CN107988174A - A kind of rabies-canine distemper-the strain of canine parvovirus gene recombined virus, construction method and its application - Google Patents

Abstract

The invention discloses a rabies-canine distemper-canine parvovirus gene recombinant virus strain, a construction method and an application thereof. The invention carries out gene rearrangement on the full-length genome of the virus through homologous recombination, gene replacement and gene insertion methods, and constructs a rearranged rabies virus genome plasmid with the canine distemper G gene and the canine parvovirus VP2 gene, named: rabies-dog Distemper‑canine parvovirus, pcDSRV ₉ ‑NG‑eGFP+CDV N‑PM+VP2‑L genomic plasmid; rabies‑distemper‑canine parvovirus, rSRV ₉ +CDV N+ rescued by reverse genetics The VP2 gene recombinant virus, the gene nucleotide sequence of the recombinant rabies virus is SEQ ID NO:1. The recombinant gene virus of the present invention can be used for the development of triple oral vaccines of rabies, canine distemper and parvovirus for canines and other wild animals to prevent three kinds of infectious diseases.

Description

A kind of rabies-canine distemper-canine parvovirus gene recombinant virus strain and construction method and its application

technical field

The invention relates to the field of biological virus vaccine research, in particular to a rabies-canine distemper-canine parvovirus gene recombinant virus strain, a construction method and application thereof.

Background technique

Rabies virus belongs to Rhabdoviridae, and its genome encodes five structural proteins: RNA-dependent RNA polymerase (L), nucleoprotein (N), phosphoprotein (P), matrix protein (M), outer membrane protein (G ), G protein and N protein are the main antigens of rabies virus, which can stimulate the body to produce corresponding antibodies and cellular immunity. Rabies is mainly transmitted through animal bites or scratches. The virus is transmitted along the peripheral nerves to the nerve center, resulting in cell function damage and the death of infected animals. At present, vaccine immunization is the most effective means to prevent and control the disease, relying on vaccine immunization can reduce the occurrence of rabies.

Therefore, it is extremely important to understand and master the relevant information of rabies virus, among which, in order to increase the expression of the antigen gene G gene, and avoid affecting the expression of rabies immunogenicity due to the insertion of foreign genes. At present, the research on rabies is mostly limited to one virus of rabies, but there are relatively few studies on viruses constructed by gene recombination of multiple antigens.

Contents of the invention

In view of this, the embodiment of the present invention provides a recombinant rabies virus strain, a construction method and its application, the main purpose of which is to construct a rearranged rabies virus with a canine distemper G gene and a canine parvovirus VP2 gene for the development of Rabies-distemper-parvovirus triple oral vaccine.

In order to achieve the above object, the present invention mainly provides the following technical solutions:

On the one hand, the embodiment of the present invention provides a rabies-canine distemper-canine parvovirus gene recombinant virus strain, which is a rearranged rabies virus with a canine distemper G gene and a canine parvovirus VP2 gene, named: rabies - canine distemper-canine parvovirus gene recombinant virus, the gene nucleotide sequence of the recombinant virus is SEQ ID NO:1.

On the other hand, the embodiment of the present invention provides a method for constructing the above-mentioned rabies-canine distemper-canine parvovirus gene recombinant virus strain, the method comprising the following steps:

Amplify the N, P, M, G, and L genes of the rabies SRV ₉ virus respectively;

The Gibson Assembly connection method was used for recombinant gene connection, the G gene of the SRV ₉ virus was transferred from the fourth position of the original genome sequence to the second position of the viral genome, and then the eGFP reporter gene was inserted into the G and P genes through restriction sites In between, a pcDSRV _9- NG-eGFP-PML gene recombination vector was constructed;

Amplify the CDV N gene in the canine distemper pMD18-CDV N recombinant plasmid by PCR method;

The end restriction site of the eGFP gene of the pcDSRV ₉ -NG-eGFP-PML recombinant vector was digested by enzyme digestion, followed by Hpa I digestion, and the canine distemper CDV N gene was constructed into pcDSRV ₉ -NG-eGFP by Gibson Assembly ligation - On the PML recombinant vector, a pcDSRV ₉ -NG-eGFP+CDV N-PML recombinant vector was constructed;

