CN111690619A

CN111690619A - Recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes and construction and application thereof

Info

Publication number: CN111690619A
Application number: CN202010312089.0A
Authority: CN
Inventors: 琚春梅; 陈美静; 何淑菲; 颜志斌; 吴晓燕; 纪秋云
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-04-20
Filing date: 2020-04-20
Publication date: 2020-09-22

Abstract

The invention discloses a recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes, which is a pseudorabies recombinant virus PRV-AH-gE^‑/gI^‑Deleting part of TK gene from the deleted strain, and inserting gC gene at gE/gI gene deletion position; the deleted partial TK gene sequence is shown as SEQ ID NO: 1; the gC gene sequence is shown in SEQ ID NO. 2. The rPRV-AH-TK for bi-expressing gC protein^‑/gI^‑/gE^‑/gC⁺The vaccine can better cause the generation of neutralizing antibodies and can provide better protection effect for immunized mice. The recombinant virus constructed by the invention can be used as a new vaccine candidate strain to provide more choices for controlling the current domestic pseudorabies epidemic situation, and has a larger application prospect.

Description

Recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes and construction and application thereof

Technical Field

The invention relates to the technical field of porcine pseudorabies attenuated live vaccines, in particular to a recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of gC gene, and construction and application thereof.

Background

Pseudorabies is an acute infectious disease caused by pseudorabies virus (PRV), and the main clinical symptoms include fever, nervous system symptoms, respiratory symptoms and the like. In addition to PRV infection in pigs, cattle, sheep, dogs and cats, a number of wild animals can be infected with the disease, with pigs being the natural host for PRV. To date, there is no effective drug for pseudorabies, and vaccination remains an effective means of preventing and controlling pseudorabies. The developed vaccines at home and abroad include inactivated vaccines, attenuated vaccines and gene-deleted vaccines. Because the early domestic and external inactivated vaccines and attenuated vaccines have some defects and the gene editing technology is gradually mature, the genetic engineering vaccines become hot spots for research.

After 1990, PRV gene deletion vaccines are applied in large scale in China, and epidemic situations are effectively controlled on the whole except for accidental occurrence in individual areas. However, since 2011, new pseudorabies epidemic situations have appeared in more than 20 provinces such as Henan, Hebei and Jiangsu. According to the report, epidemic situation also appears in a plurality of pig farms immunized with Bartha-k61 strain gene-deleted vaccines, which indicates that the Bartha-k61 vaccine with double deletion of attenuated gE/gI which is used in China can not provide effective protection for immunized pigs, so that in order to effectively control the prevalence of PRV variants, the development of more efficient vaccines aiming at the PRV variants is urgently needed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of gC genes.

The invention also aims to provide a construction method of the recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of the gC gene.

The invention further aims to provide application of the recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes.

The above object of the present invention is achieved by the following technical solutions:

a recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC gene is a pseudorabies recombinant virus PRV-AH-gE^-/gI^-Deleting part of TK gene from the deleted strain, and inserting gC gene at gE/gI gene deletion position; the deleted partial TK gene sequence is shown as SEQ ID NO: 1; the gC gene sequence is shown in SEQ ID NO. 2.

The invention takes the PRV AH epidemic strain separated from the Anhui pig farm in 2013 as the parent pig, and the PRV-AH-gE strain is obtained^-/gI^-Deleting about 800bp of TK gene on the basis of the deleted strain, and inserting a gC expression box at the gE/gI gene deletion position to obtain PRV-AH-TK^-/gE^-/gI^-/gC⁺A strain. I.e. PRV-AH-TK^-/gE^-/gI^-/gC⁺The recombinant strain aims to further induce body humoral immunity and cellular immunity through double expression of gC, and provides more effective protection for immune pigs.

The invention also provides a construction method of the recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes, which comprises the following steps:

s1, designing 2 pairs of primers TK-LA1-F/R and TK-RA1-F/R by taking a PRV ZJ01 strain PRV AH-China-2013 strain as a template according to a PRV ZJ01 strain gene sequence, and respectively amplifying a left arm fragment LA1 and a right arm fragment RA1 which are positioned at two sides of a TK gene and can be used for homologous recombination; then connected with a pMD18-T vector to construct a recombinant plasmid pMD-LA1-RA1 (TK);

s2, using a plasmid pEGFP-N1 as a template, designing a primer Sosoo-EGFP-F/R to amplify an EGFP expression box, and constructing a transfer plasmid pMD-LA1-EGFP-RA1(TK) for expressing the EGFP;

s3, recombining the product obtained in the step S2Plasmid pMD-LA1-EGFP-RA1(TK) transfects BHK-21 cells, then inoculates PRV-AH-gE^-/gI^-The deletion strain is screened to generate fluorescent recombinant virus to obtain rPRV-AH-Tk^-/gI^-/gE^-/EGFP⁺(TK) strain; using the same method, with rPRV-AH-gI^-/gE^-/EGFP⁺(TK) is a parent strain and a plasmid pMD-LA1-RA1(TK) undergoes homologous recombination and non-fluorescent recombinant virus is screened to obtain rPRV-AH-TK^-/gI^-/gE^-A strain;

s4, amplifying an EGFP expression cassette by taking pEGFP-N1 as a template to construct a pMD-LA-EGFP-RA (gE) plasmid; rPRV-AH-TK^-/gI^-/gE^-Homologous recombination of the strain and the plasmid pMD-LA-EGFP-RA (gE), and then screening and obtaining rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(gE) strain.

