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African swine fever fusion protein containing endotoxin as well as preparation method and application thereof

An African swine fever and fusion protein technology, applied in the field of vaccines, can solve problems such as weak immunogenicity, and achieve the effects of good effect, simple preparation method and good safety

Active Publication Date: 2020-10-09
河南省生物工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention provides an African swine fever fusion protein containing endotoxin. According to the research of the inventor, the endotoxin in the African swine fever fusion protein can enhance the effect of humoral and cellular immunity, and it can still be effective at a higher content. Ensure the safety of African swine fever fusion protein to solve the problem of weak immunogenicity of African swine fever subunit vaccine in the prior art

Method used

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  • African swine fever fusion protein containing endotoxin as well as preparation method and application thereof
  • African swine fever fusion protein containing endotoxin as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1.1 Amino acid sequence-SEQ ID NO.1 of African swine fever fusion protein:

[0026] MTKPRKK MEVIFKTDLRSSSQVVFHAGKKKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSEN IHEKNDNETNECTSKKEQEPSSEVPKDSKKQYGKAPDFKKTIYGTPLKEEEKEV KKMDSEFFQPVYPRHYGECLSPVTTPSFFSTHMYKKFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTKKLRQRNTYTHKDLENSLKKMASGGAFCLIANDGKADKIKKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDKKHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLKKVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDAKKTYQRTRALVGSSSSGSSSGHHHHHHHH

[0027] 1.2 Provide SEQ ID NO.1 to Sangon Biotech (Shanghai) Co., Ltd., and the company will synthesize the nucleotide sequence encoding SEQ ID NO.1 according to the amino acid sequence, and transform the synthesized nucleotide sequence into pET28a- ASFV plasmid.

[0028] 1.3 Expression of recombinant protein

[0029] Using CaCl 2...

Embodiment 3

[0053] Preparation of African swine fever vaccine: Take 1128 ml of No. 7 white oil, add 720 ml of Span-80, and mix well; then weigh 24 grams of aluminum stearate, fully dissolve the above reagents with a colloid mill, mix well and autoclave bacteria, which is the oil phase. The African swine fever fusion protein purified in Example 2 was aseptically filtered and diluted to 0.6 mg / ml with sterile normal saline, and 500 ml of the solution was added to 1,000 ml of the above-mentioned sterile oil phase, and stirred at 10,000 r / min for 2- 5min, get the African swine fever vaccine, spare.

Embodiment 4

[0054] Embodiment 4 The African swine fever vaccine prepared in embodiment 3 is used for immunizing animals

[0055] 4.1 Immunization method: Divide 30 pigs into 10 groups for immunization, inject intramuscularly in the neck behind the ears, and inject 0.5mL of the African swine fever vaccine prepared in Example 3 in the left and right ears respectively; On the 14th day (the first immunization and the second immunization were performed on the 1st day and the 15th day respectively), blood was collected from the anterior vena cava on the 14th and 28th day for subsequent detection.

[0056] Grouping situation: There are 9 groups in the test group, 3 pigs in each group; 1 group in the control group, 3 pigs; pigs in different groups were treated with African swine fever vaccine made from African swine fever fusion protein containing different endotoxin contents. Immunization, the specific groups are shown in Table 1 below:

[0057] Table 1

[0058] group 1 2 3 4 5 ...

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Abstract

The invention relates to the technical field of vaccines, in particular to an African swine fever fusion protein containing endotoxin as well as a preparation method and an application of the Africanswine fever fusion protein. The content of endotoxin in the African swine fever fusion protein containing endotoxin is 50-12800 EU / mg. Researches of the inventor discover that endotoxin in the Africanswine fever fusion protein can play a role in enhancing humoral and cellular immunity, so that the problem of weak immunogenicity of African swine fever subunit vaccines in the prior art is solved toa certain extent.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to an African swine fever fusion protein containing endotoxin and its preparation method and application. Background technique [0002] At present, the research and development of African swine fever vaccine mainly focuses on preparing subunit vaccines with better safety by selecting common proteins with stable immunogenicity such as p30, p54, p72, CD2v, p12, p54, p17 and p30. However, the immunogenicity of the prepared subunit vaccine is relatively weak, and it is not easy to trigger cellular immune response, which affects the use effect of the vaccine. For example, the literature "Modern adjuvants do not enhance the efficacy of an inactivated African swine fever virus vaccine preparation" (Sandra et al., "Vaccine" 32 (2014) 3879-3882) showed that the addition of B cell activators to the African swine fever vaccine showed Antibody-Dependent Enhancement (ADE). A total of 15 hybri...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C07K1/18A61K39/12A61K39/39A61P31/20
CPCC07K14/005A61K39/12A61K39/39A61P31/20C12N2710/12022C07K2319/40C12N2710/12034A61K2039/6087A61K2039/575A61K2039/57A61K2039/552Y02A50/30
Inventor 王云龙李玉林张怡青王继创王国强程蕾王敏王运从
Owner 河南省生物工程技术研究中心
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