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Recombinant rabies virus carrying two genes G and gene VP2 and application of recombinant rabies virus

A rabies virus and gene technology, applied to recombinant rabies virus and its application fields, can solve the problems of attenuated strain mutation, harm to dogs and wild animals, and immune failure.

Inactive Publication Date: 2016-12-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the effect of the attenuated vaccine is ideal, it can resist CPV infection to a certain extent, but the attenuated strain may mutate and become stronger, causing great harm to dogs and wild animals
Traditional vaccines are easily affected by maternal antibodies and lead to immunization failure. There are reports of CPV outbreaks in dog groups after vaccination at home and abroad.

Method used

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  • Recombinant rabies virus carrying two genes G and gene VP2 and application of recombinant rabies virus
  • Recombinant rabies virus carrying two genes G and gene VP2 and application of recombinant rabies virus
  • Recombinant rabies virus carrying two genes G and gene VP2 and application of recombinant rabies virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Screening of CPV epidemic strains

[0061] 1. Process and extract nucleic acid from suspected CPV samples collected clinically, and perform PCR amplification.

[0062] (1) See Table 1 for primer sequences

[0063] CPV detection primers CPV1 / CPV2 refer to the literature of Liu Zhonghua et al. (2003). By comparing the VP2 gene sequence with accession number M74849 published in GenBank, two pairs of primers VP2.F / VP2-1.R and VP2-2.F / VP2.R were designed with Primer Premier 5.0 (PREMIER Biosoft International) software.

[0064] Table 1

[0065]

[0066] (2) Utilize primer CPV1 / CPV2, carry out PCR amplification according to the system shown in Table 2:

[0067] Table 2

[0068]

[0069] (3) Take the positive samples detected by PCR with primers CPV1 / CPV2, and perform PCR amplification with primers VP2.F / VP2-1.R and VP2-2.F / VP2.R respectively. The system is shown in Table 3.

[0070] table 3

[0071]

[0072](4) According to the instruction manual of t...

Embodiment 2

[0078] Example 2 Cloning of CPV strain VP2 gene into HEP-Flury

[0079] 1. Construct the recombinant plasmid pHEP-Flury (VP2), see the strategy figure 2 shown.

[0080] (1) Utilize primer VP2.F / VP2.R (see Table 1) to amplify the VP2 gene (sequence shown in SEQ ID NO.1) of the CPV strain selected in the embodiment 1, VP2.F and VP2.R The primers are expected to amplify with a length of 1755bp (the amplification reaction system is shown in Table 5), and respectively introduce BsiWI and NheI restriction sites.

[0081] table 5

[0082]

[0083] Add the above reagents in turn to the PCR tube.

[0084] The PCR reaction conditions were: 95°C for 5min; 25 cycles of 94°C for 30s, 52°C for 30s, and 68°C for 2min; and finally extended at 68°C for 10min.

[0085] (2) After the reaction, 5 μL of PCR products were taken for electrophoresis detection on 1.0% agarose gel. Obtain a specific DNA electrophoresis band, the size is about 1755bp, which is consistent with the experiment exp...

Embodiment 3

[0099] Example 3 Construction of recombinant plasmid pHEP-Flury (dG-VP2) using recombinant plasmid pHEP-Flury (VP2)

[0100] 1. Use the recombinant plasmid pHEP-Flury (VP2) constructed in Example 2 to construct pHEP-Flury (dG-VP2). For the construction scheme, see Figure 5 .

[0101] (1) Using the pHEP-Flury (VP2) plasmid as a template, using the upstream primer pDV2.F (the sequence is 5'-CGG GGTACC ATGGTTCCTCAGGTTCT-3') and downstream primer pDV2.R (sequence: 5'-GCCTGCAGTTTTATTCCATATTATTC-3') for PCR amplification.

[0102] In the upstream primer pDV2.F, the sequence of the underlined part is the enzyme cutting site of Acc65I, which is the homologous enzyme with BsiWI. The sequence below Acc65I is the initiation codon ATG of the ORF box of the G gene.

[0103] The position of the downstream primer in the ORF box of VP2 gene is 281-305, and the position of PstI in the ORF box of VP2 gene is 269-274.

[0104] The pair of primers used the pHEP-Flury (VP2) plasmid as a temp...

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Abstract

The invention discloses a recombinant rabies virus rHEP-Flury(dG-VP2) carrying two genes G and a gene VP2. According to the recombinant virus, a rabies virus HEP-Flury strain serves as a framework, the VP2 gene shown as SEQ ID NO.1 is inserted into HEP-Flury, an additional gene G of the rabies virus is inserted, and the recombinant plasmid pHEP-Flury(dG-VP2) carrying the two genes G and the gene VP2 is obtained; finally, rescuing screening is carried out, and the recombinant rabies virus rHEP-Flury(dG-VP2) is obtained. The recombinant rabies virus rHEP-Flury(dG-VP2) carries the two genes G and expresses VP2 protein, a high rabies virus resisting antibody and canine parvovirus VP2 resisting antibody level can be generated through immune induction, the cost of vaccines for dogs can be reduced, and very good application and popularization prospects are achieved.

Description

technical field [0001] The invention belongs to the field of molecular biological immunology. More specifically, it relates to a recombinant rabies virus carrying two G genes and VP2 genes and its application. Background technique [0002] Rabies (Rabies), caused by rabies virus (RV), is a severe zoonotic disease characterized by infecting the central nervous system, and the fatality rate is almost as high as 100% once the onset occurs. Asia and Africa are areas with a high incidence of rabies. Domestic animals such as cats and dogs are the main hosts of RV. About 99% of patients get sick after being bitten or scratched by these animals. Rabies is still a serious threat to people's lives in these areas Important infectious diseases for safety. my country is a high-incidence area of ​​rabies, and the number of cases is second only to India, ranking second in the world. With the increase of domestic pets, the control and prevention of rabies in our country still has a long ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C12N7/01C12N15/66A61K39/295A61K39/12A61K39/205A61P31/14A61P31/20
CPCA61K39/12A61K2039/5252A61K2039/5254A61K2039/5256C07K14/005C12N7/00C12N15/66C12N2750/14022C12N2750/14034C12N2760/20121C12N2760/20134
Inventor 郭霄峰施赫赫罗均牛学锋
Owner SOUTH CHINA AGRI UNIV
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