S5, using the recombinant plasmid pgC-N1 as a template, designing a primer Sosoo-gC-F/R PCR amplification P_CMVInserting a gC-SV40polyA expression cassette into a transfer plasmid pMD-LA-RA (gE) to construct a plasmid pMD-LA-gC-RA (gE); rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(gE) strain and plasmid pMD-LA-gC-RA (gE) homologous recombination, screening and obtaining rPRV-AH-TK^-/gI^-/gE^-/gC⁺A strain;

the nucleotide sequences of the primer TK-LA1-F/R and TK-RA1-F/R, Sosoo-EGFP-F/R, Sosoo-gC-F/R are sequentially shown as SEQ ID NO. 3-10.

Preferably, said PRV-AH-gE^-/gI^-The construction method of the deletion strain comprises the following steps:

s1, according to a PRV ZJ01 strain gene sequence, using a pseudorabies virus variant PRV AH-China-2013 strain as a template, designing 2 pairs of primers gE-LA-F/R and gE-RA-F/R, and respectively amplifying a left arm fragment LA and a right arm fragment RA which are positioned at two sides of PRV gI and gE and can be used for homologous recombination; then connecting with a pMD18-T vector to construct a recombinant plasmid pMD-LA-RA (gE);

s2, using a plasmid pEGFP-N1 as a template, designing a primer EGFP-F/R to amplify an EGFP expression cassette, and constructing a transfer plasmid pMD-LA-EGFP-RA (gE) for expressing the EGFP;

s3, mixingThe recombinant plasmid pMD-LA-EGFP-RA (gE) prepared in the step S2 is used for transfecting BHK-21 cells, then PRV-AH strains are inoculated, and rPRV-AH-/gI is obtained by screening fluorescence recombinant viruses^-/gE^-/EGFP⁺(gE) strain; by the same method, with rPRV-AH-/gI^-/gE^-/EGFP⁺(gE) is parental strain and plasmid pMD-LA-RA (gE) to generate homologous recombination and screen non-fluorescent recombinant virus to obtain PRV-AH-gE^-/gI^-A strain.

The nucleotide sequences of the primers gE-LA-F/R and gE-RA-F/R, EGFP-F/R are shown in SEQ ID NO. 11-16.

The invention also requests to protect the application of the recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes in preparing a pseudorabies virus vaccine.

The invention also requests a porcine pseudorabies virus gene deletion vaccine, which comprises the recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes.

Preferably, the porcine pseudorabies virus gene deletion vaccine further comprises an adjuvant.

The invention uses rPRV-AH-TK^-/gI^-/gE^-/gC⁺、rPRV-AH-TK^-/gI^-/gE^-At 10⁷TCID₅₀The dose of (a) was inoculated to each of 4-week-old Kunming mice. The results showed no death of the mice and no clinical symptoms of PRV, indicating 10⁷TCID₅₀Is a safe dose for virus inoculation of 4 week old Kunming mice.

With rPRV-AH-TK^-/gI^-/gE^-/gC⁺、rPRV-AH-TK^-/gI^-/gE^-Immunizing Kunming mice of 4 weeks, wherein the immunizing dose is respectively 10⁵TCID₅₀And 10⁶TCID₅₀Meanwhile, a commercial vaccine control group is set up. 10 for 21d after prime and 28d after prime⁶TCID₅₀PRV strains were challenged. The results show that rPRV-AH-TK at the same immune dose^-/gI^-/gE^-/gC⁺Immune group is rPRV-AH-TK^-/gI^-/gE^-Neutralizing antibodies were high (p) in mice of the immunized group<0.05). The toxic action-counteracting effect is shown in 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺And 10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺The virus attack protection rate on the mice is 75.0 percent, and the mice are the immune groups with the highest virus attack protection rate in all the immune groups. Shows that the rPRV-AH-TK of the gC protein is doubly expressed^-/gI^-/gE^-/gC⁺The vaccine can better cause the generation of neutralizing antibodies and can provide better protection effect for immunized mice. The recombinant virus constructed by the invention can be used as a new vaccine candidate strain to lay a foundation for controlling the current domestic pseudorabies epidemic situation.

Compared with the prior art, the invention has the following beneficial effects:

the invention takes PRV AH-China-2013 strain as parent strain, and obtains a recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of gC gene by first construction. The rPRV-AH-TK for bi-expressing gC protein^-/gI^-/gE^-/gC⁺The vaccine can better cause the generation of neutralizing antibodies and can provide better protection effect for immunized mice. The recombinant virus constructed by the invention can be used as a new vaccine candidate strain to provide more choices for controlling the current domestic pseudorabies epidemic situation, and has a larger application prospect.

Drawings

FIG. 1 shows a recombinant virus PRV-AH-TK of the present invention^-/gE^-/gI^-/gC⁺The construction route of (1).

FIG. 2 shows the result of PCR identification of TK deletion of each strain of the invention. M: DL5000 DNA marker; 1: rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(TK)；2：rPRV-AH-TK^-/gI^-/gE^-；3： rPRV-AH-TK^-/gI^-/gE^-/gC⁺(ii) a 4: PRV-AH; 5: and (5) negative control.

FIG. 3 shows the PCR amplification results of gC expression cassette and gE deletion for each strain. M: DL5000 DNA marker; 1: rPRV-AH-TK^-/gI^-/gE^-；2：rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(gE)；3： rPRV-AH-TK^-/gI^-/gE^-/gC⁺(ii) a 4: PRV-AH; 5: and (5) negative control.

FIG. 4 shows a recombinant virus PRV-AH-TK^-/gI^-/gE^-/gC⁺The result of the genetic stability test of (2). M: DL5000 DNASMarker; 1. 2 and 3 are TK-EGFP-F/

R amplification generation

1, 10 and 15 rPRV-AH-TK respectively^-/gI^-/gE^-/gC⁺(ii) a 4: negative control; 5. 6, 7 gE1-F/R amplification of 1, 10, 15 rPRV-AH-TK^-/gI^-/gE^-/gC⁺(ii) a 8: amplifying PRV-AH by gE 1-F/R; 9: negative control; 10. 11 and 12 are gE-F/R amplified

generation

1, 10 and 15 recombinant viruses; 13: and (5) negative control.

