AU2021100454A4

AU2021100454A4 - Expression Vector of Goat Poxvirus Vaccine Strain

Info

Publication number: AU2021100454A4
Application number: AU2021100454A
Authority: AU
Inventors: Chuangfu Chen; Zhiru Guo; Youwen Li; Feng Wei
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-04-15
Anticipated expiration: 2029-01-25

Abstract

The present invention provides an expression vector for expressing foreign protein in recombinant goat poxvirus (GTPV). Exogenous gene is inserted to the downstream of VV I1L promoter or VV P11 promoter in the expression vector, and 12 recombinant GTPV with expressed foreign protein are obtained by homologous recombination with parental strain of GTPV. The flanking sequence in the vector that can be homologous recombination with parental strain of GTPV is vaccine strain of GTPV G14-STV-44-55, containing TK gene or IFNR- y gene or RR small subunit. Using these expression vectors, recombinant GTPV, expressing different foreign proteins, can be constructed by different methods. Moreover, these recombinant viruses can be used as recombinant live vector vaccines for disease prevention and treatment, as well as for the study on the expression, processing, structure and function of different bioactive proteins. 1/4 " Ned A"p Xbol Netd 7.5K gpt Xhol Noti T4 7 ,I Figure 1 The cloning process of the reporter gene GPT and VV7.5 promoter. TK(24Q0) Y - MR(2600 b) RRASE (26 ) Figure 2 The cloning process of TK gene, RR small subunit gene and y-IFN receptor gene.

Description

1/4 Ned

" A"p

Xbol Netd 7.5K gpt Xhol Noti T4

7 ,I

Figure 1 The cloning process of the reporter gene GPT and VV7.5 promoter.

TK(24Q0) Y - MR(2600 b) RRASE (26 )

Figure 2 The cloning process of TK gene, RR small subunit gene and y-IFN receptor

gene.

Expression Vector of Goat Poxvirus Vaccine Strain

TECHNICAL FIELD

[01] The invention provides an expression vector for expressing foreign protein in recombinant GTPV, belonging to the field of biotechnology.

BACKGROUND

[02] With the development of modern molecular biology, people can manipulate the genes of various organisms and master the technology of cloning and recombining DNA in bacterial plasmids, which greatly expand the way people make vaccines, thereby a variety of genetic vaccines to prevent and control animal diseases are developed and virus vector live vaccine is one of them.

[03] Up to now, many kinds of biologically active proteins have been expressed in recombinant poxvirus (VV), and the obtained recombinant VV expressing various pathogens has become a recombinant live vector vaccine for preventing and treating various infectious diseases and even tumours of human and animals. However, there is a wide host range of VV infection and side effects of isolated individual inoculation with VV. Especially, after vaccinating with VV, the individuals with immune deficiency will produce viremia and have the risk of spreading the virus. Therefore, the safety of VV-based recombinant vaccine has aroused more and more concerns. As a result, other poxviruses with higher host specificity are used as live vaccine vectors gradually.

[04] The genome structure and replication characteristics of Capripoxvirushave many similarities with VV, so the strategy of constructing VV vector can be used to construct CPV vector. The LSDV expression vector constructed by Taylor et al, has been recombined to express Rabies virus glycoprotein gene and hemagglutinin gene, which can protect mammals from the attack of corresponding pathogens.

[05] The development of a bivalent or multivalent live Capripoxvirus vaccine, taking attenuated vaccine strain of Capripoxvirus as vector, against foot-and-mouth disease, bluetongue disease and other major diseases that seriously threaten the sheep industry or other foreign diseases can achieve the goal of preventing two or more diseases by once immunization without increasing the cost of epidemic prevention. In addition, it is not only beneficial to the local animal husbandry production, but also of great significance to improve the comprehensive ability of animals in epidemic areas to resist foreign diseases and ensure national biosafety.

SUMMARY

[06] The purpose of the present invention is to construct the expression vector of GTPV vaccine strain G14-TSV44-55, and develop recombinant vaccines of various viruses, bacteria and parasites with GTPV as a living vector, so as to enable GTPV to express antigen proteins of other pathogens. Further, GTPV bivalent or multivalent live vaccine is constructed to achieve the goal of once injection to prevent two or more diseases, thereby reducing the production cost of animal husbandry, alleviating labour intensity and improving economic benefits.

[07] The scheme of the invention is realized as follows.

[08] An expression vector for expressing foreign protein in recombinant GTPV is characterized in that in the expression vector, exogenous gene is inserted to the downstream of VV I1L promoter (<210>10 in sequence list) or VV P11 promoter (<210>11 in sequence list) and 12 recombinant GTPVs with expressed foreign protein are obtained by homologous recombination with parental strain of GTPV.

