RU2008355C1 - Fragment of dna, coding synthesis of glycoprotein of rabies g virus, recombinant plasmidal dna pvg18-1, coding glycoprotein of rabies g virus, isolate of bacterium excherichia coli is producent of glycoprotein of rabies g virus - Google Patents

Abstract

FIELD: genetic engineering. SUBSTANCE: rabies virus (isolate "Vnukovo-32") was reproduced in cell culture of kidneys of golden hamster. Virions in cultural fluid were concentrated by means of ultrafiltration through special filters and then settled by ultracentrifugation. Virional RNA was separated by means of guanidine-thiocyanate and used for synthesis of cDNA gene of glycoprotein G. cDNA was amplified by means of specific primers according to method of polymerase chain reaction (PCR). Amplified gene of glycoprotein G was cloned in cells E. coli by means of procaryotic vector pUC-19. Sequence of nucleotides and amino acids was detected according to method of Senger. Gene of glucoprotein G was recloned into expressing procaryotic vector pUC-18. Expressed viral protein showed specific immunogenic activity. EFFECT: possibility of use in construction of vaccines against rabies and diagnostic preparations.

Description

 The invention relates to biotechnology, in particular to genetic engineering, and is a DNA fragment encoding the synthesis of rabies G virus glycoprotein G, a recombinant plasmid DNA and a strain containing this recombinant plasmid, a producer of the structural protein G glycoprotein G. The invention can be used to create vaccines and diagnostics against rabies.

 Rabies is one of the oldest diseases known to man. The etiological agent of rabies is a virus that is widespread among warm-blooded animals. Successful treatment of a deadly disease is associated with the creation of the Pasteur vaccine in 1885. To date, it has been developed on the basis of an inactivated virus, however, the cost of such vaccines during systematic use is quite high and, in addition, there is always a risk that, if the virus is not sufficiently inactivated, it can cause rabies during vaccination.

 It is known that the protective effect of vaccines is associated with the surface protein of the coat of rabies virus (protein G [1]). Also, it was found that isolated protein G is able to protect animals from rabies diseases [2].

 Cloning of the cDNA corresponding to the rabies glycoprotein gene of the ERA virus strain was first reported by Anillionis A. and Wunner W. H. et al. [3], and for the CVS-Yelverton E. strain [4].

 A known DNA fragment encoding the rabies virus antigen glycoprotein [5]. The DNA fragment presented in US patent N 4393201, has several disadvantages, it is not suitable for expression of the viral protein due to homopolymer end sequences at the 5 'and 3'-ends of the fragment; the gene contains a mutation that occurred during the synthesis of cDNA with virionic mRNA, which affects the immunogenic and protective properties of protein G (in mature glycoprotein at position 8, leucine is encoded instead of proline).

 A known method of obtaining a DNA fragment encoding the synthesis of glycoprotein G, described in the above US patent [5]. The known method includes culturing rabies virus infected cells, isolating and purifying matrix RNA from them using oligo (dT) cellulose and sucrose gradient ultracentrifugation followed by synthesis of single-stranded and double-stranded cDNA, suturing of homopolymer ends and cloning in E. coli cells. The disadvantages of this method include the difficulty of isolating messenger RNAs, the inability to obtain a pure fraction of virionic mRNAs, the specific synthesis of cDNA, the need to synthesize a second strand, suturing non-specific homopolymer ends and, therefore, conducting a large screening of the obtained clones to search for a clone containing a DNA fragment encoding the synthesis glycoprotein G. The DNA fragment obtained by this method for further use, for example, expression of the viral protein G, is unsuitable and requires laborious genetic engineering x rearrangements - replacement homopolymer end sequences recognizable by restriction endonucleases; replacing the nucleotide sequences encoding the amino acid leucine at position 8 with the sequence encoding the amino acid proline.

 Recombinant DNA is known that encodes the rabies virus glycoprotein G gene [5]. The disadvantages of this recombinant DNA include the fact that it is cloned at the homopolymer ends and is not suitable for cloning without special, laborious genetic engineering rearrangements in the recombinant DNA and viral protein expression.

 Due to the fact that rabies virus does not inhibit the synthesis of cellular RNA during cell infection, and the synthesis of viral mRNA makes up no more than 5% of total cellular RNA synthesis, it is very difficult to clone the mRNA of the glycoprotein G gene. In both cases, the authors had to carry out complex methodological work on the isolation of viral mRNA from infected cells, their cloning and identification of clones containing the glycoprotein G gene.