FIG. 5 is a growth curve of the virus.

FIG. 6 shows the result of RT-PCR amplification of recombinant viruses. M: DL 2000DNA Marker; 1. 2 and 3 are TK-EGFP-F/R amplified rPRV-AH-TK respectively^-/gI^-/gE^-/gC⁺，rPRV-AH-TK^-/gI^-/gE^-PRV-AH; 4: negative control; 5. 6 and 7 are gE1-F/R amplified rPRV-AH-TK^-/gI^-/gE^-/gC⁺， rPRV-AH-TK^-/gI^-/gE^-PRV-AH; 8: and (5) negative control.

FIG. 7 is a Western Blot assay of recombinant viral gC-His fusion protein.

FIG. 8 shows the result of detecting neutralizing antibodies in the sera of immunized mice. Group A vs group C: a first step of; group A vs group E: a first step of; group A vs group E: (ii) a; group D vs group E: a first step of; group B vs group D: a first step of; group C vs group D: a first step of; group A vs group B: p < 0.05;. p < 0.01;. p < 0.001;. p < 0.0001).

FIG. 9 shows the protection effect of immune mice against toxic challenge.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Materials used in the following examples PRV AH-China-2013 (PRV-AH for short) was a domestic PRV newly circulating strain isolated and preserved in the pre-laboratory, and was now preserved in the microbiological research laboratory of the veterinary medical institute of agriculture university, south China, BHK-21 cells were purchased from ATCC cell bank, E.coli DH5 α engineering bacteria were purchased from TaKaRa company, pEGFP-N1 fluorescent plasmid was purchased from Clontech company, pMD^TM18-T Vector Cloning Kit was purchased from TaKaRa.

The experimental animals were SPF-grade female Kunming mice 6 weeks old, purchased from southern medical university laboratory animal center.

The recombinant virus PRV-AH-TK of the invention^-/gE^-/gI^-/gC⁺The scheme of (2) is shown in FIG. 1, and a transfer plasmid pMD-LA-EGFP-RA (gE) for expressing EGFP was constructed by amplifying an EGFP expression cassette using pEGFP-N1 as a template. Transfecting a plasmid pMD-LA-EGFP-RA (gE) into a BHK-21 cell by using a liposome method, then inoculating an rPRV-AH strain, and screening a fluorescent recombinant virus to obtain the rPRV-AH-gI^-/gE^-/EGFP⁺(gE). By the same method, rPRV-AH-gI^-/gE^-/EGFP⁺Homologous recombination is carried out on parent strains and plasmids pMD-LA-RA (gE) and non-fluorescent recombinant viruses are screened to obtain rPRV-AH-gI^-/gE^-. Knocking out TK gene by the same method to obtain recombinant virus PRV-AH-TK^-/gI^-/gE^-。

And then, using pEGFP-N1 as a template to amplify the EGFP expression cassette, and constructing a pMD-LA-EGFP-RA (gE) plasmid. By homologous recombination, rPRV-AH-TK^-/gI^-/gE^-Homologous recombination occurs between the strain and the plasmid pMD-LA-EGFP-RA (gE), and then rPRV-AH-TK is obtained by screening^-/gI^-/gE^-/EGFP⁺(gE). pgC-N1 constructed from the EGFP gene of the gC gene coding region in place of the pEGFP-N1 plasmid was amplified in the pre-laboratory using PRV AH strain as a template, and a His tag was introduced into the 5' end of the gC coding sequence (CDS) to detect the inserted gC protein. Amplification of P by PCR_CMVA gC-SV40polyA expression cassette is inserted into a transfer plasmid pMD-LA-RA (gE) to construct a plasmid pMD-LA-gC-RA (gE). rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(gE) and plasmid pMD-LA-gC-RA (gE) homologous recombination, screening to obtain rPRV-AH-TK^-/gI^-/gE^-/gC⁺。

EXAMPLE 1 construction of recombinant vectors

1. Construction and identification of transfer vectors pMD-LA1-RA1(TK) and pMD-LA1-EGFP-RA1(TK)

(1) According to the gene sequence of a pseudorabies virus ZJ01 strain (GI:32187339) in GenBank login, a Primer (TK-LA1-F/R) of the upper arm and a Primer (TK-RA1-F/R) of the lower arm of a PRV TK coding region are respectively designed and amplified by using Primer 5.0 software, an EcoR I enzyme cutting site is introduced into the 5 ' end of the TK-LA1-F, a Pst I enzyme cutting site and an EcoR V enzyme cutting site are introduced into the 5 ' end of the Primer TK-RA1-F, a Hind III enzyme cutting site is introduced into the Primer TK-RA 1-R5 ', the Primer information is shown in Table 1, and the primers are synthesized by Scophthal technologies Limited.

TABLE 1 amplification of TK-LA/RA primer sequences

(2) PRV TK homologous arm LA1 is amplified by taking the extracted PRV-AH genomic DNA as a template and adopting TK-LA1-F/TK-LA1-R primers, and PRV TK homologous arm RA1 is amplified by adopting TK-RA1-F/TK-RA1-R primers. The homology arm LA1 and the pMD18-T vector are connected through TA cloning, and then transformation screening is carried out to obtain a recombinant plasmid pMD-LA 1-T; carrying out double enzyme digestion on the recombinant plasmid pMD-LA1-T and RA1 fragments by using restriction enzymes Pst I and Hind III respectively, carrying out connection transformation on enzyme digestion products, screening to obtain a recombinant plasmid pMD-pMD-LA 1-RA1(TK), and carrying out double enzyme digestion identification on the purified recombinant plasmid by using restriction enzymes Hind III and PstI again.

(3) The plasmid pMD-LA1-RA1(TK) was linearized by a single cut with the restriction enzyme Pst I and fragments were recovered.