The VP2 gene in the canine parvovirus pMD19-T-VP2 recombinant plasmid was amplified by PCR;

Carry out BsiWI digestion at the end of the M gene restriction site in the pcDSRV ₉ -NG-eGFP+CDV N-PML recombinant vector by enzyme digestion, and insert the amplified VP2 gene into pcDSRV ₉ -NG-eGFP by Gibson Assembly ligation + Between the ML genes of the CDVN-PML vector, a pcDSRV ₉ -NG-eGFP+CDV N-PM+VP2-L gene recombination vector was constructed;

Recombinant rabies-distemper-canine parvovirus, rSRV ₉ + CDV N + VP2 gene recombinant virus, was rescued by reverse genetic method.

In yet another aspect, the embodiment of the present invention provides the application of the above-mentioned recombinant rabies virus strain in the preparation of medicaments for preventing rabies.

In another aspect, the embodiment of the present invention provides a vaccine composition, which contains the above-mentioned recombinant rabies virus strain.

Compared with prior art, the beneficial effect of the present invention is:

The present invention uses the Gibson Assembly connection method to rearrange the genes of rabies, canine distemper and canine parvovirus, and constructs the rearranged rabies virus genome plasmid with the canine distemper N gene and the canine parvovirus VP2 gene, namely pcDSRV ₉ -NG -eGFP+CDV N-PM+VP2-L gene recombination vector, and then rescued rabies-canine distemper-canine parvovirus by reverse genetic method, namely rSRV ₉ +CDV N+VP2 gene recombination virus.

The gene recombinant virus constructed by the present invention can increase the expression of rabies virus antigen gene G, avoid the influence of rabies immunogenic gene expression due to insertion of foreign genes, and can simultaneously express canine distemper and canine parvovirus immunogenic genes. Therefore, the successful construction of the recombinant virus can be applied to the development of "rabies-distemper-canine parvovirus triple gene recombinant vaccine" and "rabies-canine distemper-canine parvovirus triple gene recombinant oral vaccine". It is used for the immunization and prevention of rabies, canine distemper and canine parvovirus disease in dogs, pet dogs and special breeding canids such as foxes and raccoon dogs; it can also be used for feeding wild animals to prevent three kinds of infections the spread of disease.

Description of drawings

Fig. 1 is a block diagram of technical steps for constructing rabies-distemper-canine parvovirus provided by the embodiment of the present invention.

Detailed ways

In order to further illustrate the technical means and effects of the present invention to achieve the intended invention purpose, the following preferred embodiments will describe the specific implementation, technical solutions, features and effects of the application according to the present invention in detail as follows. Specific features, structures, or characteristics of the various embodiments described below may be combined in any suitable manner.

Embodiment 1 (construct rabies-canine distemper-canine parvovirus)

The construction of rabies-distemper-canine parvovirus gene recombinant plasmid is shown in Figure 1:

Amplify the N, P, M, G, and L genes of the rabies SRV ₉ virus respectively;

The Gibson Assembly connection method was used for recombinant gene connection, the G gene of the SRV ₉ virus was transferred from the fourth position of the original genome sequence to the second position of the viral genome, and then the eGFP reporter gene was inserted into the G and P genes through restriction sites In between, a pcDSRV _9- NG-eGFP-PML gene recombination vector was constructed;

Amplify the CDV N gene in the canine distemper pMD18-CDV N recombinant plasmid by PCR method;

The end restriction site of the eGFP gene of the pcDSRV ₉ -NG-eGFP-PML recombinant vector was digested with Hpa I by enzyme digestion, and the canine distemper CDV N gene was constructed into pcDSRV ₉ -NG-eGFP- by using the Gibson Assembly ligation method On the PML recombinant vector, construct pcDSRV ₉ -NG-eGFP+CDV N-PML recombinant vector;

The VP2 gene in the canine parvovirus pMD19-T-VP2 recombinant plasmid was amplified by PCR;

Carry out BsiW I enzyme digestion on the M gene end restriction site in the pcDSRV ₉ -NG-eGFP+CDV N-PML recombinant vector by enzyme digestion, and insert the amplified VP2 gene into the pcDSRV ₉ -NG- Between the ML genes of the eGFP+CDV N-PML vector, a pcDSRV ₉ -NG-eGFP+CDV N-PM+VP2-L gene recombination vector was constructed;

Rabies-canine distemper-canine parvovirus, rSRV ₉ + CDV N + VP2 gene recombinant virus, was rescued by reverse genetic method.

