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Apx I C gene deleted mutant strain of porcine infectious actinobacillus pleuropneumonia, construction method, vaccine and application

A gene deletion, pleuropneumonia technology, applied in the biological field, can solve problems such as vaccines that are only suitable for basic research, do not meet biosafety requirements, and have no cross-protection

Inactive Publication Date: 2012-08-01
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, attenuated mutant strains are constructed by inserting the resistance marker gene into the target gene through homologous recombination. Although it is easy to screen the mutant strains in the resistance selective medium, these mutant strains do not meet biological safety because they contain resistance markers. requirements, and therefore cannot be used for vaccine production but only for basic research
In the past few decades, the pathogenesis of porcine infectious pleuropneumonia and related virulence factors have been studied more and more deeply, but until now there is still no safe, reliable vaccine that can provide effective cross-protection

Method used

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  • Apx I C gene deleted mutant strain of porcine infectious actinobacillus pleuropneumonia, construction method, vaccine and application
  • Apx I C gene deleted mutant strain of porcine infectious actinobacillus pleuropneumonia, construction method, vaccine and application
  • Apx I C gene deleted mutant strain of porcine infectious actinobacillus pleuropneumonia, construction method, vaccine and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Construction of gene deletion mutant strain SW1ΔIC

[0067] Escherichia coli DH5α, BL21, Bacillus subtilis PKC01 strain, Actinobacillus pleuropneumoniae serotype 5 K17 strain and SW1 strain were purchased from China Veterinary Drug Control Institute.

[0068] Escherichia coli was cultured in LB liquid or solid medium, and ampicillin (Amp) or kanamycin (Kan) with a final concentration of 100 μg / ml was added according to different needs; infectious pleuropneumonia actinic rods were cultured in TSB liquid Cultured in medium or TSA solid medium, and added NAD at a final concentration of 10 μg / ml.

[0069] pBluescriptⅡSK+, pVAX1, PCR product cloning vector pMD19-TSimple were purchased from Treasure Bioengineering (Dalian) Co., Ltd.

[0070] Plasmid mini-extraction kit and DNA gel recovery kit were purchased from OMEGA Company. Taq DNA polymerase and DNA Marker were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Various restriction endonuclease...

Embodiment 2

[0122] Example 2. Study on the biological characteristics of the gene deletion strain SW1ΔIC

[0123] (1) Growth characteristics test

[0124] Inoculate the single colonies of the mutant strain (SW1ΔIC) and the parental strain (SW1) in TSB liquid medium for overnight culture, then inoculate 50 μL of the above bacterial liquid into 50 mL of TSB liquid medium for culture, and wait until the OD 600 When the value is 0.13, start to measure, take a sample every 1h, and read its OD with a nucleic acid protein analyzer 600 value, through each time point OD 600 value to compare their growth rates. According to OD 600 The growth curves of parental strains and mutant strains were plotted, and the growth characteristics of the two were compared.

[0125] The growth curves of mutants and parental strains are as follows: Figure 21 shown. It can be seen from the growth curve that the growth of the mutant strain and the parent strain are basically consistent, which indicates that the ...

Embodiment 3

[0132] Example 3. Preparation of Deletion Strain (SW1ΔIC) Attenuated Vaccine

[0133] The genetic stability test was carried out on the constructed APP serotype 5 mutant strains, and the deletion mutant strains were streak-cultured on a TSA plate (the same below) containing 0.01% NAD and cultured at 37°C until obvious colonies were grown. Bacterial colonies were continuously subcultured, and the generation was defined as 10 generations, and the strains of each generation were inoculated into TSB culture fluid containing 0.01% NAD (the same below) to observe whether the bacteria grew, and at the same time, the bacteria of each generation were identified by PCR. The 475bp band after the deletion can still be amplified at the 10th generation, and heredity is stable. Inoculate the gene-deleted strain into a test tube containing 5mL of TSB culture medium and recover for 6-8h, then inoculate it on a TSA plate and incubate overnight at a constant temperature of 37°C, pick a well-grow...

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Abstract

The invention discloses a resistance marker-free Apx I C gene deleted mutant strain of porcine infectious actinobacillus pleuropneumonia. The number of the vaccine candidate strain is No. SW1delta I C. The strain is preserved in China Center for Type Culture Collection (CCTCC) on August 31, 2011, with a preservation number of CCTCC M 201130. The activating gene C of the toxin Apx I is deleted by means of genetic engineering, and the size of the deleted fragment is 475bp. The gene deleted strain constructed in the invention has genetic stability, and cannot reverse to virulence. In the invention, the gene deleted strain is also utilized to prepare a gene deleted attenuated vaccine. Meanwhile, immune efficacy tests of laboratory rats and piglets prove that the vaccine can induce good immune protection.

Description

technical field [0001] The present invention relates to porcine infectious actinobacillus pleuropneumoniae ApxIC gene deletion mutant strain, construction method, vaccine and application, and belongs to the field of biotechnology. Background technique [0002] Porcine infectious pleuropneumonia is a highly contagious and fatal respiratory disease of pigs caused by Actinobacillus pleuropneumoniae (APP). Exist widely in all the pig-raising countries in the world, especially in Europe and the United States, which has caused huge economic losses to the pig industry and seriously hindered the healthy development of the world's pig industry. Due to the large number of serotypes of Actinobacillus pleuropneumoniae and the prevalence of different serotypes in different countries and regions, it has brought great difficulties to the prevention and control of the disease. At present, the incidence rate of this disease in my country is still increasing year by year, and the positive ra...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/74A61K39/02A61P31/04C12R1/01
Inventor 曹三杰文心田王国镔黄小波华方根文翼平谢文浩李建赵勤刘琼
Owner SICHUAN AGRI UNIV
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