(4) According to the primer design principle of Sosoo seamless cloning technology, a primer Sosoo-EGFP-F/Sosoo-EGFP-R is designed, a base motif with the length of 15-25 bp consistent with that of two ends of a linearized vector pMD-LA-RA (TK) is introduced into the 5' end of the primer, the specific primer sequence is shown in Table 2, and the primer sequence is synthesized by Populidae biology company.

TABLE 2 primer sequences for amplification of CMV-EGFP-polyA

Fluorescent plasmid pEGFP-N1 is taken as a template to amplify fluorescent protein gene segment CMV-EGFP-polyA.

(5) Seamless cloning of pMD-LA1-RA1(TK) and CMV-EGFP-polyA fragments, which is described in Sosoo seamless cloning reagent specification of Scirridae biotechnology company; and transforming, screening, extracting and carrying out enzyme digestion identification on the seamless cloning product to obtain a transfer vector pMD-LA1-EGFP-RA1 (TK).

2. Construction and identification of transfer vectors pMD-LA-EGFP-RA (gE) and pMD-LA-RA (gE)

(1) Primers (gE-LA-F/R) for amplifying the upper arm and the lower arm of PRV gI and gE coding regions (gE-RA-F/R) were designed using Primer 5.0 software, respectively, based on the gene sequence of strain ZJ01 (GI:32187339) of pseudorabies virus in GenBank accession. By referring to the sequence of fluorescent plasmid pEGFP-N1 (GI:1377911) registered in GenBank, primers EGFP-F/R amplification EGFP expression cassette was designed, EcoR V and pmei cleavage sites were introduced into the upstream primer, and EcoR V cleavage sites were introduced into the downstream primer. The information of the primers is shown in Table 3, and the primers were synthesized by Oncology Biotechnology Ltd.

TABLE 3 gE/gI deletion homology arms gE-LA and gE-RA amplification primer sequences

(2) PRV gE/gI homologous arm LA is amplified by using extracted PRV-AH genomic DNA as a template and adopting gE-LA1-F/gE-LA1-R primers, and PRV gE/gI homologous arm RA is amplified by adopting gE-RA-F/TK-RA-R primers. The homologous arm LA and the pMD18-T vector are connected through TA cloning, and then transformation screening is carried out to obtain a recombinant plasmid pMD-LA-T; carrying out double enzyme digestion on the recombinant plasmid pMD-LA-T and the RA fragment by using restriction enzymes Pst I and Hind III respectively, carrying out connection transformation on enzyme digestion products, screening to obtain a recombinant plasmid pMD-pMD-LA-RA (gE), and carrying out double enzyme digestion identification on the recombinant plasmid pMD-LA-RA (gE) by using EcoR I, Pst I, Hind III and Pst I again for the purified recombinant plasmid.

(3) (3) using a plasmid pEGFP-N1 as a template, adopting an EGFP-F/R primer to amplify the EGFP expression cassette, recovering PCR products, using EcoR V to respectively enzyme-cut the EGFP expression cassette recovery product and a recombinant plasmid pMD-LA-RA (gE), dephosphorylating the enzyme-cut products, then connecting, transforming and screening to obtain a transfer vector pMD-LA-EGFP-RA (gE). Plasmid pMD-LA-EGFP-RA (gE) was identified by double digestion with restriction enzymes Hind III and BamH I.

3. Construction and identification of transfer vector pMD-LA-gC-RA

(1) According to the Sosoo seamless cloning design principle, a primer Sosoo-gC-F/Sosoo-gC-R is designed, a base sequence with 15-25 bp consistency at two ends of a linearized vector pMD-LA-RA (gE) is introduced into the 5' end of the primer, the specific primer sequence is shown in Table 4, and the primer is synthesized by the Protechincal Biotechnology company.

TABLE 4 primer sequences for amplification of CMV-gC-polyA

(2) Primers were designed to amplify the PRV gC coding and a His sequence was introduced at the 5' end. Replacement of the EGFP gene of the pEGFP-N1 plasmid by the gC His series yielded a pgC-N1 plasmid (see patent CN 109750007A). The recombinant plasmid pgC-His-N1 is used as a template, and a gC protein gene fragment CMV-gC-polyA is amplified by using a primer Sosoo-gC-F/Sosoo-gC-R.

(3) The plasmid pMD-LA-RA (gE) is subjected to single-enzyme linearization by using a restriction enzyme EcoR I, the pMD-LA-RA (gE) and CMV-gC-polyA fragments are seamlessly cloned, and the seamless cloning refers to the Sosoo seamless cloning reagent instruction; and transforming, screening, extracting and carrying out enzyme digestion identification on the seamless cloning product to obtain a transfer vector pMD-LA-gC-RA (gE).

Example 2 construction and purification of recombinant viruses

1. Recombinant virus PRV-AH-gI^-/gE^-/EGFP⁺(gE) preparation of

(1) Transfer vector pMD-LA-EGFP-RA (gE) endotoxin removal

The method is described in the instruction of OMEGA endotoxin removal kit.

(2) Homologous recombination of transfer vector pMD-LA-EGFP-RA (gE) with PRV-AH

The transfer vector pMD-LA-EGFP-RA (gE) was linearized with restriction enzyme PstI and transfected according to the Lipofectamine2000Regent Liposome nucleic acid transfection reagent product instructions. After incubation for 4h, discarding the incubation solution, washing the cells with DMEM or PBS for 3 times to prevent the influence of the residual liposome on the cell growth, inoculating the recombinant virus PRV-AH in a virus content of 0.01M.O.I., supplementing 500 μ L of cell maintenance solution, placing at 37 deg.C with 5% CO₂The cells in the cell culture box are continuously cultured. Observing the pathological change condition of the cells at intervals of hours, harvesting the viruses after the cells are completely pathological changed, repeatedly freezing and thawing for 2-3 times, and storing at-80 ℃.