Finally, the rabies-canine distemper-canine parvovirus, that is, the recombinant rabies virus strain, is obtained after transformation, screening of positive clones, enzyme digestion, electrophoresis and sequencing identification. The nucleotide sequence of its gene is SEQ ID NO:1.

Embodiment 2 (the recombinant virus of embodiment 1 is rescued by reverse genetic method)

The constructed rabies-distemper-canine parvovirus (pcDSRV ₉ -NG-eGFP+CDV N-PM+VP2-L) recombinant plasmid was combined with pcDNA3.1-N and pcDNA3.1-P by reverse genetics , pcDNA3.1-M, pcDNA3.1-G and pcDNA3.1-L helper plasmids were co-transfected into BSR cells at a certain concentration by lipofection, and the rabies-distemper-canine parvovirus "rSRV" was rescued ₉ +CDV N+VP2 Gene Recombinant Virus", and RT-PC, indirect immunofluorescence detection.

(1) Oral immunization of dogs with recombinant rabies virus

Nine dogs were randomly divided into groups. The first group was the control group injected with the commercially available canine pentavalent inactivated vaccine, and the injection dose was 1mL/dog; the second group was the oral immunization test of compound adjuvant + rSRV ₉ + CDV N + VP2 gene recombinant virus group, the feeding dose was 5mL for dogs and 4mL for cats (log7.0FFD ₅₀ ); the third group was compound adjuvant + "rSRV ₉ + CDV N + VP2" gene recombinant virus subcutaneous injection test group, and the injection dose for dogs was 5mL /Only. Orally and subcutaneously injected on 0d, 7d, 14d respectively, 3mL blood per mouse was collected on 0d and 7d, 14d, 28d, 58d and 118d after immunization, left at room temperature for 1-2h, centrifuged at 5000rpm/min for 5min, and serum was drawn; synchronously The saliva of the dogs was collected and stored in a -20°C refrigerator for later use.

(2) Detection method for serum IgG antibody of dogs immunized with rabies gene recombinant virus

5 mL of blood was collected from the anterior radial vein of the canine forelimb, centrifuged at 5000 rpm/min for 5 min, and the serum was separated. The detection method of canine serum IgG antibody is strictly in accordance with the instructions of "Canine Serum Rabies-specific IgG Antibody ELISA Quantitative Detection Kit".

By detecting and quantitatively analyzing the blood-specific anti-rabies IgG, canine distemper IgG, and canine parvovirus IgG antibody production levels of immunized dogs, and comparing the immune effects of commercially available canine five-inactivated vaccines, to determine rabies Neutralizing IgG antibody immune effect of "rSRV ₉ + CDV N + VP2" gene recombinant virus against rabies virus, canine distemper virus and canine parvovirus.

(3) Detection method for serum IgA antibody of dogs immunized with rabies gene recombinant virus

Insert the oral test paper into the dog's oral cavity from the corner of the dog's mouth, let the dog chew the cotton swab on the test paper head, take it out after the cotton swab is fully wet, use tweezers to remove the cotton swab and put it into a centrifuge tube containing 800ul PBS buffer solution, and place it on the shaker Fully oscillate, 5000rpm/min, centrifuge for 5min, and absorb the supernatant; the detection method of dog oral mucosal IgA antibody is strictly in accordance with the instructions of "Canine Rabies Specific IgA Antibody ELISA Detection Kit".

By detecting and quantitatively analyzing the levels of saliva-specific anti-rabies secretory SIgA, canine distemper SIgA, and canine parvovirus SIgA antibodies in immunized dogs, and comparing the immune effects of commercially available canine pentavalent inactivated vaccines, the results are as follows: To determine the immunization effect of SIgA antibody against rabies virus, canine distemper virus and canine parvovirus in saliva of rabies "rSRV ₉ + CDV N + VP2" gene recombinant virus.