 A known strain of E. coli x1776 cells into which the rabies virus glycoprotein G gene has been cloned (US Pat. No. 4,393,201). However, data on the expression of viral protein G are not presented in the patent.

 The problem to which the invention is directed, is to obtain a gene encoding the synthesis of rabies glycoprotein G virus, cloning the synthesized gene in Escherichia coli cells and obtaining a producer strain of rabies glycoprotein G.

The essence of the proposed invention is as follows:
The essence of the DNA fragment encoding the rabies virus G glycoprotein G is that it has a size of 1.6 kb. contains the cleavage sites for the restriction enzymes BamHl and Pstl at the beginning and end of the gene, 1 Hindlll restriction enzyme cleavage site, located at a distance of 92 bp from the BamHl-terminus, a Pvull restriction enzyme cleavage site located at a distance of 130 bp from the BamHl-terminus, the Xhol restriction enzyme cleavage site, located at a distance of 514 bp from the BamHl-terminus, the Sphl restriction enzyme cleavage site, located at a distance of 728 bp from the BamHl-terminus, Clal restriction enzyme cleavage site, located at a distance of 1458 bp from the bamhl-end. The fragment contains at the beginning of the ATG gene a codon that initiates protein synthesis, and a TGA codon at the end of the gene that terminates protein synthesis. In total, the fragment encodes the synthesis of 505 amino acids.

 To obtain a DNA fragment encoding rabies virus glycoprotein G, a method is used which consists in culturing the virus of the Vnukovo-32 strain in a cell culture, concentrating and purifying the virus by ultrafiltration and ultracentrifugation, isolation of virion RNA in the presence of guanidine thiocyanate with a phenol-chloroform mixture followed by precipitation with a reference, cDNA synthesis is then carried out using the reverse transcriptase enzyme and a primer complementary to the glycoprotein G gene sequences, and amplification of the glycoprotein G gene by polymerase chain reaction [6] using specific primers containing sequences recognized by the restriction endonucleases BamHl and Pstl and complementary to the ends of the G protein gene.

 The essence of the recombinant plasmid DNA PBG18-1 encoding the synthesis of glycoprotein G, having a size of 4, 2 T. p. , consists in the fact that it contains the plasmid DNA of the pUC18 vector of 2.6 bp in size. consisting of a fragment of plasmid pBR322 with a size of 2186 bp , section of the LacZ operon with a size of 445 bp and polylinker, and Bam H1-Pst1-DNA fragment containing the glycoprotein G gene with a size of 1.6 T. p.

 Like m. plasmids PVG18-1 equal to 2.8 MDa.

 The plasmid DNA PVG18-1 contains unique restriction sites — EcoRl, Sacl, Kpnl, Smal, Xmal, BamHl, Pstl, Clal, Pvul, Xhpl, and 2 Hindlll sites. The position of all these restriction sites is shown in table. 1.

The plasmid contains the following genes:
bla gene for beta-lactamase synthesis and ampicillin resistance;
lacZ gene for the synthesis of beta-galactosidase;
a gene encoding glycoprotein synthesis;
lacZ promoter providing transcription of the glycoprotein G.

 Synthesis of glycoprotein occurs under the control of the lacZ promoter.

 A physical map of the recombinant plasmid PVG 18-1 is shown in FIG. 1; in FIG. 2 is a diagram of a polymer chain reaction (PCR).

 The strain producing rabies glycoprotein G is obtained by transformation of E. coli DN-5 cells using recombinant plasmid PVG18-1.

 The bacterial strain Escherichia coli DN-5, containing the plasmid PVG18-1, was deposited in the All-Union Collection of Microorganisms at the IBPM Academy of Sciences of the USSR and contained in it under the number VKM CR-355D.

 The strain is characterized by the following features.

 Morphological signs: cells are straight, rod-shaped, motile, have peritrichous flagella, gram-negative, non-spore.

 Cultural traits: cells grow well on commonly used culture media. On 1.5% Difco nutrient agar, the colonies are smooth, gray, shiny, the edges are even, cloudy. When grown in liquid media, meat peptone broth and L-broth form a uniform intense turbidity.

Physiological-biochemical characteristics: optimal cultivation temperature 37 ^C, pH 6.8-7.5. As a carbon source, many carbohydrates, organic acids, alcohols are used. Mineral salts in the ammonium or nitrate form, organic compounds in the form of peptone, tryptone, amino acids are used as a source of nitrogen.

