Riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and preparation method thereof

A technology of outer membrane protein and Reeseri bacteria, which is applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problem of difficult balance between antigen content and protective power, and achieve extensive cross-protection effect, convenient purification, and low production cost

Active Publication Date: 2012-07-18
兆丰华生物科技(南京)有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccine immunization prevention is an important means to control colibacillosis at present, but the commonly used monovalent serotype inactivated vaccines are only resistant to the same type of pathog

Method used

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  • Riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and preparation method thereof
  • Riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and preparation method thereof
  • Riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of the prokaryotic expression strain of R. anatipestifer RA1, RA2 type outer membrane protein

[0042] (1) Design of primers

[0043] Specific primers were designed according to the OMPA gene sequence of R. anatipestifer ATCC11845 strain (Accession No. AF104937) in Genebank. The upstream primer introduced the BamH I restriction site, and the downstream primer introduced the Xho I restriction site.

[0044] Its primer sequence is as follows:

[0045] RA-OMPA-F: GCT GGATCC ATGGGTAAAGAATTTATG

[0046] RA-OMPA-R:AGT CTCGAG TCTTACAAGAAGAGGACGCTT

[0047] (2) Genome extraction

[0048] A, Streak inoculation of R. anatipestifer RA1 type, RA2 type bacterial classification on the tryptone soybean agar (TSA) plate containing 2% newborn bovine serum, cultivate 24h in 37 ℃ of incubators;

[0049] B. Pick a single colony and inoculate it in the Martin Broth liquid medium, and shake it on a shaker at 37°C overnight;

[0050] C. Transfer the bacterial so...

Embodiment 2

[0066] Example 2 Avian pathogenic Escherichia coli O 78 Construction of Outer Membrane Protein Prokaryotic Expression Strain

[0067] (1) Design of primers

[0068] Specific primers were designed according to the OMPA gene sequence of Escherichia coli K12W3110 strain (accession number AP009048) in Genebank, the upstream primer introduced the BamH I restriction site, and the downstream primer introduced the XhoI restriction site.

[0069] Its primer sequence is as follows:

[0070] E-OMPA-F: AT GGATCC GCTCCGAAAGATAAC

[0071] E-OMPA-R: ATA CTCGAG TTAAGCCTGCGGCTGAGT

[0072] (2) Genome extraction

[0073] A. Avian pathogenic Escherichia coli O 78 Streak inoculation of strains on MB agar plate, culture at 37°C for 36h;

[0074] B. Pick a single colony and inoculate it in MB liquid medium, and shake it on a shaker at 37°C overnight;

[0075] C. Transfer the bacterial solution to a 1.5mL eppendorf tube, centrifuge at 8000r / min for 2min, discard the supernatant, add 500μL ...

Embodiment 3

[0091] Example 3 Induced expression and mass production of R. anatipestifer RA1, RA2 outer membrane protein prokaryotic expression strains

[0092] (1) Induced expression of prokaryotic expression positive bacteria of R. anatipestifer RA1 and RA2 outer membrane proteins screened

[0093] Inoculate the preserved bacterial solution into 5mL LB medium with Kan resistance at a ratio of 1:1000 by volume, and inoculate two tubes for each bacterium, one for induction and the other for non-induced control, and at the same time inoculate One tube of empty plasmid bacteria was used as a control. Incubate overnight at 37°C to OD 600nm When the value is about 0.4, add IPTG to the induction tube and the empty plasmid control tube to a final concentration of 1mmol / L, and do not add IPTG to the non-induced control tube. After culturing at 37°C for 4 hours, take out 1mL bacterial solution from each tube and put it in 1.5mL eppendorf every half hour. Label the tube, centrifuge at 12000rpm, d...

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Abstract

The invention belongs to the veterinary biotechnical field, in particular to a riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine and a preparation method thereof. The riemerella anatipestifer-escherichia coli outer membrane protein bivalent vaccine is prepared by emulsifying an antigen and oil adjuvant according to the volume ratio of 1:1-1:5, wherein the antigen is a riemerella anatipestifer RA1 outer membrane protein and/or a riemerella anatipestifer RA2 outer membrane protein and an avian pathogenic escherichia coli 078 outer membrane protein. As proved by an animal experiment, a vaccine immune animal prepared by adopting the method has high vaccine safety and a remarkable immune effect, a high-titer antibody can be generated inside the animal, and the attack of the same type of riemerella anatipestifer and a plurality of serum escherichia coli can be resisted.

Description

technical field [0001] The invention relates to a dual vaccine of R. anatipestifer and Escherichia coli outer membrane protein and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] R. anatipestifer (RA) disease, also known as duck infectious serositis, is one of the most serious infectious diseases to the duck industry. It is more common in 1-8 week-old ducklings, especially 2-3 week-old ducklings. Susceptible, the mortality rate is generally between 5% and 75%, and the mortality rate can reach more than 90% when the environment is harsh or mixed with other diseases. The RA serotypes are complex, and there is a lack of cross-protection among the serotypes. RA1 and RA2 accounted for more than 74% of the isolated strains, and they were the main serotypes currently prevailing in my country. In view of the fact that conventional drugs are prone to drug resistance, the control effect on RA is not ideal, and there is basically no c...

Claims

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Application Information

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IPC IPC(8): A61K39/116A61P31/04C12N15/31C12N15/70C07K14/195C07K14/245A61K39/02A61K39/108
Inventor 杜金玲牟巍赵亚荣黄瑜程龙飞郭书豪傅光华卢会英王勇鹣施少华陈红梅
Owner 兆丰华生物科技(南京)有限公司
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