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Recombinant haemophilus parasuis immunoprotecive antigen HbpA2 and preparation method thereof

A technology for recombining Escherichia coli and recombinant bacteria, applied in the field of genetic engineering, can solve the problems of poor protection of Haemophilus parasuis and only 50% immune protection effect, and achieve the prevention of Haemophilus parasuis disease and clinical application prospects Good, high antibody level results

Active Publication Date: 2015-03-11
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent application with the publication number CN103288934A discloses a method for recombinantly expressing the HbpA gene (Gene ID: 7276898) by means of genetic engineering. However, the recombinant HbpA protein prepared by it can not protect mice from Haemophilus parasuis infection. Good, its immune protection effect is only 50%

Method used

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  • Recombinant haemophilus parasuis immunoprotecive antigen HbpA2 and preparation method thereof
  • Recombinant haemophilus parasuis immunoprotecive antigen HbpA2 and preparation method thereof
  • Recombinant haemophilus parasuis immunoprotecive antigen HbpA2 and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 Preparation of Haemophilus parasuis immunoprotective antigen HbpA2 of the present invention

[0036] 1. Recombinant expression

[0037] 1.1 Cloning of HbpA2 gene and construction of expression vector

[0038] 1.1.1 Primer design and synthesis

[0039]According to the HbpA2 gene sequence of Haemophilus parasuis (Gene ID: 7278927), a pair of primers were designed with biological software Premier Primer5.0 to amplify the HbpA2 gene.

[0040] Upstream primer: 5'-AGTACTCCGACAAATACATTGGTCAACTGT-3';

[0041] Downstream primer: 5'-CTCGAGTTAAGGCTTCAGACTTACGCCATA-3';

[0042] 1.1.2 PCR amplification of HbpA2 gene

[0043] Using the genomic DNA of Haemophilus parasuis CVCC3361 as a template, PCR amplification was carried out according to the conventional method, the amplification system was 25 μL, and 4 replicates were performed. Amplification system:

[0044] Material

volume

genome template

1μL

Primer (upstream, downstream)

2...

experiment example 1

[0131] Experimental example 1 In vivo protective effect of recombinant protein HbpA2 of the present invention

[0132] 1. Experimental method

[0133] 1.1 Determination of the median lethal dose (LD50) of Haemophilus parasuis CVCC3361

[0134]1.1.1 Determination of LD0 and LD100

[0135] 10 mice were used as a group, and each mouse was intraperitoneally injected with live bacterial solution 1×10 9 CFU (LD0 estimated in the pre-experiment), count the death of the mice after 7 days, if two or more died, the challenge dose was reduced by 0.1×10 9 CFU, if all survive, the attack dose will increase by 0.1×10 9 CFU until only one mouse in a group dies, then the adjacent low dose is LD0. Obtain LD100 by the same method.

[0136] 1.1.2 Determination of the challenge dose of the experimental group

[0137] The mice were randomly and equally divided into 7 groups, with 10 mice in each group, of which 7 groups were control groups, and groups 1-6 were test groups. Calculate the r v...

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Abstract

The invention discloses a nucleotide sequence as shown in SEQ ID NO:1 and discloses a recombinant vector and recombinant bacteria containing the recombinant bacteria, a recombinant HbpA2 protein coded by the nucleotide sequence as shown in the SEQ ID NO:1 and a preparation method thereof. The recombinant HbpA2 protein has good immunogenicity and protection genicity. The HbpA2 generated after immunization has high specific antibody level and can remarkably protect attacks of mouse anti-haemophilus parasuis serum type-5 strain with strong virulence. It shows that the recombinant protein HbpA2 is a protective antigen for haemophilus parasuis and can be made into a vaccine.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant Haemophilus parasuis immunoprotective antigen HbpA2 and a preparation method thereof. Background technique [0002] Haemophilus parasuis (Hps) belongs to the genus Haemophilus (Pasteurellaceae) and is a small gram-negative bacillus with various forms. It colonizes the upper respiratory tract of healthy pigs and belongs to the normal flora , Under certain conditions such as immunosuppression, it will cause Glasser's dis-ease, which is mainly characterized by fibrinous polyserositis, meningitis, and arthritis. In recent years, due to the rapid development of large-scale breeding in pig farms , especially in the intensive farming of factories, the swine gerbil disease has become popular and broke out in the world, showing sudden mortality, high morbidity and fatality rate, seriously endangering the development of pig industry, and causing huge economic losses. [00...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/285C12N1/21C12R1/19
Inventor 文心田曹三杰周鹏文翼平黄小波伍锐张禄滑代科何绿琴丁玲强
Owner SICHUAN AGRI UNIV
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