 Resistance to antibiotics, shows resistance to ampicillin (100-150 μg / ml).

Genetic features: -genotype: F ^- , recAl, endAl, gyrA96, thi, usdR17, supE44, relAl, L;
The presence of the plasmid PVG18-1 in the E. coli strain containing the DNA fragment, the rabies virus glycoprotein G gene, is confirmed by checking its resistance to ampicillin, as well as by isolating and analyzing plasmid DNA using blot hybridization.

Strain Storage conditions: the strain is stored in 50% glycerol at ^-20 ° C for years, or in the freeze dried state at -70 ^C. (Protective Environment - milk).

 Example 1. Construction of a DNA fragment containing the gene of rabies virus glycoprotein G.

 For cloning of the glycoprotein gene, the Vnukovo-32 rabies virus strain, previously not used for genetic engineering studies, is used [7]. The virus is grown either in a culture of primary cells of the Syrian hamster kidney cells, or in a culture of transplantable stromal cells of monkey kidneys (line 4647). Infection of cells is carried out by introducing into a suspension of cells or a monolayer of the virus at the rate of 0.005 LD / 50 per cell. The first harvest of the virus is carried out after 96 hours of incubation. The obtained collection of virus-containing liquid is clarified through "nuclear" filters with d = 1.9-2.0 μm, after which it is concentrated on a pelicon cassette 50-60 times.

Virions from the concentrated culture fluid was precipitated by centrifugation (25000 rev / min, for 2 hours at ⁴ ° C). The precipitate is homogenized in 5 volumes of a 4 M solution of guanidine thiocyanate, and then extracted with a mixture of phenol: chloroform (1: 1), chloroform and the aqueous phase is precipitated with 0.5 volumes of ethanol. The remaining salts of guanidine thiocyanate are washed with 70% ethanol and reprecipitated with ethanol. Thus obtained virionic RNA is used for the synthesis of cDNA. The reaction mixture in a volume of 50 μl contains the following components: 1 μn virion RNA, 1 μg of primer to the 5'-end of the glycoprotein gene (5'GGATCCAGGAAAGATGGTTCCTCAGGCTCTCCTGTTTG 3 '), 10 mm Tris-HCl pH 8.3; 10 mm MgCl ₂ , 140 mm KCl, 1 mm each of the four triphosphates, 50 units reverse transcriptase of avian myeloblastosis virus. Incubations are for 2 hours at 42 ^C. For hydrolysis of RNA was then incubated for 1 h at 65 ° ^C and precipitated with 3 volumes of ethanol. The obtained cDNA is used to synthesize a DNA copy of the glycoprotein G gene by PCR (Polymerase chain reaction), FIG. 2. The incubation mixture contains 67 mm Tris-HCl pH 8.4; 2 mM MgCl ₂ , 16.6 mM (NH ₄ ) ₂ SO ₄ ; 10 mM 2ME; 200 μM each of deoxytriphosphates, 200 μg / ml BSA, 1 μM 5'-primer (used for cDNA synthesis), 1 μM 3'-primer (5'GCTGCAGCAAGGGGAGGTGATCTTCAGACTTGGATCGT 3 '), 10-100 pg cDNA. The incubation mixture was heated at 94 C ^for 7 min, then incubated for 3 min at ⁵⁶ ° C, centrifuged for 10 s, was added 2 .mu.l (7.6 units. Tag1-polymerase) and 50 ul was layered on top of vaseline oil. Incubated at 70 ^about 10 minutes The following parameters are used for DNA amplification: 1 min at 94 ^{° C,} 4 min at 65-67 ° ^C. The amplification cycle was repeated 25-30 times. Complete amplification 15 minute synthesis at 67 ^C. The mixture is extracted with chloroform, and the amplification products were separated by electrophoresis in 1% agarose gel. DNA fragments corresponding to the expected molecular weight were excised from the gel, eluted, treated with the appropriate restriction enzymes (BamHl; Pst1).

 PRI me R 2. Cloning of the glycoprotein G gene in E. coli cells.

