Genetically engineered rabies virus vaccine with recombination-defective adenovirus carrier

A genetically engineered vaccine, replication-deficient technology, applied in the field of recombinant vaccines, can solve the problems of unclear efficacy of pathological changes

Inactive Publication Date: 2003-02-19
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the GP protein of CVS-N2c rabies virus is only expressed at a low level in neuronal cells, which causes pathological changes and its efficacy as a subunit vaccine for the prevention and control of rabies is still unclear

Method used

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  • Genetically engineered rabies virus vaccine with recombination-defective adenovirus carrier
  • Genetically engineered rabies virus vaccine with recombination-defective adenovirus carrier
  • Genetically engineered rabies virus vaccine with recombination-defective adenovirus carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. Example 1 Construction of rabies virus CVS-N2c GP gene recombinant replication-defective adenovirus of the present invention A. Immunogen

[0018] The brain tissue of Kunming mice with typical rabies symptoms was taken, and Trizol reagent (Gibco BRL) was added to extract total RNA, and the GP gene cDNA of rabies virus was obtained by reverse transcription polymerase chain reaction, which was cloned into the vector pUC18 and sequenced for double-stranded DNA. For its sequence, see SEQ ID NO: 1 in the sequence listing. B. Build process

[0019] The GP gene of the CVS-N2c strain was subcloned from its cloning vector into the adenovirus shuttle vector pAdShuttle-CMV to obtain a recombinant shuttle plasmid, such as figure 1 (A). It was confirmed by restriction analysis that a single copy of the foreign gene was inserted. Subsequently, this recombinant shuttl...

Embodiment 2

[0022] When the 293 cells grew to 90% saturation, the recombinant virus rAdCVSGP was inoculated at a multiplicity of infection m.o.i=5-10. After 3-4 days, when the cell lesion reaches 70-90%, the cell solution is collected in a centrifuge tube, centrifuged at 2000rpm for 15min to harvest the cells, and the supernatant is frozen for future use. The cells were freeze-thawed and lyzed four times in a dry ice / 37°C water bath, centrifuged at 5000rpm for 15min, and the supernatant of the lysate was added to the upper layer of the CsCl density gradient centrifugation medium. Centrifuge at 35000rpm for 2h. Carefully suck out the virus band with a pipette, add it to the dialysis band and dialyze against PBS with stirring. After dialysis was complete, freeze at -20°C. Example 3 Mice intracerebral challenge-immune protection experiment

Embodiment 3

[0022] When the 293 cells grew to 90% saturation, the recombinant virus rAdCVSGP was inoculated at a multiplicity of infection m.o.i=5-10. After 3-4 days, when the cell lesion reaches 70-90%, the cell solution is collected in a centrifuge tube, centrifuged at 2000rpm for 15min to harvest the cells, and the supernatant is frozen for future use. The cells were freeze-thawed and lyzed four times in a dry ice / 37°C water bath, centrifuged at 5000rpm for 15min, and the supernatant of the lysate was added to the upper layer of the CsCl density gradient centrifugation medium. Centrifuge at 35000rpm for 2h. Carefully suck out the virus band with a pipette, add it to the dialysis band and dialyze against PBS with stirring. After dialysis was complete, freeze at -20°C. Example 3 Mice intracerebral challenge-immune protection experiment

[0023] 12-14g of clean or ordinary Kunming mice were centrifuged with CsCl density gradient to obtain purified virus for intraperitoneal or subcutane...

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Abstract

A genetically engineered rabies virus vaccine with the E1 and E3 deficient recombinant defective human adenovirus as carrier is disclosed. The rabies virus coated glucoprotein encoding gene and the element to regulate it for efficient expression are carried in the E1 region. The said encoding gene comes from the rabies virus CVS-N2C with strong neurovirulence.

Description

technical field [0001] The invention relates to a recombinant vaccine for preventing animal rabies. Background technique [0002] Rabies is a fatal zoonotic infectious disease caused by rabies virus infection, which is characterized by the invasion of the central nervous system. Rabies occurs widely all over the world, and my country is a high-incidence area of ​​rabies. Prophylaxis with rabies virus vaccine is an effective way to prevent and control the occurrence of rabies. Rabies virus vaccines are gradually developed from early nerve tissue vaccines. At present, primary cell culture vaccines and passage cell refined and purified vaccines are widely used. These vaccines have good immune effects, but due to high production costs, transportation and storage must rely on low temperature. "Cold chain", administration methods and other factors make it inconvenient to apply to the immunization of various wild or domestic animals such as dogs and foxes, which are storage hosts...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/205A61P31/12C12N15/09C12N15/861
Inventor 王树惠李文辉
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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