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Recombinant rabies virus carrying interleukin 6 gene and application thereof

A rabies virus and gene technology, applied in recombinant rabies virus and its application fields, can solve problems such as damage to the body

Inactive Publication Date: 2017-07-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as an inflammatory factor, IL6 overexpression or use will cause excessive inflammatory response and damage the body

Method used

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  • Recombinant rabies virus carrying interleukin 6 gene and application thereof
  • Recombinant rabies virus carrying interleukin 6 gene and application thereof
  • Recombinant rabies virus carrying interleukin 6 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 Amplification of canine IL6 gene

[0065] 1. Collect inguinal lymph node and spleen samples from the dead Chinese garden dog, process the samples, extract nucleic acid, and perform RT-PCR amplification.

[0066] (1) See Table 1 for primer sequences

[0067] Design primers for canine IL6 amplification, design primers IL6-F / IL6-R (containing Bsi WI and Pst I restriction endonuclease sites and protective bases).

[0068] Table 1

[0069]

[0070] (2) Utilize random primers and Oligo(dT), carry out cDNA synthesis according to the system shown in Table 2:

[0071] Table 2

[0072]

[0073] (3) Using the above-mentioned cDNA synthesized by reverse transcription as a template, PCR amplification was performed using primers IL6-F / IL6-R. The system is shown in Table 3.

[0074] table 3

[0075]

[0076] (4) According to the instruction manual of the gel extraction kit (HiPure Gel Pure DNA Micro Kit) from Magen Company, the PCR product was gel-cut, recov...

Embodiment 2

[0082] Example 2 Cloning of canine IL6 gene into HEP-Flury

[0083] 1. Construction of recombinant plasmid pHEP-CaIL6

[0084](1) Utilize primer IL6-F / IL6-R (see Table 1) to amplify the canine IL6 gene (sequence shown in SEQID NO.1) that obtains in embodiment 1, IL6-F / IL6-R primer predicts amplification The length is 642bp (the amplification reaction system is shown in Table 5), and Bsi WI and Pst I restriction sites are respectively introduced.

[0085] table 5

[0086]

[0087]

[0088] Add the above reagents in turn to the PCR tube.

[0089] The PCR reaction conditions were as follows: 98°C for 2min; 30 cycles of 98°C for 10s, 62°C for 20s, and 72°C for 1min; and finally extended at 72°C for 10min.

[0090] (3) The IL6 gene amplified by PCR was recovered and purified by gel cutting, and the purified IL6 gene and pHEP-3.0 plasmid (recombinant plasmid carrying the full-length cDNA of HEP-Flury strain) were restricted by BsiWI and PstI nucleic acid Endonuclease is pr...

Embodiment 3

[0098] Example 3 Rescue and Screening of Recombinant Virus rHEP-CaIL6 Using Recombinant Plasmid pHEP-CaIL6

[0099] 1. The transfection and virus screening methods were carried out with reference to the methods of Inoue (2003) and Guo Xiaofeng et al. (2006).

[0100] Transfection:

[0101] (1) Prepare BHK-21 cells: the cell density of 12-well cell culture plate is 1×10 5 cells / well, the culture medium contains 10% fetal bovine serum; culture at 37°C for 12-16 hours until the cells are 60%-80% confluent. Wash cells once with 800 μL 1×PBS upon transfection.

[0102] (2) Exploring the ratio of the auxiliary plasmid

[0103] The groping ratio of recombinant plasmid pHEP-CaIL6 and helper plasmids pH-N, pH-P, pH-L and pH-G is shown in Table 8:

[0104] Table 8

[0105] pHEP-CaIL6 pH-N pH-P pH-L pH-G 1 1.00μg 0.30μg 0.175μg 0.05μg 0.075μg 2 1.00μg 0.30μg 0.155μg 0.07μg 0.075μg 3 1.00μg 0.25μg 0.155μg 0.02μg 0.075μg ...

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Abstract

The invention discloses a recombinant rabies virus rHEP-CaIL6 carrying an immune enhancement factor interleukin (IL) 6 gene and application thereof. The recombinant virus takes rabies virus HEP-Flury strain as the skeleton, the IL6 gene of canine is inserted to a position between the G and L gene of HEP-Flury to obtain a recombinant plasmid pHEP-CaIL6, and finally saving screening is carried out to obtain the recombinant rabies virus strain rHEP-CaIL6. The recombinant virus carries the immune enhancement factor, can enhance the immune response and induce the production of higher rabies virus neutralizing antibody, thus better protecting the body from resisting the attack of lethal rabies virus, also can produce a neutralizing antibody with protective ability at a low dose, and lowers the cost of canine vaccines. Moreover, the IL6 gene is recombined into the rabies virus, thus achieving stable expression of IL6 protein, also avoiding the overexpression thereof, and overcoming the defect that excessive IL6 can cause pathological injury.

Description

technical field [0001] The invention belongs to the technical field of molecular biological immunology. More specifically, it relates to a recombinant rabies virus carrying an immune enhancing factor interleukin 6 gene 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/66A61K39/205A61P31/14
CPCA61K39/12C07K14/5412C12N7/00C12N15/66C12N2760/20121C12N2760/20134
Inventor 郭霄峰吴玉婷罗均张博越田钦
Owner SOUTH CHINA AGRI UNIV
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