(3) Recombinant virus PRV-AH-gI^-/gE^-/EGFP⁺(gE) empty class screening

Mixing the harvested viruses, and continuously diluting 100 μ L of virus solution by 10 times, i.e. 10^-1～10^-5And (4) diluting. Get 10^-3、10^-4、10^-5The dilution was used to inoculate BHK-21 cells with 100. mu.L of each virus solution. After 12h, the cells were observed for pathology and development of fluorescence under a fluorescence microscope. Due to the recombinant virus PRV-AH-gI^-/gE^-/EGFP⁺(gE) fluorescent proteins can be produced in the cells after infection of the cells and green fluorescence can be observed under a fluorescence microscope. The parent strain does not observe fluorescence under a fluorescence microscope, so that the recombinant virus PRV-AH-TK can be distinguished by using a fluorescent marker^-/EGFP⁺(TK) and parental strain PRV-AH. And (3) searching for the marked fluorescent lesion in the virus inoculation hole, sucking the lesion in a super clean bench, and repeatedly freezing and thawing for 2-3 times and storing at-80 ℃.

Ten times of dilution is carried out on the virus liquid after repeated freeze thawing, namely 10^-1～10^-5Diluting to obtain 10^-3、10^-4、10^-5Diluting, respectively taking 100 μ L of virus liquid, inoculating to BHK-21 cells, and placing at 37 deg.C with 5% CO₂The cell culture box of (1) was incubated for 1 h. Then the culture medium was aspirated off, the cells were washed 2-3 times with PBS, covered with a cell culture medium containing 2% serum supplemented with 1% low-melting agarose, and placed at 37 ℃ in 5% CO₂Cultured in a cell culture box. Observing and marking under a fluorescence microscope after 12h, sucking the fluorescent disease spots on a super clean bench, and repeatedly freezing and thawing. Multiple rounds of selection were performed with reference to the previous selection procedure until all viruses in the cell plate fluoresced, thereby obtaining purified rPRV-AH-gI^-/gE^-/EGFP⁺(gE) strain.

2. Recombinant virus PRV-AH-gI^-/gE^-Preparation of

As above, PRV-AH-gI^-/gE^-/EGFP⁺(gE) strain and pMD-LA-RA (gE) homologous recombination to obtain rPRV-AH-gI^-/gE^-。

3. Recombinant virus PRV-AH-TK^-/gI^-/gE^-/EGFP⁺Preparation of (TK)

As above, PRV-AH-gI^-/gE^-Homologous recombination of the strain and pMD-LA1-EGFP-RA1(TK) to obtain rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(TK)。

4. Recombinant virus PRV-AH-TK^-/gI^-/gE^-Preparation of

As above, PRV-AH-TK^-/gI^-/gE^-/EGFP⁺(TK) strain and pMD-LA1-RA1(TK) are subjected to homologous recombination to obtain rPRV-AH-TK^-/gI^-/gE^-。

5. Recombinant virus PRV-AH-TK^-/gI^-/gE^-/EGFP⁺Preparation of (gE)

As above, PRV-AH-TK^-/gI^-/gE^-Obtaining rPRV-AH-TK by homologous recombination of strains and pMD-LA-EGFP-RA (gE)^-/gI^-/gE^-/EGFP⁺(gE)。

6. Recombinant virus PRV-AH-TK^-/gI^-/gE^-/gC⁺Preparation of (gE)

As above, PRV-AH-TK^-/gI^-/gE^-/EGFP⁺(gE) strain and pMD-LA-gC-RA (gE) homologous recombination to obtain rPRV-AH-TK^-/gI^-/gE^-/gC⁺(gE)。

7. Recombinant virus PRV-AH-TK^-/gI^-/gE^-/gC⁺(gE) PCR identification

Designing a primer gE-F/R to identify the gI/gE deletion gene of the recombinant virus and the inserted gC expression cassette, designing a primer TK-EGFP-F/R to identify the TK deletion gene of the recombinant virus, wherein the specific primer sequence is shown in Table 5, and the primer is synthesized by the Oncology company.

TABLE 5 identification of Strain primer sequences

Designing primers on two sides of a TK gene deletion site to identify TK deletion, taking DNA extracted from a recombinant strain as a template, and performing PCR amplification identification by adopting the primer TK-EGFP-F/R. The results are shown in FIG. 2, rPRV-AH-TK with the fluorescent protein expression cassette inserted^-/gI^-/gE^-/EGFP⁺The amplified fragment of The (TK) strain is about 2159 bp in size, rPRV-AH-TK^-/gI^-/gE^-Strain and rPRV-AH-TK^-/gI^-/gE^-/gC⁺The amplified fragments of the strains are all about 329bp, while the amplified fragment of the PRV-AH strain without deletion of TK gene is about 1128bp, which is consistent with the expectation. Identification of rPRV-AH-TK at the Gene level^-/gI^-/gE^-Strain and rPRV-AH-TK^-/gI^-/gE^-The TK gene has been correctly deleted in the/gC + strains.

Designing primers on two sides of the deletion of gE/gI to identify the deletion of gE/gI and the insertion of a gC expression cassette, taking DNA extracted from a recombinant strain as a template, and adopting a primer TK-EGFP-F/R to carry out PCR amplification identification. The results are shown in FIG. 3, rPRV-AH-TK^-/gI^-/gE^-The PCR amplified fragment of the strain is 1049bp, and the PCR amplified fragment of the PRV-AH strain is 3732bp, which indicates that the gI/gE gene is correctly deleted. And with rPRV-AH-TK^-/gI^-/gE^-The strain is a parent strain and is subjected to homologous recombination to obtain rPRV-AH-TK^-/gI^-/gE^-/gC⁺The PCR amplification fragment size of the strain is about 3897bp, which is consistent with the expected result, and the correct insertion of the gC expression cassette into the gI/gE gene deletion position is preliminarily proved.