DNA fragments treated with BamHl and Pstl restriction enzymes are ligated with the prokaryotic vector pUC-19 treated with similar restriction enzymes. A 50 μl ligase mixture contains: 0.5 μg DNA fragment, 0.5 μg pUC-19 plasmid DNA, 50 mM Tris-HCl pH 7.6; 10 mM MgC1 ₂ , 10 mM 2-mercaptoethanol, 100 μg / ml BSA, 100 units Phage T-4 DNA ligases. Incubation is carried out at 8 ° C. ^for 16 hours. 25 μl of the ligase mixture is used for transformation. Competent E. coli cells (strain DH-5) are prepared by the standard method described in the manual (Maniatis T., Fritsch E., Sambrook J., Molecular Cloning, 1984, pp. 240-241). Clones were selected on a selective medium containing ampicillin (50 μg / ml) and X-gal (20 μg / ml). Colorless colonies are grown, recombinant plasmid DNA is isolated and analyzed for the presence of an insert by electrophoresis in a 1% gel. Clones containing the inserted DNA fragment were analyzed using restriction endonucleases Pst1, BamHl. The hydrolysis products are analyzed by electrophoresis on a 1% agarose gel. Clones containing a DNA fragment corresponding to the size of the glycoprotein G gene are further analyzed. Analysis of clones carrying recombinant DNA by Sanger sequencing [8] confirms the presence of the nucleotide sequence of the rabies virus glycoprotein gene (clones PVG19-1; PVG69-3 and PVG12-4). Clone PVG19-1 contains the nucleotide sequence of the glycoprotein G gene 1 to 1647.

 PRI me R 3. Obtaining a producer strain of glycoprotein G rabies virus.

Clone PVG19-1, containing the whole glycoprotein G gene, is not suitable for the expression vector, since the Lac promoter is located at the end of the gene. To obtain an expression vector, the gene is cloned into the pUC18 plasmid at the polylinker sites (BamH1-Pst 1). Clone PVG18-1 expressing the immunogenic active rabies virus glycoprotein G is obtained. For expression of the recombinant protein E. coli cells DN-5 (3-5 ml) were incubated in LB medium overnight at 37 ° ^C are then added 100 ml of fresh LB medium and incubated at ³⁷ ° C until the cell density D = 0,5- 0.8. For induction of LacZ promoter, IPTG was added to 2 mM and the cells incubated further at ³⁷ ° C for 4-5 hours. The cells were harvested by centrifugation at 5000 rev / min, 10 min, 0-4 ° ^C. Resuspend in 10 ml cold on ice lysis buffer (0.2 M Tris-HCl pH 7.5; 0.2 M NaCl, 0.01 M Mg acetate, 0.01 M 2-ME, 5% glycerol). To complete lysis and reduce the viscosity of the solution, the cells are treated on an ultrasonic disintegrator 8-10 times for 10-20 s at 22 kHz. Then the homogenate is centrifuged at 5000-10000 rev / min, 30 min, 4 ° ^C. The pellet was dissolved in 10 ml of a solution containing 6M guanidinium chloride and 1% 2-ME. The homogenate is dialyzed overnight against 100 volumes of PBS buffer. The antigenic activity of the isolated protein is investigated by enzyme immunoassay (ELISA). The results are presented in table. 2.

 Thus, the proposed method for producing a DNA fragment allows to obtain a fragment encoding the synthesis of a complete protein G of rabies virus, containing unique restriction sites at the ends of the DNA fragment, allowing you to quickly and efficiently clone it into any genetic engineering system used to create vaccines and diagnostics against rabies.

 The proposed recombinant plasmid PVG18-1, containing the rabies virus glycoprotein G gene, allows to obtain the E. coli strain VKM CR-355D, producer of the rabies virus protein G. The resulting strain can be used to create rabies diagnosticums. (56) 1. Dietzshold B; Wictor T. Y.; Wunner W. H. and Varrichio A; Virology, 1983, 124, 330-337.

 2. Wunner W. H. Dietzschold B, Curtis P. Y., Wictor T. Y., Y. gen. Virol. 1983, 64, 1649-1656.

 3. Anillionis A., Wunner W. H., Curtis P. Y., Nature, 1981, 294, 275-278.

 4. Yelverton E., Norton Sh. , Obiveski Y. H., Goeddel D. V., Sciense, 1983, 219, 614-620.

5. US patent N 4393201, CL C 07 H 21/04, "DNA which codes for glycoprotein of ERA-strain rabies virus."
6. Appelhans H., Biotchnology Education, 1991, vol. 2, N. 1, pp. 31-35.