[09] In these expression vectors, the flanking sequence that can be homologous recombination with parental strain of GTPV is vaccine strain of GTPV G14-STV-44 , containing TK gene (<210>1 in sequence list) or IFNR- y gene (<210>7 in sequence list) or RR small subunit (<210>4 in sequence list).

[010] The promoters for regulating exogenous genes in these vectors are artificially synthesized.

[011] The exogenous genes expressed in these vectors can be antigen genes of viruses, bacteria and parasites, pathogenic genes, tumor-related antigen genes, immune regulatory genes of humans, livestock and poultry, and immune genes of other animals.

[012] The advantages and positive effects of the invention are as follows.

[013] 1. The expression vector with TK gene as flanking sequence of poxvirus strain can not only be homologous recombined with its parents, but also with other GTPV strains. Therefore, recombinant GTPV strains with different biological characteristics can be constructed as required.

[014] 2. The recombinant virus constructed by the expression vector containing GPT reporter gene can be screened out by harvesting diseased cells in the presence of mycophenolic acid, sub-culturing and purifying. Further, they can be detected by PCR at the same time.

[015] With these expression vectors, multiple recombinant GTPV for expressing different exogenous proteins can be constructed for different purposes. These recombinant viruses can be used as recombinant live vector vaccines for the prevention and treatment of different diseases and can also be used as research tools to study the relationship between expression, processing, structure and function of different bioactive proteins.

BRIEF DESCRIPTION OF THE FIGURES

[016] Figure 1 shows the cloning process of the reporter gene GPT and VV7.5 promoter. The method is to digest plasmid PLSEG with XhoI and NotI at the same time, recover the small fragment and connect it to PBS-T vector, and identify the direction of VV 7.5 promoter by nucleotide sequence determination method.

[017] Figure 2 shows the cloning process of TK gene, RR small subunit gene and y-IFN receptor gene. The specific method is to amplify the above three genes and flanking sequences on virus genome by PCR, recover the target fragment by agarose gel electrophoresis, and connect it to PGM-T vector, and identify its correctness by nucleotide sequencing.

[018] Figure 3 shows the cloning process of the reporter gene promoter composite DNA fragment by connecting the promoter and the reporter gene in series. Particularly, four reporter gene promoter composite DNA fragments are obtained by PCR amplification from plasmid PBS-7.5-GPT by using overlapping primers containing VV I1L or VV P11 promoters. The target fragment is recovered by agarose gel electrophoresis and ligated to PGM-T vector, and its correctness is identified by nucleotide sequencing.

[019] Figure 4 shows the process of inserting promoter reporter gene composite DNA fragment into TK gene. Specifically, plasmid PGM-TK is digested with Acc65 I, and then recovered, then the end of plasmid PGM-TK is filled with Klenow Fragment and dephosphorylated with alkaline phosphatase. The plasmid containing four complex DNA fragments is digested with ECOR I, and small fragments are recovered and filled, and then ligated with PGM-TK.

[020] Figure 5 shows the process of inserting promoter reporter gene complex into RR gene. Particularly, the plasmid PGM-RR is digested with BglII, the end is filled with Klenow Fragment and dephosphorylated with alkaline phosphatase. The plasmid containing four complex DNA fragments is digested with ECOR I, and the small fragments are recovered and filled, and then ligated with PGM-RR.

[021] Figure 6 shows the process of inserting the promoter reporter gene complex into the y-IFN receptor gene. Particularly, the plasmid PGM-y-IFNR is digested with Pme I, and recovered and dephosphorylated with alkaline phosphatase. The plasmid containing four complex DNA fragments is digested with ECOR I, and the small fragments are recovered and filled with Klenow Fragment to connected with PGM-TK.

[022] Wherein, the TK gene in the present invention refers to thymidine kinase gene, which is composed of 534bp. TK gene of GTPV vaccine strain is located in the central conserved region of its genome, and its nucleotide sequence is shown as < 222> (936) ~ (1469) in < 210>1 in the sequence list.

[023] RR gene in the present invention refers to RNA reductase gene, including 966 bp. RR gene of GTPV vaccine strain is located in the terminal region of its genome, and its nucleotide sequence is shown as < 222 > (932) ~ (1897) in < 210>4 in the sequence list.

[024] The y-IFNR gene in the present invention refers to the y-interferon receptor gene, which contains 828 bp. The y-IFNR gene of Chinese GTPV vaccine strain is located in the terminal region of its genome, and its nucleotide sequence is shown as < 222 > (833) ~ (1670) in < 210>7 in the sequence list.