Embodiment 3 (identification of the biological characteristics of the recombinant virus constructed in embodiment 1)

The constructed rabies "rSRV ₉ + CDV N + VP2" gene recombinant virus was observed by electron microscope to observe the morphological characteristics of the recombinant virus; the virus _titer and The virus virulence was detected; the antigenicity of the exogenous protein in the virus liquid was qualitatively and quantitatively analyzed by SDS polyacrylamide gel electrophoresis, BCA protein quantification method and Western-blot experiment; the recombinant virus was immunized with experimental animals (mice and dogs) ) Through double-antibody sandwich ELISA, T lymphocyte transformation test and ontology animal challenge test, the immune titer and immune protection of rabies-canine distemper-canine parvovirus "rSRV ₉ + CDV N + VP2 gene recombinant virus" were tested detection.

Through the above detection method, the virus morphology changes of the "rSRV ₉ + CDV N + VP2" gene recombinant virus, the genetic characteristics of virus proliferation, the recombinant virus immune protein genes (rabies N gene, canine distemper N gene, canine parvovirus VP2 Quantitative analysis of the expression and production levels of gene) and its immunogenicity verification, as well as changes in the level of immune regulation of the body's cells, to comprehensively evaluate the immune effect of the recombinant virus.

Embodiment 4 (identification of the genetic stability of the recombinant virus constructed in embodiment 1)

The recombinant virus was continuously cultured in BHK-21 cells for 30 generations, and the TCID ₅₀ was measured for each culture passage, and the virus virulence change curve was drawn to observe the change of the virus virulence of the recombinant virus in continuous passage.

After 30 passages of virus cells were continuously passaged, TCID ₅₀ detection was performed on the virus cultured in each passage of cells, and the change of virus titer in 30 passages was plotted as a "virus titer change curve" to determine the recombinant virus in the cells. genetic stability.

Embodiment 5 (identification of the biosafety of the recombinant virus constructed in embodiment 1)

The dogs were immunized with the constructed rabies "rSRV ₉ + CDV N + VP2 gene recombinant virus", the saliva, feces and urine of the immunized animals were collected regularly, and BHK cells were inoculated, and the indirect immunofluorescence test and PCR specific amplification method were used to inoculate the immune group. The chemical test is used to detect the in vitro detoxification of immunized animals. This method passes the expected half-year biosafety test to identify the recombinant virus after oral immunization, in vitro detoxification, virus distribution in metabolic pathways, and virus pollution in the living environment, so as to identify the recombinant virus. Virus use safety.

The present invention provides the application of the recombinant rabies virus strain prepared in Example 1 in the preparation of medicaments for preventing rabies.

The present invention provides a vaccine composition, which contains the recombinant rabies virus strain prepared in Example 1.

The gene recombinant virus constructed by the present invention can increase the expression of rabies virus antigen gene G, avoid the influence of rabies immunogenic gene expression due to insertion of foreign genes, and can simultaneously express canine distemper and canine parvovirus immunogenic genes. Therefore, the successful construction of the recombinant virus can be applied to the development of "rabies-distemper-canine parvovirus triple gene recombinant vaccine" and "rabies-canine distemper-canine parvovirus triple gene recombinant oral vaccine". It is used for the immunization and prevention of rabies, canine distemper and canine parvovirus disease in dogs, pet dogs and special breeding canids such as foxes and raccoon dogs; it can also be used for feeding wild animals to prevent three kinds of infections the spread of disease.

The gene nucleotide sequence of the recombinant rabies virus strain constructed by the present invention is SEQ ID NO: 1, as follows:

