A kind of trivalent genetic engineering subunit vaccine composition for preventing duck infectious serositis and preparation method thereof

An infectious and serositis technology, applied in the direction of genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve ducklings infected with infectious duck serositis, high safety requirements, immunogenic Poor sex and other problems, to achieve good immune effect, reduce production costs, small immune dose effect

Active Publication Date: 2022-07-19
扬州优邦生物药品有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the commercially available vaccines with approval numbers in my country are all type 1, type 2 bivalent and type 1, type 2, and type 7 trivalent inactivated vaccines, and there are no type 1, type 2, and type 10 vaccines on sale. Urgent need to develop a type 1, type 2, type 10 trivalent vaccine
In addition, currently only inactivated vaccines are commercialized in my country. In production, ducklings are generally injected with duck infectious serositis inactivated vaccines when they are 7 days old. It takes 7 days or longer to produce an effective protective immune response after vaccination. time, so an immune blank period is formed during this period, making ducklings susceptible to infectious duck serositis at an early stage
The immunogenicity of inactivated vaccines is poor, requiring multiple doses of booster, and the safety requirements in the production process are high, resulting in high production costs

Method used

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  • A kind of trivalent genetic engineering subunit vaccine composition for preventing duck infectious serositis and preparation method thereof
  • A kind of trivalent genetic engineering subunit vaccine composition for preventing duck infectious serositis and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of recombinant baculovirus

[0046] 1. Construction of transfer vector: truncation and multiple expression of antigenic proteins TbdR1, SIP, GldG, OmpA and HA of L. anatipestifer, the obtained protein fragments TbdR1-T3, SIP-T3, GldG-T3, OmpA -T3, HA-T3, CAMP and TrpB were entrusted by Anhui General Bio for gene synthesis, and connected to the commercial vector pFastBac I to obtain transfer vectors RA-A, RA-B, RA-C, RA-D, RA-E, RA-F and RA-G.

[0047] 2. Construction of recombinant baculovirus: The transfer vectors RA-A to RA-G synthesized in step 1 were respectively transferred into E. coli DH10 Bac competent cells, and positive clones were selected for PCR identification with M13 primer.

[0048] M13-F: TGTAAAACGACGGCCAGT

[0049] M13-R: CAGGAAACAGCTATGAC

[0050] The PCR reaction system was (total volume 25 μL): DNA template 0.5 μL, M13-F and M13-R 0.5 μL, DNA polymerase 12.5 μL and sterile water 11 μL.

[0051] PCR reaction conditions wer...

Embodiment 2

[0054] Example 2: Preparation of recombinant protein

[0055] 1. Recombinant baculovirus amplification: Inoculate the f1 generation recombinant baculoviruses rRA-A, rRA-B, rRA-C, rRA-D, rRA-E, rRA-F and rRA-G obtained in Example 1 respectively Insect cells sf9 were cultured at 27°C for 4 days, the culture was collected, and the supernatant was centrifuged to obtain the f2 generation recombinant baculovirus;

[0056] 2. Identification of expressed proteins:

[0057] (1) The f2-generation recombinant baculoviruses rRA-A~rRA-G obtained in step 1 were respectively inserted into insect cells sf9 at an inoculum of MOI=5~10, cultured at 27°C for 4 days, the culture was collected, centrifuged to remove The supernatant obtained recombinant proteins TbdR1-T3, SIP-T3, CAMP, GldG-T3, OmpA-T3, TrpB and HA-T3;

[0058] (2) SDS-PAGE identification: The supernatant obtained in step 2 was subjected to SDS-PAGE electrophoresis; after electrophoresis, after staining and decolorization, it was ...

Embodiment 3

[0061] Example 3: Vaccine Preparation

[0062] 1. Inactivation: The TbdR1-T3, SIP-T3, CAMP, GldG-T3, OmpA-T3, TrpB and HA-T3 recombinant proteins prepared in Example 2 were added to the inactivation tank, and the final concentration was 0.2%. ~0.5% inactivator BEI, inactivated at 37°C for 24h.

[0063] 2. Semi-finished product inspection

[0064] (1) Sterility test: carry out sterility test according to the appendix of the current "Chinese Veterinary Pharmacopoeia".

[0065] (2) Determination of protein content: The protein content was detected by BCA method.

[0066] (3) Inactivation test: The inactivated recombinant proteins were respectively inserted into insect cells sf9, and cultured at 27°C for 72 hours. No lesions were observed, and the inactivation test was determined to be qualified.

[0067] 3. Preparation of vaccine vaccine composition:

[0068] The semi-finished protein antigen after passing the inspection is used for vaccine preparation (each liquid component...

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Abstract

The invention discloses a trivalent genetic engineering subunit vaccine composition for preventing duck infectious serositis and a preparation method thereof, belonging to the field of veterinary biological products. The trivalent genetic engineering subunit vaccine composition for preventing duck infectious serositis provided by the present invention includes TbdR1-T3, SIP-T3, CAMP, GldG-T3, OmpA-T3, TrpB and HA-T3 recombinant proteins. The duck infectious serositis trivalent genetic engineering subunit vaccine of the invention has good immunization effect and small immunization dose, and can effectively prevent diseases caused by different serotypes of L. anatipestifer. The preparation method of the invention is simple, can prepare a large amount of antigenic proteins of L. anatipestifer, has short time consumption, high expression amount, greatly reduces the production cost, is beneficial to large-scale production, and fills up the existing commercial duck infectivity. Blanks of trivalent vaccine types 1, 2 and 10 in serositis vaccines.

Description

technical field [0001] The invention relates to a trivalent genetic engineering subunit vaccine composition for preventing duck infectious serositis and a preparation method thereof, belonging to the field of veterinary biological products. Background technique [0002] Duck infectious serositis, also known as Riemerella anatipestifer, is a bacterial infectious disease caused by Riemerella anatipestifer (RA), which is one of the main diseases that endanger the duck industry. The disease was first reported by American scholars Hendrickson and Hilbert in 1932. In 1936, the disease occurred in a commercial duck farm in Illinois, USA. It was once called "duck sepsis". After scientific research, the disease was named "infectious serositis". Leibovitz proposed the use of the name "Riemerella anatipestifer infection". Since the first isolation of Riemerella anatipestifer from duck farms in the suburbs of Beijing in 1982 by Guo Yupu and others in my country, the disease has occurr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/116A61K39/02A61P31/04C12N15/866C07K14/195
CPCA61K39/0208A61P31/04C12N15/86C07K14/195A61K2039/552A61K2039/70C12N2710/14043C12N2800/105
Inventor 李甜甜丁国伟叶正琴徐萍魏荣荣潘晨陈林中日陈森贾丁菡李琛荣雪路
Owner 扬州优邦生物药品有限公司
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