a predecessor to the new polypeptide capable of restoring dichloroindophenol and oxidized glutathione to DNA that encodes this polypetide, farmkompanijam comprising the polypeptide, monoclonal antibodies against the indicated polypeptide

The invention relates to genetic engineering, in particular to a technology for obtaining high-yielding strains Eserichia coli - produced recombinant human proteins used in modern medicine as thrombolytic agents

The invention relates to the feeding of material into the cells of the body, namely, the supply of genetic material to living tissue

The invention relates to methods of introducing foreign genetic material into bacteria using vectors in particular to methods of introducing foreign genes into the genomes of gram-negative microorganisms

The invention relates to biotechnology and concerns gumanitarnogo immunoglobulin specific for the protein L-selectin person

The invention relates to agriculture and can be used in breeding, seed production, genetics and physiology of crops

The invention relates to molecular biology and medicine and can be used for rapid detection of allele with a mutationF508 gene transmembrane regulatory protein cystic fibrosis (TRBM) for mass screening of samples

The invention relates to biotechnology and can be used to produce recombinant epidermal growth factor (human) (CAFR)

The invention relates to the field of medicine
Up!