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Avian infectious brunchitis virus s1 recombinant protein antigen, preparation method and uses

A technology of bronchitis and chicken infectivity, applied in the biological field, can solve the problems of weak cross protection, danger, weak seedlings and easy to disperse toxins, etc., and achieve the effect of good immunogenicity and strong immunogenicity

Inactive Publication Date: 2006-04-26
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Practice has shown that these three types of vaccines all have deficiencies to a certain extent. The reason is that there are many serotypes of the mycoviruses and lack or only weak cross-protection between each serotype. These three types of vaccines can only target one serotype. type, the occurrence and prevalence of the disease cannot be effectively controlled
In addition, the immune effect of the inactivated vaccine is not good, and the attenuated vaccine is easy to disperse the virus and may undergo genetic recombination with the wild virus to produce new mutant strains, so there is a potential danger
The research on nucleic acid vaccine is still in its infancy, and it is impossible to popularize and apply it in the short term, and its immune effect and safety still need further in-depth research

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  • Avian infectious brunchitis virus s1 recombinant protein antigen, preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Truncated expression and purification process of IBV S1 protein antigen;

[0031] 1. IBV ZJ971 (M41, J) culture and RNA extraction

[0032] The seed virus of ZJ971 (M41, J) strain was inoculated into 9-11 day-old SPF chicken embryos via the allantoic cavity route, incubated in a 37°C incubator, and the allantoic fluid of chicken embryos was collected between 24h-72h, and impurities were removed after rough separation. The supernatant was precipitated with 30% PEG6000 (containing 7.85% NaCl), suspended with TEN (pH 8.0), and RNA was extracted according to the instructions of the Trizol kit; the extracted RNA was used for RT-PCR.

[0033] 2. RT-PCR of ZJ971-S1D (M41-S1D, J-S1D) gene

[0034] 1. Reverse transcription of IBV ZJ971 (M41, J)

[0035]Take the above viral RNA, add 12μmol / L IBV ZJ971-S1D (M41-S1D, J-S1D) downstream primer 1μl, incubate at 70°C for 5 minutes, take it out and put it on ice; then add 5×reaction buffer 4μl, 20U / μl Rnasin 1μl, 10mM dNTPs 2μl, mix ...

Embodiment 2

[0062] The specific process of truncating and expressing the S1 protein of three strains of chicken infectious bronchitis virus as a diagnostic antigen:

[0063] After inducing a large amount of expression of the recombinant bacteria, the bacteria were collected by centrifugation and resuspended with lysate Buffer B (pH8. 5 Dilute the Tris-Cl coating buffer and coat the enzyme-labeled reaction plate, first incubate at 37°C for 2 hours, and then absorb at 4°C for 16-18 hours. Wash with PBST 3 times, 10 minutes each time. Block with PBST containing 5% skimmed milk powder for 2 hours, and wash as above. The IBV positive serum to be tested and the negative serum of SPF chickens were serially diluted with PBS to 2 times respectively and added to the wells, reacted at 37°C for 1 hour, and washed as above. The goat anti-chicken IgG secondary antibody (1:3000) labeled with horseradish peroxide was added to each well, reacted for 1 hour, and washed as above. Then add TMB diluted wit...

Embodiment 3

[0065] Three truncated recombinant S1 protein antigens have strong immunogenicity and can be used to prepare IBV genetically engineered subunit protein vaccines:

[0066] The mouse monoclonal antibody prepared by using these three truncated recombinant S1 protein antigens and IBV were co-inoculated with SPF chicken embryos aged 9 to 11 days, and at the same time, an experimental control group was set up to inoculate IBV alone, with 6 chicken embryos in each group, and the results of inoculation were significant In the mouse-derived monoclonal antibody group, 4 eggs were effectively protected, while chicken embryos in the experimental control group developed characteristic lesions or died of infectious bronchitis, which strongly confirmed that the expressed recombinant protein antigen has a strong immunogenicity. It can produce neutralizing antibodies that can neutralize IBV infection, so that the truncated recombinant S1 protein antigen can be used to prepare IBV genetically en...

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Abstract

The present invention discloses one kind of recombinant protein antigen for chicken infectious bronchitis virus (IBV) S1 and its preparation process and use. These nucleotide sequences are shown in SEQ ID No. 1~3. The present invention provides expression method of pronucleus encoding 3 truncated S1 proteins, can express the amino end 288 bp and 309 bp part segments of S1 protein of three IBV strains separately, and provides the purification method of the S1 protein expressing products of the three IBV strains. During detection with IBV positive serum, the expressed partial S1 protein has excellent antigen reaction, so that the present invention may be used as diagnosis antigen used in different detection methods for detecting IBV infection or immunologically produced antibody. In addition, the expressed partial S1 protein may be sued in preparing monoclonal antibody and polyclonal antibody for detecting IBV antigen and preparing subunit vaccine.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a chicken infectious bronchitis virus S1 recombinant protein antigen, a preparation method and an application thereof. Background technique [0002] Infectious bronchitis is an acute, highly contagious disease of poultry caused by infectious bronchitis virus (IBV), a single-stranded positive-sense, non-segmented RNA virus that is One of the main pathogens currently endangering the poultry industry in the world. Since it was discovered in the United States in 1931, more than 100 strains and more than 30 serotypes of infectious bronchial viruses have been isolated, and there are large differences in their tissue tropism and virulence. Illness poses great difficulties. At present, the main measure to prevent and control the disease is to immunize chicken flocks with inactivated vaccines and live attenuated vaccines. Practice has shown that these three types of vaccines all have defic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C12N15/09G01N33/535
Inventor 周继勇吴建祥胡金强张德勇
Owner ZHEJIANG UNIV
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