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Rabies virus ERA attenuated vaccine mutant strain as well as preparation method and live rabies vaccine

A technology of rabies virus and attenuated vaccine, which is applied in the biological field and can solve problems such as animal diseases

Active Publication Date: 2014-06-25
北京中联康生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional live attenuated vaccines may cause disease in vaccinated animals due to residual virulence or in vivo reproduction variation, which has certain risks

Method used

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  • Rabies virus ERA attenuated vaccine mutant strain as well as preparation method and live rabies vaccine
  • Rabies virus ERA attenuated vaccine mutant strain as well as preparation method and live rabies vaccine
  • Rabies virus ERA attenuated vaccine mutant strain as well as preparation method and live rabies vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Reverse genetics plasmid construction

[0047] According to the ERA sequence in Genebank, primers were designed, and the full-length sequence was synthesized in four segments (R1-R4). Hammerhead ribozyme (Ham) was introduced into R1, G protein signal peptide partial deletion and 333 amino acid mutation were introduced into R3, and hepatitis D ribozyme was introduced into R4.

[0048] 1F: GTCACT GGTAAC TGG TGTTAAGCGTCTGATGAGTCCGTGAGGACG

[0049] AAACTATAGGAAAGGAATTCCTATAGTC ACGCTTAACAACCAGATCAAAG

[0050] (KpnI / Ham) (SEQ ID NO: 3)

[0051] 1R: CTGCAG GTTTTTTTCATG (PstI) (SEQ ID NO: 4)

[0052] 2F: CCCGGG AGGCAACACC (Sam I) (SEQ ID NO: 5)

[0053] 2R: ACTAGT TAATAGTTTTTTTCAC (SpeI) (SEQ ID NO: 6)

[0054] 3F: CTGCAG AACATCCTCAAAAGACTCAAGGAAAGATGCAGGCTCTC (PstI) (SEQ ID NO: 7)

[0055] 3R: CCCGGG GTTTTTTTTCAAAAAGAACCCCCC (Sam I) (SEQ ID NO: 8)

[0056] 4F: ACTAGT CATTAGATCAGAAG (SpeI) (SEQ ID NO: 9)

[0057] 4R: GCGGCCGC GCCCTCCCTTAGCCATCCGAGTGGG...

Embodiment 2

[0066] virus recovery

[0067] When BHK-21 cells were cultured in MEM complete medium with 10% newborn bovine serum to 90% confluence in a 6-well plate, the liposome 2000 method was used to co-transfect the following plasmids: 4ug / well of pBlueERA and 4 kinds of helper plasmids pBlueN( 2ug / well), pBlueP (lug / well), pBlueL (lug / well), pT7 (4ug / well). After transfection, the cells were placed in 5% CO 2 Discard the supernatant after incubating at 37°C for 4-6 hours in an incubator, supplement MEM complete medium with 5% newborn bovine serum, and incubate at 37°C, 5% CO 2 After culturing in the incubator for 72 hours, the cells were frozen and thawed three times, the supernatant was collected by centrifugation, and the recovered virus was obtained by filtering with a filter membrane with a particle size of 0.22 μm.

Embodiment 3

[0069] Titration of virus after recovery

[0070] BHK-21 cells were cultured in a 24-well plate to a single layer, and the recovered virus was diluted 10 times to infect the BHK-21 cells. 37℃5%CO 2 After culturing in an incubator for 72 hours, fix in 80% acetone at -20°C for half an hour, wash with PBS and stain with FITC-labeled rabies virus N protein antibody at 37°C for 60 minutes. Fluorescent wells were counted with a fluorescence microscope after washing with PBS. The results showed that the virus was amplified in BHK-21 cells after recovery, and the virus titer reached 6×10 7 TCID 50 / ml.

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Abstract

The invention relates to a rabies virus ERA attenuated vaccine mutant strain as well as a preparation method and a live rabies vaccine, wherein the rabies virus ERA attenuated vaccine mutant strain is capable of expressing and coding a mutant glycoprotein, and the mutant glycoprotein has the mutations that an amino acid in the second site of a signal peptide is missing, an amino acid in the third site of the signal peptide is missing, the amino acid in the 333rd is mutated into glutamic acid from arginine, a psi pseudogene region is missing, and the gene G of the glycoprotein is inserted before the gene M. From the above, the rabies virus ERA attenuated vaccine mutant strain is safer, more efficient, and also lower in cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a rabies virus ERA attenuated vaccine mutant strain, a preparation method thereof and a live rabies vaccine. Background technique [0002] Rabies is a zoonotic viral infectious disease. Once a person develops the disease, the fatality rate is 100%. In the past two years, the incidence and death of human rabies in my country have exceeded 3000 people per year, and the epidemic situation is on the rise. Humans are primarily infected with rabies virus through the bite of a rabid domestic or wild animal. Therefore, controlling domestic or wild rabies infection can not only reduce the mortality of these animals, but also effectively control human infection with rabies virus, which is an effective means of preventing rabies. [0003] The main means of controlling animal infection with rabies virus is to cast orally attenuated live attenuated vaccine. All currently...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/85A61K39/205A61P31/14C12R1/93
Inventor 李耀东刘俊生刘延亭郑杰熊炜高杨
Owner 北京中联康生物科技股份有限公司
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