Example 3 recombinant Virus PRV-AH-TK^-/gI^-/gE^-/gC⁺Analysis of genetic stability

rPRV-AH-TK^-/gI^-/gE^-/gC⁺Inoculating to BHK-21 cells, harvesting virus after cytopathic effect is complete, repeatedly freezing and thawing, and inoculating to new BHK-21 cells, and repeating the process for 15 generations. And respectively taking virus liquid harvested from the 1 st generation, the 10 th generation and the 15 th generation to extract virus DNA. Primers were designed to identify the inserted gC expression cassette, TK-deleted primers and gE-deleted primers. And (3) performing PCR amplification identification by using the extracted DNA as a template, and performing electrophoresis detection on a PCR product by using 1.0% agarose gel.

As shown in FIG. 4, the TK-EGFP-F/R amplification product fragments are all about 329bp, which is consistent with the TK deletion amplification fragment. The amplification of gE1-F/R did not have the band of interest, indicating that the gE gene had been deleted. The gE-F/R amplified gC expression cassettes inserted into the gE position, and the amplification products were all about 3897bp, consistent with the expectation. Thus the recombinant virus PRV-AH-TK^-/gI^-/gE^-/gC⁺The genetic stability is good.

EXAMPLE 4 plotting of one-step growth curves of recombinant viruses

Recombinant virus rPRV-AH-TK^-/gI^-/gE^-/gC⁺、rPRV-AH-TK^-/gI^-/gE^-And PRV-AH cells with the growth density of 80-90% are inoculated according to the virus amount of 0.01MOI, and after incubation for 1h, the culture solution is discarded. Washing cells with DMEM for 2 times, supplementing cell maintenance solution, and standing at 37 deg.C with 5% CO₂Cultured in a cell culture box. 1 time of virus collection is carried out every 4 hours before 24 hours, 1 time of virus collection is carried out every 8 hours between 24-72 hours, and virus collection is started after 1 hour of virus collection. 3 replicates were made at each time point, and titers were determined after repeated freeze-thawing of the collected virus at each time point, and virus growth curves were plotted.

The results are shown in FIG. 5, rPRV-AH-TK^-/gI^-/gE^-/gC⁺、rPRV-AH-TK^-/gI^-/gE^-The growth kinetics of the strain is similar to that of a PRV-AH parent strain, and no obvious difference exists, which indicates that the inserted gC gene or the deleted TK, gE and gI genes do not influence the growth characteristics of the virus. The virus titer increased more rapidly before 24h and slowly after 24h to the plateau.

Example 5 RT-PCR detection of mRNA levels of TK/gE Gene of recombinant Virus

(1) Extraction of Total RNA from cells

rPRV-AH-TK^-/gI^-/gE^-/gC⁺、rPRV-AH-TK^-/gI^-/gE^-And PRV-AH virus is inoculated on BHK-21 cells, when the cytopathic effect reaches 90%, the total RNA of the cells is extracted, and the operation method refers to the instruction of the OMEGA total RNA extraction kit.

(2) Reverse transcription of RNA

The extracted total RNA was reverse transcribed and DNA removed using the reverse transcription reagents golden RT6 cDNA Synthesis Kit and gDNA Remover from Oncorks.

(3) PCR detection of TK/gE of recombinant viral cDNA

According to TK/gE sequence analysis of PRV prevalent in China on GenBank, primers TK1-F/R and gE1-F/R for amplifying TK/gE conserved sequence are designed by using Primer 5.0, the Primer sequences are shown in the following table 6, and the primers are synthesized by Pomaceae Biotech company.

TABLE 6 RT-PCR primer sequences

PCR amplification was performed using the reverse transcribed cDNA as template, primers TK1-F/R and gE 1-F/R.

The results are shown in FIG. 6, and rPRV-AH-TK was amplified with primer TK1-F/R^-/gI^-/gE^-/gC⁺And rPRV-AH-TK^-/gI^-/gE^-Without a band, the PRV-AH virus amplified band is about 221bp in size. The recombinant virus rPRV-AH-TK amplified by using a primer gE1-F/R^-/gI^-/gE^-/gC⁺And rPRV-AH-TK^-/gI^-/gE^-There was no band of interest, while the band amplified by PRV-AH virus was about 292bp in size. The deletion of TK and gE genes is further verified.

Example 6 recombinant Virus PRV-AH-TK^-/gI^-/gE^-/gC⁺Western Blot detection of inserted gC gene

(1) Extraction of Total cellular protein

Recombinant virus PRV-AH-TK^-/gI^-/gE^-/gC⁺、PRV-AH-TK^-/gI^-/gE^-And PRV-AH is inoculated on BHK-21 cells, and when the cytopathic effect reaches 90%, the total cell protein is extracted

(2) SDS-PAGE gel electrophoresis

(3) Rotary film

(4) Sealing of

(5) Primary antibody incubation

Diluting the primary antibody according to the instruction of His mouse primary antibody of Biyunsian, placing the protein membrane in the diluted primary antibody for incubation, and incubating for 1h on a shaking bed at room temperature, wherein the incubation can be carried out overnight at 4 ℃ under the condition of poor effect. The internal reference was made with beta-actin primary antibody, as described above.

(6) Incubation with secondary antibody

The dilution was performed at 1:1000 with reference to the specification of horseradish peroxidase-labelled goat anti-mouse secondary antibody from Biyunnan. The washed protein membrane was incubated with secondary antibody for 1h on a slow shaker at room temperature.

(7) Protein detection

Preparing a BeyoECL Plus color development solution, sucking the color development solution and dropping the color development solution on a protein membrane, incubating for 1min, performing protein detection on a gel imaging system, and storing an exposure image.