[025] The VV I1L and VV P11 promoters in the present invention are identified and published abroad and synthesized by Shanghai bioengineering company, and their nucleotide sequences are respectively as < 210>10 and < 210>11 in the sequence lost.

[026] 7.5 promoter refers to the early and late promoter of vaccinia virus 7.5.

[027] The GPT reporter gene in the present invention refers to the xanthine guanine phosphoribosyl transferase gene of Escherichiacoli.

DESCRIPTION OF THE INVENTION

[028] Construction of recombinant virus

[029] 1. Determination of virus PFU

[030] Monolayer cells of sheep testis were prepared and sub-cultured with 10 cell/ml in a 12-well cell culture plate with 3ml per well. The cells were cultured until they grew into a monolayer. The culture solution was sucked off, and the cells were washed with PBS twice. The GTPV virus were diluted 10 times with PBS at a gradient of 10: 1-10: 5, then each well was inoculated with virus diluent 3001, and one well was set as cell control, and adsorbed at 37C for 1 ~ 1.5 h. Then it was added to MEM

culture medium containing 2% calf serum to culture for 72 ~ 96 h, and the culture medium was sucked off. Washing twice with PBS, then each hole was covered with 1 ml Formalin crystal violet dye solution (preparation method: every 50 ml was added with 10 ml of 5% crystal violet, 37.5 ml of 0.85% NaCl, 1 ml of formaldehyde, and 1.5 ml of H20) for dyeing at room temperature for 30 min, and the dye solution was sucked off. After rinsing twice with distilled water, the number of plaque and PFU of virus stock solution were calculated.

[031] 2. Construction of recombinant expression plasmid

[032] The expression vector was digested with restriction endonuclease Xho I, and then the sticky end of the digested linear vector was filled, which was then dephosphorylated. And then connecting it with the target gene, and the expression plasmid was screened and identified, and then the plasmid DNA was extracted and purified. Transfecting DNA into sheep testicular cells.

[033] 3. Screening methods of recombinant viruses

[034] (1) Preparation of screening solution. Taking a certain amount of DMEM and adding 2.5% of calf serum, 100 U/ ml of penicillin-streptomycin, 20-30 ug/ml of mycophenolic acid (MPA), 230-280 ug/ml of xanthine, 10-20 ug/ml of hypoxanthine, 1-3 ug/ml of aminopterinum and 1-3 ug/ml of thymine.

[035] (2) Preparation of LT cell monolayer for screening recombinant GTPVD expressing GPT in a 12-well cell culture plate. The cultured LT cells were digested with 0.25% pancreatin, the centrifuged cells were suspended with screening solution, and the cells were added into the 12-well cell culture plate with a cell volume of 1x 10' cell/ml, 1ml per well. After that, they were cultured in an incubator of 5%CO2at 37C for 16-24 h until the cells grew up to 70-80%.

[036] (3) The virus harvested after co-transfection of vector with GPT as reporter gene and GTPV vaccine strain was inoculated into sheep testis cells with 300 ~ 500 PFU per well or inoculated with 1/10 of the volume of screening solution. After mixing, culturing in an incubator at 37C with 5% CO2, observing once a day for 14 days continuously. Then collecting the culture medium with cytopathic effects, and centrifuging to remove cell debris. Then using cell lysate to purify the protein and identify the obtained recombinant virus.

[037] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, in keeping with the broad principles and the spirit of the invention described herein.

[038] The present invention and the described embodiments specifically include the best method known to the applicant of performing the invention. The present invention and the described preferred embodiments specifically include at least one feature that is industrially applicable

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. An expression vector for expressing foreign protein in recombinant GTPVs,

characterized in that in the expression vector, exogenous gene is inserted to the

downstream of VV I1L promoter (<210>10 in sequence list) or VV P11 promoter

(<210>11 in sequence list) and 12 recombinant GTPV with expressed foreign protein

are obtained by homologous recombination with parental strain of GTPV.

2. The expression vector as stated in Claim 1, characterized in that the flanking

sequence in the vector that can be homologous recombination with parental strain of

GTPV is vaccine strain of GTPV G14-STV-44-55, containing TK gene (<210>1 in

sequence list) or IFNR- y gene (<210>7 in sequence list) or RR small subunit (<210>4

in sequence list).

3. The expression vector as stated in Claim 1, characterized in that the promoters for

regulating exogenous genes in these vectors are artificially synthesized.

4. The expression vector as stated in Claim 1, characterized in that the exogenous genes

expressed in these vectors can be antigen genes of viruses, bacteria and parasites,

pathogenic genes, tumour-related antigen genes, immune regulatory genes of humans,

livestock and poultry, and immune genes of other animals.