 7. Selimov M. A. Modern achievements in the field of rabbiology. M.: VNIIMI, 1987, p. 69.

 8. Sanger F., Miklen S., Coukson A. R., Proc. Nat. Acad. Sci. USA 1977, 74, 5463-5467.

Claims (2)

1. A DNA fragment encoding the synthesis of glycoprotein G of rabies virus, the size of 1.6 T. p. obtained by isolation of virionic RNA from rabies virus and synthesis of 1 cDNA chain using specific primers followed by gene amplification, with the following sequence:
GGATCCAGGAAAG ATG GTT CCT CAG GCT CTC CTG TTT GTA CCC CTT CTG GTT TTT
MET Val Pro Gln Ala Leu Leu Phe Val Pro Leu Leu Val Phe
56 CCA TTG TGT TTT GGG AAA TTC CCT ATT TAC ACG ATA CCA GAC
Pro Leu Cys Phe Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp
98 AAG CTT GGT CCC TGG AGC CCG ATT GAC ATA CAT CAC CTC AGC
Lys Leu Gly Pro Trp Ser Pro Ile Asp Ile His His Leu Ser
140 TGC CCA AAC AAT TTG GTA GTG GAG GAC GAA GGA TGC ACC AAC
Cys Pro Asn Asn Leu Val Val Glu Asp Glu Gly Cys Thr Asn
182 CTG TCA GGG TTC TCC TAC ATG GAA CTT AAA GTT GGA TAC ATC
Leu Ser Gly Phe Ser Tyr MET Glu Leu Lys Val Gly Tyr Ile
224 TTA GCC ATA AAA ATG AAC GGG TTC ACT TGC ACA GGC GTT GTG
Leu Ala Ile Lys MET Asn Gly Phe Thr Cys Thr Gly Val Val
266 ACG GAG GCT GAA AAC TAC ACT AAC TTC GTT GGT TAT GTC ACA
Thr Glu Ala Glu Asn Tyr Thr Asn Phe Val Gly Tyr Val Thr
308 ACC ACG TTC AAA AGA AAG CAT TTG CGC CCA ACA CCA GAT GCA
Thr Thr Phe Lys Arg Lys His Leu Arg Pro Thr Pro Asp Ala
350 TGT AGA GCC GCG TAC AAC TGG AAG ATG GCC GGT GAC CCCAGA
Cys Arg Ala Ala Tyr Asn Trp Lys MET Ala Gly Asp Pro Arg
392 TAT GAA GAG TCT CTA CAC AAT CCG TAC CCT GAC TAC AGC TGG
Tyr Glu Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr Ser Trp
434 CTT CGA ACT GTA AAA ACC ACC AAG GAG TCT CTC GTT ATC ATA
Leu Arg Thr Val Lys Thr Thr Lys Glu Ser Leu Val Ile Ile
476 TCT CCA AGT GTA GCA GAT TTG GAC CCA TAT GAC AGA TCC CTT
Ser Pro Ser Val Ala Asp Leu Asp Pro Tyr Asp Arg Ser Leu
518 CAC TCG AGG GTC TTC CCT AGC GGG AAG TGC TCA GGA GTA GCG
His Ser Arg Val Phe Pro Ser Gly Lys Cys Ser Gly Val Ala
560 GTG TCT TCT ACC TAC TGC TCC ACT AAC CAC GAT TAC ACC ATT
Val Ser Ser Thr Tyr Cys Ser Thr Asn His Asp Tyr Thr Ile
602 TGG ATG CCC GAG AAT CCG AGA CTA GGG AAG TCT TGT GAC ATT
Trp MET Pro Glu Asn Pro Arg Leu Gly Lys Ser Cys Asp Ile
644 TTT ACC AAT AGT AGA GGG AAG AGA GCA TCC AAA GGG AGT GAG
Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser Glu
686 ACT TGC GGC TTT GTA GAT GAA AGA GGC CTA TAT AAG TCT TTA
Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu
728 AAA GGA GCA TGC AAA CTC AAG TTA TGT GGA GTT CTC GGA CTT
Lys Gly Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu
770 AGA CTT ATG GAT GGA ACA TGG GTC GCG ATG CAA ACA TCA AAT
Arg Leu MET Asp Gly Thr Trp Val Ala MET Gln Thr Ser Asn
812 GAA ACC AAA TGG TGC CCT CCC GAT CAG TTG GTG AAC CTG CAC
Glu Thr Lys Trp Cys Pro Pro Asp Gln Leu Val Asn Leu His
854 GAC TTT CGC TCA GAC GAA ATT GAG CAC CTT GTT GTA GAG GAG
Asp Phe Arg Ser Asp Glu Ile Glu His Leu Val Val Glu Glu