The results are shown in FIG. 7, for the recombinant virus PRV-AH-TK^-/gI^-/gE^-/gC⁺Three bands of 92KDa, 77KDa and 55KDa are detected, the sizes of the bands are consistent with the apparent protein size generated by the factor for predicting the glycosylation of gC, and no protein is detected by other strains by using His primary antibody, which indicates that the recombinant virus PRV-AH-TK is indicated^-/gI^-/gE^-/gC⁺The inserted gC expression cassette is capable of normally expressing gC protein.

Example 7 recombinant Virus safety experiments

10 SPF Kunming mice, 4 weeks old, were randomly divided into two groups of 5 mice each. One group of mice injected with rPRV-AH-TK^-/gI^-/gE^-/gC⁺Another group of mice was injected with rPRV-AH-TK^-/gI^-/gE^-The virus content is 10⁷TCID₅₀All mice were injected intramuscularly in the hind leg and were continuously observed for symptoms and death in 14d mice.

Results show that 10⁷TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺And rPRV-AH-TK^-/gI^-/gE^-Mice were inoculated and observed continuously for 14d, with no mice dead and no typical symptoms of PRV. Description of the preferred embodiments 10⁷TCID₅₀Is rPRV-AH-TK^-/gI^-/gE^-/gC⁺And rPRV-AH-TK^-/gI^-/gE^-Safe dose to Kunming mice, the minimum safe dose of recombinant virus to Kunming mice is higher than 10⁷TCID₅。

Example 8 mouse immunization and challenge experiments

(1) 56 SPF Kunming mice, 4 weeks old, were randomly divided into 7 groups, numbered A-G. A. Group B mice immunize rPRV-AH-TK^-/gI^-/gE^-Vaccine with immunization dose of 10⁵TCID₅₀、10⁶TCID₅₀. C. Group D mice immunize rPRV-AH-TK^-/gI^-/gE^-/gC⁺Vaccine with immunization dose of 10⁵TCID₅₀、10⁶TCID₅₀. Group E is commercial attenuated live vaccine control group (HB 2000 produced by pre-family organism), and is immunized according to the instruction, and the immunization dose of each mouse is not less than 10⁵TCID₅₀. Group F served as challenge control. Group G was DMEM as a negative control. Carrying out second immunization after 3 weeks of first immunization, and attacking the mice in other groups except DMEM group after 4 weeks of second immunization with the dose of attacking the mice being 10⁶TCID₅₀PRV-AH. To follow the detection of neutralizing antibodies in miceNeed carry out the orbit blood sampling to the mouse in the room, the blood sampling time is: 1 week before immunization, 2 weeks after immunization, 4 weeks, 6 weeks, 7 weeks. After challenge, mice were observed for symptoms and death for 14d, and were dissected 14d later.

(2) The collected blood of the mouse was placed in a 37 ℃ incubator for 1 hour and then transferred to a 4 ℃ refrigerator overnight. After the blood separates out serum as much as possible, centrifuging for 10min at 4000r/min, transferring the separated serum to a new centrifugal tube, inactivating for 30min at 56 ℃, and detecting serum antibody by using a neutralization experiment.

(3) Neutralization test

The level of neutralizing antibodies in mice was determined by the fixed virus-diluted serum method.

Immunized mice were bled at the orbital week 2, week 4, week 6 and week 7 after the primary immunization and tested for serum neutralizing antibodies using a neutralization assay. From the measured neutralizing antibody levels at each time point, a line graph is plotted, as shown in fig. 8, showing that the antibody levels gradually increased after the first immunization, rapidly increased after the boost immunization, and reached the highest level at week 6, which is the best time period for analyzing vaccine variability at week 6. Neutralization antibodies generated between the 6 th week vaccines were analyzed for differences using Graphpad software. 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺Immune group antibody level ratio 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-High, significantly different (p) in the immune group<0.0001)。10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺ Immune group ratio 10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-High antibody level with significant difference (p)<0.5)。10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺The antibody titer of the immunized group was not significantly different from that of the commercial vaccine (p)<0.5)。 10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-And 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺The antibody titers did not differ significantly (p)<0.01)。10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺The highest antibody level in the immune group was 1: 168.

Post-secondary immunization 10 at 28d⁶TCID₅₀The PRV-AH strain attacks the virus on the mice, and the symptoms and death condition of the mice after attacking the virus are observed for 14 days continuously. The results are shown in FIG. 9, where the mice in the challenge control group all died at challenge 3d, 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺Immunization groups and 10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺Immune groups were compared with rPRV-AH-TK^-/gI^-/gE^-The immune group has higher toxic challenge protection rate, and all rPRV-AH-TKs^-/gI^-/gE^-/gC⁺The immune group has higher protection rate than the commercial vaccine immune group. 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺And 10⁶TCID₅₀rPRV-AH-TK^-/gI^-/gE^-Has comparable neutralizing antibody levels, and 10⁵TCID₅₀rPRV-AH-TK^-/gI^-/gE^-/gC⁺A higher protection rate can be provided.

Sequence listing

<110> southern China university of agriculture

<120> recombinant pseudorabies virus TK/gI/gE deletion strain with double-copy gC genes, and construction and application thereof

<141>2020-04-20

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acacgcgcct ggtcccgctc ttcgggcccg cggtcgaggg cccgcccgag atgacggtcg 180

tctttgaccg ccacccggtg gccgcgacgg tgtgcttccc gctggcgcgc ttcatcgtcg 240

gggacatcag cgcggcggcc ttcgtgggcc tggcggccac gctgcccggg gagccccccg 300

gcggcaacct ggtggtggcc tcgctggacc cggacgagca cctgcggcgc ctgcgcgccc 360

gcgcgcgcgc cggggagcac gtggacgcgc gcctgctcac ggccctgcgc aacgtctacg 420

ccatgctggt caacacgtcg cgctacctga gctcggggcg ccgctggcgc gacgactggg 480

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gcccgcgcga cgaccccgag ctccaggaca ccctcttcgg cgcgtacaag gcgcccgagc 600

tctgcgaccg gcgcgggcgg ccgctcgagg tgcacgcgtg ggcgatggac gcgctcgtgg 660

ccaagctgct gccgctgcgc gtctccaccg tcgacctggg gccctcgccg cgcgtctgcg 720

ccgcggccgt ggcggcgcag gcgcgcggca tggaggtgac ggagtccgcg tacggcgacc 780

acatccggca gtgcgtgtgc 800

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cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 180

caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 240

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cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 360

ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 420

tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 480

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tttttaaaac cgcgatgggg tgacgggggg gccattcgca cgcgccatgg cctcgctcgc 780

gcgtgcgatg ctcgcgctgc tggcgctcta cacggcggcc atcgccgcgg cgccgtcgtc 840

cacgacggcg ctcggcacga cgcccaacgg gggcgggggc ggcaacagca gcgcgggcga 900

gctctcgccc tcgccgccct cgacgcccga gcccgtctcg gggacgacgg gggccgcggc 960

ctccacgccc gccgccgtct cgacgccccg ggtcccgccg ccctcggtct cgcgccggaa 1020

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gcgcttccgc tcgcccgacg ccgaccccga gtactttgac gagcccccgc gcccggagct 1260

cccgcgggag cggctcctct tcagctccgc caacgcctcc ctcgcccacg cggacgcgct 1320

cgcctccgcc gtcgtcgtcg agggcgagcg cgcgaccgtc gccaacgtct cgggcgaggt 1380

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cgtccgcgac tactacccgc ggcgcagcgt gcgcctgcgc tggttcgcgg acgagcaccc 1620

ggtggacgcc gccttcgtga ccaacagcac cgtggccgac gagctcgggc gccgcacgcg 1680

cgtctccgtg gtgaacgtga cgcgcgcgga cgtcccgggc ctcgcggccg cggacgacgc 1740

ggacgcgctc gcgccgagcc tgcgctgcga ggccgtgtgg taccgcgaca gcgtggcctc 1800

gcagcgcttc tccgaggccc tgcgccccca cgtctaccac ccggcggcgg tctcggtgcg 1860

cttcgtcgag ggcttcgccg tctgcgacgg cctctgcgtg cccccggagg cgcgcctcgc 1920

ctggtccgac cacgccgccg acaccgtcta ccacctcggc gcctgcgccg agcaccccgg 1980

cctgctcaac gtgcggagcg cccgcccgct gtcggacctc gacgggcccg tcgactacac 2040

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tcgcgtccgg ttgtttgttt gtgcgtgtgt gtgtgagctc tcgcgcgtcc ccgggccccg 2340

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ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa 2580

cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg 2640

ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 2700

tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata 2760

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Claims

1. A recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of gC genes is characterized in that the pseudorabies recombinant virus PRV-AH-gE is^-/gI^-Deleting part of TK gene from the deleted strain, and inserting gC gene at gE/gI gene deletion position; the deleted partial TK gene sequence is shown as SEQ ID NO: 1; the gC gene sequence is shown in SEQ ID NO. 2.

2. The method for constructing a recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of a gC gene as claimed in claim 1, comprising the following steps:

s3, transfecting the recombinant plasmid pMD-LA1-EGFP-RA1(TK) prepared in the step S2 to BHK-21 cells, and then inoculating PRV-AH-gE^-/gI^-The deletion strain is screened to generate fluorescent recombinant virus to obtain rPRV-AH-Tk^-/gI^-/gE^-/EGFP⁺(TK) strain; using the same method, with rPRV-AH-gI^-/gE^-/EGFP⁺(TK) is a parent strain and a plasmid pMD-LA1-RA1(TK) undergoes homologous recombination and non-fluorescent recombinant virus is screened to obtain rPRV-AH-TK^-/gI^-/gE^-A strain;

S5, using the recombinant plasmid pgC-N1 as a template, designing a primer Sosoo-gC-F/R PCR amplification P_CMVA gC-SV40polyA expression cassette is inserted into a transfer plasmid pMD-LA-RA (gE) to construct a plasmid pMD-LA-gC-RA (gE); rPRV-AH-TK^-/gI^-/gE^-/EGFP⁺(gE) strain and plasmid pMD-LA-gC-RA (gE) homologous recombination, screening and obtaining rPRV-AH-TK^-/gI^-/gE^-/gC⁺A strain;

3. The method of claim 2, wherein the PRV-AH-gE is present^-/gI^-Construction method of deletion strainThe method comprises the following steps:

s2, amplifying an EGFP expression cassette by using a plasmid pEGFP-N1 as a template, and constructing a transfer plasmid pMD-LA-EGFP-RA (gE) for expressing the EGFP;

s3, transfecting the recombinant plasmid pMD-LA-EGFP-RA (gE) prepared in the step S2 to BHK-21 cells, then inoculating a PRV-AH strain, and obtaining rPRV-AH-/gI by screening a fluorescence-emitting recombinant virus^-/gE^-/EGFP⁺(gE) strain; by the same method, with rPRV-AH-/gI^-/gE^-/EGFP⁺(gE) is parental strain and plasmid pMD-LA-RA (gE) to generate homologous recombination and screen non-fluorescent recombinant virus to obtain PRV-AH-gE^-/gI^-A strain.

4. Use of the recombinant pseudorabies virus TK/gI/gE deletion strain with double copies of gC gene as claimed in claim 1 in the preparation of a pseudorabies virus vaccine.

5. A porcine pseudorabies virus gene deletion vaccine, characterized by comprising the recombinant pseudorabies virus TK/gI/gE deletion strain of the double-copy gC gene of claim 1.

6. The porcine pseudorabies virus gene deletion vaccine according to claim 5, further comprising an adjuvant.