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Method for constructing haemophilus parasuis genome library and screened immune protein

A technology of Haemophilus suis and genome library, applied in the direction of microorganism-based methods, chemical library, genetic engineering, etc., can solve the problems of lagging gene function research, high production cost, and protein antigen barriers

Inactive Publication Date: 2011-11-23
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the ELISA detection kit for Haemophilus parasuis cannot detect all 15 serotypes at one time. Generally, HPS whole bacteria is used as the antigen. Since HPS is a fastidious bacteria, the production cost is high.
Although the full sequence of the HPS genome has been tested at present, the research on gene function is relatively lagging behind, which sets obstacles for the development of protein antigens

Method used

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  • Method for constructing haemophilus parasuis genome library and screened immune protein
  • Method for constructing haemophilus parasuis genome library and screened immune protein
  • Method for constructing haemophilus parasuis genome library and screened immune protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The establishment of embodiment 1 bacterial SH08-54P genome library

[0042] 1.1 Bacterial genomic DNA extraction

[0043] Inoculate the freeze-dried SH08-54P on the prepared chocolate agar plate, place it in a constant temperature incubator at 37°C, and after cultivating overnight, pick a single colony and inoculate it in a bacterial bottle containing BHI liquid medium containing factor V, and place it in The liquid culture was carried out on a constant temperature air shaker at 37°C. After a period of time, the liquid bacterial culture was expanded and used for bacterial genome extraction (QIAGEN genome extraction kit was used), and the genome concentration was quantified with a spectrophotometer to ensure that the total amount was greater than 100ug.

[0044] The ratio of OD260nm / OD280nm of SH08-54P total DNA measured by Bio-Rad nucleic acid and protein analyzer was 1.712, which indicated that the purity of genomic DNA was high. The genome concentration is 207ug / ml...

Embodiment 2

[0064] Immunological Screening of Example 2 Genome Library

[0065] 2.1 Preparation of rabbit anti-SH08-54P serum

[0066] (1) Preparation of SH08-54P crude antigen

[0067] Inoculate the freeze-dried and preserved SH08-54P strain on the prepared plate and place it in a constant temperature incubator at 37°C. After culturing overnight, pick a single colony and place it in a bacterial bottle containing BHI liquid medium containing factor V, and place it at 37°C The constant temperature air shaker is used for liquid culture, and after a period of time, the liquid bacterial culture is expanded for culture. Centrifuge at 8000r / min at 4°C for 5min, discard the supernatant, wash 3 times with 20mL PBS, centrifuge at 8000r / min again at 4°C for 5min, discard the supernatant, resuspend the pellet in PBS, freeze and thaw at -20°C 3 times. Measure its concentration, aliquot into small portions, and store at -20°C for later use.

[0068] (2) Preparation of rabbit anti-SH08-54P serum

...

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Abstract

The invention belongs to the field of biological engineering, and provides a method for constructing a haemophilus parasuis genome library. The method comprises the following steps of: extracting bacterial genome DNA of haemophilus parasuis with collection number CCTCC NO. M2010063, performing enzyme digestion on the haemophilus parasuis bacterial genome, connecting enzyme digestion fragments of the haemophilus parasuis bacterial genome and vectors and transforming, and performing polymerase chain reaction (PCR) identification and positive cloning. The haemophilus parasuis with collection number CCTCC NO. M2010063 is screened by using the genome library, antiserum is prepared in the screening process, enzyme-linked immunosorbent assay (ELISA) is performed on the antiserum, and the library is repeatedly screened by using the immune serum to obtain 5 positive clones. The positive clones obtained by repeated screening provide a choice for researching antigenic peptide for ELISA detection, and 15 serum type protein antigens of the haemophilus parasuis can be detected at one time.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to Haemophilus parasuis, in particular to a method for constructing a genome library of Haemophilus parasuis and an immune protein for screening. Background technique [0002] Haemophilus parasuis (HPS) mainly causes polyserositis in pigs. In recent years, the pathogen has led to an increasing trend in the mortality of weaned piglets. Haemophilus parasuis is usually an opportunistic pathogen that colonizes the upper respiratory tract, but under certain conditions, it can cause Grass disease, which is characterized by severe serositis, including pleurisy, pericarditis, peritonitis, etc., which manifests as For severe respiratory disease. At present, respiratory infectious diseases, which are mainly transmitted through the respiratory tract, have leapt to the top of the three major systemic infectious diseases. The emergence of immunosuppressive diseases such as post-weaning piglet mul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/70C12N15/55C12N15/54C12N15/31C12N15/63C12R1/21C12R1/19
Inventor 刘佩红王建葛菲菲万世平沈莉萍徐锋杨显超
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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