GCTAGC (Nhe1) TGTTAAGCGTCTGATGAGTCCGTGAGGACGAAACTATAGGAAAGGAATTCCTATAGTC (Ham RZ sequence) ACGCTTAACAACCAGATCAAAGAAAA AACA (transcription start signal for N gene) AAAAAAA (transcription stop signal for N gene) CT (spacer sequence) AACA (transcription start signal for G gene)TCCCTCAAAAGACTCAAGGAAAG ATG (G gene Start codon) AAAAAAA (transcription termination signal of N gene of G gene) CAGTTC AACACCCCCTACA (Egfp transcription initiation signal) ATG (Egfp initiation codon) TAA (EGFP termination codon) TGAAAAAAA (Egfp transcription termination signal) CTGTT AACACCCCCTACA (Cdvn start transcription signal) ATG (CDVN start codon) TAA ( CDVN stop signal) TGAAAAAAA (CDNV stop transcription signal) CT AACACCCCC (P gene transcription start signal) TGAAAAAAA (P gene transcription stop signal) CAGGC AACA ( M gene transcription initiation signal) CCACTGATAAA ATG (M gene initiation codon) TGAAAAAAA (M gene transcription termination signal) CT CAACACCCCCTACA (VP2 transcription initiation signal) ATG (VP2 initiation codon) AAA (VP2 gene transcription termination signal) CTGCTATT AACACTTCT (L基因的起始转录信号)CAACCTGAGACTTACTTCAAG ATG (L基因基因起始密码子) TGA (L基因的终止密码子)ACCGGATACTCCTGGAAGCCTGCCCATGCTAAGACTCTTGTGTGATGTATCT TGAAAAAA A (L基因的终止转录信号)CAAGATCCTAAATCTGAACCTTTGGTTGTTTGATTGTTTTTCTCATTTTTGTTGTTTATTTGTTAAGCGT GGGTCGGCATGGCATCTCCACCTCCTCG (Hdv RZ序列)

Those who are skilled in the art can choose from the prior art for the parts not covered in the embodiments of the present invention.

What is disclosed above is only the specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone familiar with the technical field can easily think of changes or replacements within the technical scope disclosed in the present invention, and should covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the above claims.

Claims (4)

1. a kind of rabies-canine distemper-canine parvovirus gene recombined virus strain, it is characterised in that it is with canine distemper G bases The relocated rabies of cause and canine parvovirus VP2 protein gene virus, is named as：Rabies-canine distemper-canine parvovirus genetic recombination Virus, the gene nucleotide series of the recombinant virus are SEQ ID NO:1.

2. the construction method of rabies-canine distemper-canine parvovirus gene recombined virus strain described in claim 1, the side Method comprises the following steps：

Respectively by rabies SRV₉N, P, M, G, L gene of virus are expanded；

Recombination connection is carried out using Gibson Assembly connection methods, by SRV₉The G genes of virus are by protogene group sequence The 4th bit transition to virus genomic second, then by eGFP reporter genes by restriction enzyme site be inserted into G and P genes it Between, it is built into pcDSRV₉- N-G-eGFP-PML gene recombined vectors；

CDV N genes in canine distemper pMD18-CDV N recombinant plasmids are expanded by PCR method；

By enzymatic cleavage methods to pcDSRV₉Hpa is carried out after the eGFP gene end restriction enzyme sites of-N-G-eGFP-PML recombinant vectors I digestions, it is using Gibson Assembly connection methods that canine distemper CDV N is gene constructed to pcDSRV₉- N-G-eGFP-PML weights On group carrier, pcDSRV is built into₉- NG-eGFP+CDV N-PML recombinant vectors；

Pass through the VP2 genes in PCR amplification canine parvovirus pMD19-T-VP2 recombinant plasmids；

By enzymatic cleavage methods to pcDSRV₉M gene ends restriction enzyme site carries out BsiW in-NG-eGFP+CDV N-PML recombinant vectors I digestions, pcDSRV is inserted into by the VP2 genes of amplification by Gibson Assembly connection methods₉-NG-eGFP+CDV N-PML Between carrier M-L genes, pcDSRV is built₉- NG-eGFP+CDV N-PM+VP2-L gene recombined vectors；

Recombinant rabies-canine distemper-canine parvovirus, i.e. rSRV is saved out by reverse genetic method₉+ CDV N+VP2 genetic recombination Virus.

3. rabies-canine distemper-canine parvovirus gene recombined virus strain described in claim 1 or 2 is preparing the mad dog of prevention Application in the medicine of disease.

4. a kind of vaccine combination, it is characterised in that the vaccine combination contains described mad described in claim 1 or 2 Dog disease-canine distemper-canine parvovirus gene recombined virus strain.