Feline infectious rhinoconjunctivitis and feline panleukopenia combined vaccine and preparation method thereof

A technology for leukopenia and rhinoconjunctivitis, applied in the fields of microorganisms and veterinary biopharmaceuticals, can solve the hidden dangers of attenuated vaccines, stimulate cellular immunity, and fail to immunize vaccines, and achieve good immune prevention effects, good safety, and immunogenicity. high sex effect

Active Publication Date: 2018-08-07
CHANGCHUN SR BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These vaccines are attenuated vaccines or inactivated vaccines, and attenuated vaccines have potential safety hazards, and inactivated vaccines cannot effectivel

Method used

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  • Feline infectious rhinoconjunctivitis and feline panleukopenia combined vaccine and preparation method thereof
  • Feline infectious rhinoconjunctivitis and feline panleukopenia combined vaccine and preparation method thereof
  • Feline infectious rhinoconjunctivitis and feline panleukopenia combined vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Preparation and identification of feline calicivirus virus-like particles

[0031]After extracting RNA from the FCV-JL2 strain, reverse transcription, and PCR sequencing, the VP1 sequence was obtained and optimized according to the insect NEWII cell preference. The codon preference index was adjusted from 0.47 to 0.98, and the GC content was adjusted from 42.6% to 57.6 %, and remove unfavorable cis-acting elements, remove mRNA prone to neck-loop regions, etc. Send the optimized sequence to the company for gene synthesis. For the optimized sequence, use primers to amplify the VP1 gene, and connect it into the pFastBac Dual vector through NotI-HindIII and SmaI-XhoI, respectively, to obtain a transfer vector carrying double-copy foreign fragments. The transfer vector was transformed into DH10Bac competent for recombination, and the recombinant baculovirus genome Bacmid carrying double-copy exogenous fragments was obtained. After Bacmid was transfected into Sf9 ...

Embodiment 2

[0039] Example 2 Preparation and Identification of Feline Panleukopenia Virus Virus-Like Particles

[0040] After sequencing the extracted genome of the FPV strain, the VP2 gene sequence was obtained and optimized according to the insect NEW II cell preference. The codon preference index was adjusted from 0.37 to 0.87, and the GC content was adjusted from 35.1% to 53.0%. Send the optimized sequence to the company for gene synthesis. For the optimized sequence, use primers to amplify the VP2 gene, and connect them into the pFastBac Dual vector through NotI-HindIII and XhoI-NheI, respectively, to obtain transfer vectors carrying double-copy foreign fragments. The transfer vector was transformed into DH10Bac competent for recombination, and the recombinant baculovirus genome Bacmid carrying double-copy exogenous fragments was obtained. After Bacmid was transfected into Sf9 cells, the recombinant baculovirus was rescued. Infecting Sf9 cells with recombinant baculovirus can produ...

Embodiment 3

[0042] Embodiment 3 cat immunity test

[0043] The above-mentioned recombinant baculoviruses were respectively inoculated into suspended Sf9 cells at MOI=0.1, cultured for 4 days, and then harvested. After freeze-thawing the Sf9 cells inoculated with FCV antigen once, centrifuge at 3000rpm for 30min to obtain the supernatant, which is the harvest solution containing FCV virus-like particles. The protein concentration was determined by BCA method and was 4.9 mg / ml. Centrifuge the Sf9 cells inoculated with FPV antigen directly at 3000rpm for 30min, discard the supernatant, and wash the cells with an equal volume of 25mM NaHCO 3 Suspend, act on ice for 30 minutes, and centrifuge at 3000 rpm for 30 minutes to obtain the supernatant, which is the harvest solution containing FPV virus-like particles. Use pig blood for the determination of hemagglutination titer, HA is 1:2 16 .

[0044] Mix the FCV virus-like particles and FPV virus-like particles prepared above at a volume of 3:...

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Abstract

The invention provides a feline infectious rhinoconjunctivitis and feline panleukopenia combined vaccine and a preparation method thereof. The feline infectious rhinoconjunctivitis and feline panleukopenia combined vaccine is prepared by: using baculovirus/insect cell expression system to produce virus-like particles of feline calicivirus and virus-like particles of feline panleukopenia, and mixing the virus-like particles of the two viruses with preservatives and adjuvants. The combined vaccine herein has the advantages of good safety, high efficiency, good production convenience, low cost and the like, and has good immuno preventive effect for feline infectious rhinoconjunctivitis and feline panleukopenia.

Description

technical field [0001] The invention relates to the field of microorganisms, genetic engineering and veterinary biopharmaceuticals, in particular to a method for producing feline infectious rhinoconjunctivitis and feline panleukopenia dual vaccine using an insect cell-baculovirus expression system. Background technique [0002] Feline infectious rhinoconjunctivitis is caused by feline calicivirus (FCV), and is clinically characterized by rhinitis, conjunctivitis, acute oral ulcers, pneumonia, and chronic gastritis. Currently, FCV is believed to have a worldwide distribution and may infect all cat species. Feline panleukopenia is caused by feline panleukopenia virus (FPV), and its clinical manifestations are characterized by sudden high fever, vomiting, diarrhea, dehydration and leukopenia in circulating blood in cats. FPV mainly infects a variety of animals such as cats and mustelids, especially young animals under 6 months of age have higher morbidity and mortality, and ar...

Claims

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Application Information

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IPC IPC(8): A61K39/23A61K39/12C12N15/40C07K14/08C12N15/866C12N7/04A61P31/14A61P31/20
CPCA61K39/12A61K2039/5258A61K2039/70A61P31/14A61P31/20C07K14/005C12N7/00C12N15/86C12N2710/14043C12N2710/14134C12N2770/16022C12N2770/16023C12N2770/16034A61K2300/00
Inventor 夏振强金宏丽石晶殷玉和赵健刘冰刘伟
Owner CHANGCHUN SR BIOLOGICAL TECH
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