896 TTG GTC AGG AAG AGA GAG GAG TGT CTG GAT GCA CTA GAG TCC
Leu Val Arg Lys Arg Glu Glu Cys Leu Asp Ala Leu Glu Ser
938 ATC ATG ACA ACC AAG TCA GTG AGT TTC AGA CGT CTC AGT CAT
Ile MET Thr Thr Lys Ser Val Ser Phe Arg Arg Leu Ser His
980 TTA AGA AAA CTT GTC CCT GGG TTT GGA AAA GCA TAT ACC ATA
Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile
1022 TTC AAC AAG ACC TTG ATG GAA GCC GAT GCT CAC TAC AAG TCA
Phe Asn Lys Thr Leu MET Glu Ala Asp Ala His Tyr Lys Ser
1064 GTC AGA ACT TGG AAT GAG ATC CTC CCT TCA AAA GGG TGT TTA
Val Arg Thr Trp Asn Glu Ile Leu Pro Ser Lys Gly Cys Leu
1106 AGA GTT GGG GGG AGG TGT CAT CCT CAT GTG AAC GGG GTG TTT
Arg Val Gly Gly Arg Cys His Pro His Val Asn Gly Val Phe
1148 TTC AAT GGT ATA ATA TTA GGA CCT GAC GGC AAT GTC TTA ATC
Phe Asn Gly Ile Ile Leu Gly Pro Asp Gly Asn Val Leu Ile
1190 CCA GAG ATG CAA TCA TCC CTC CTC CAG CAA CAT ATG GAG TTG
Pro Glu MET Gln Ser Ser Leu Leu Gln Gln His MET Glu Leu
1232 TTG GAA TCC TCG GTT ATC CCC CTT GTG CAC CCC CTG GCA GAC
Leu Glu Ser Ser Val Ile Pro Leu Val His Pro Leu Ala Asp
1274 CCG TCT ACC GTT TTC AAG GAC GGT GAC GAG GCT GAG GAT TTT
Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Ala Glu Asp Phe
1316 GTT GAA GTT CAC CTT CCC GAT GTG CAC AAT CAG GTC TCA GGA
Val Glu Val His Leu Pro Asp Val His Asn Gln Val Ser Gly
1358 GTT GAC TTG GGT CTC CCG AAC TGG GGG AAG TAT GTA TTA CTG
Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Tyr Val Leu Leu
1400 AGT GCA GGG GCC CTG ACT GCC TTG ATG TTG ATA ATT TTC CTG
Ser Ala Gly Ala Leu Thr Ala Leu MET Leu Ile Ile Phe Leu
1442 ATG ACA TGT TGT AGA AGA GTC AAT CGA TCA GAA CCT ACG CAA
MET Thr Cys Cys Arg Arg Val Asn Arg Ser Glu Pro Thr Gln
1484 CAC AAT CTC AGA GGG ACA GGG AGG GAG GTG TCA GTC ACT CCC
His Asn Leu Arg Gly Thr Gly Arg Glu Val Ser Val Thr Pro
1526 CAA ACG TGG AAG ATC ATA TCT TCA TGG GAA TCA CAC AAG AGT
Gln Thr Trp Lys Ile Ile Ser Ser Trp Glu Ser His Lys Ser
1568 GGG GGT GAG ACC AGA CTG TGA GGACTGGCCGTCCTTTCAACG
Gly Gly Glu Thr Arg Leu TER
1610ATCCAAGTCCTGAAGATCACCTCCCCTTAACTGCAG
2. Recombinant plasmid DNA pVG 18-1 encoding rabies virus G glycoprotein G, size 4.2 tp containing:
- plasmid DNA of the vector pUC 18 with a size of 2.6 T. p. consisting of a fragment of plasmid pBR 322 with a size of 2186 bp , section of the LacZ operon with a size of 445 bp and polylinker
- BamH1 - Pst1 - DNA fragment in the genome of rabies virus glycoprotein 1600 p. ,
- restriction enzyme cleavage sites:
in restriction enzyme digestion site E coR I, Sac I, Kpn I, Sma I, Xma I, BamH I, Pst I, Sph I, Gla I, Pvu I, Xho I and 2 Hind III restriction enzyme digestions,
- genetic markers;
bla - a gene that provides the synthesis of beta-lactamase and resistance to ampicillin;
- las Z - gene for the synthesis of beta-galactosidase,
- lac Z - promoter that provides transcription of the glycoprotein G gene.  3. The bacterial strain Escherichia coli VKM CR-355D - producer of rabies glycoprotein G virus.

Images