Serotype 5 swine actinobacillus pleuropneumoniae (APP) Apx I C/Apx II C double gene deleted vaccine candidate strain

A porcine pleuropneumonia and missing vaccine technology, applied in the direction of bacteria, microbe-based methods, microbiological measurement/inspection, etc., can solve the problems of vaccines that are only suitable for basic research, do not meet biosafety requirements, and have no cross-protection, and reach a broad range Market application prospect, highly targeted effect

Inactive Publication Date: 2012-11-28
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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  • Serotype 5 swine actinobacillus pleuropneumoniae (APP) Apx I C/Apx II C double gene deleted vaccine candidate strain
  • Serotype 5 swine actinobacillus pleuropneumoniae (APP) Apx I C/Apx II C double gene deleted vaccine candidate strain
  • Serotype 5 swine actinobacillus pleuropneumoniae (APP) Apx I C/Apx II C double gene deleted vaccine candidate strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Construction of double gene deletion strain SW1ΔI C / ΔII C ( image 3 )

[0030] SW1 strain, Escherichia coli DH5α, BL21, Bacillus subtilis PKC01 strain, and Actinobacillus pleuropneumoniae serotype 5 strain K17 were purchased from China Veterinary Drug Control Institute.

[0031] 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.

[0032] pBluescrIpt II SK+, pVAX1, PCR product cloning vector pMD19-TSimple were purchased from Treasure Bioengineering (Dalian) Co., Ltd.

[0033] 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. Variou...

Embodiment 2

[0110] Example 2. Research on the biological characteristics of the double-gene deletion strain SW1ΔI C / ΔII C

[0111] (1) Growth characteristics test

[0112] Inoculate the single colonies of the double-gene deletion strain (SW1ΔI C / ΔII C) 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 the parental strain and the gene deletion strain were drawn to compare the growth characteristics of the two.

[0113] The growth curves of double-gene deletion strains and parent strains are as follows: Figure 8 shown. It can be seen from the growth curve that the growth of the gene deletion strain and the...

Embodiment 3

[0120] Example 3. Preparation of double gene deletion strain (SW1ΔI C / ΔII C) attenuated vaccine

[0121] Inoculate the gene-deficient strain into a test tube containing 5mL TSB culture medium and recover for 6-8h, then streak inoculate it on a TSA plate and culture it at a constant temperature at 37°C overnight, pick a well-growing single colony and inoculate it into 100mL TSB culture on the next day Expand the culture on the base, when the OD of the bacterial solution 600 When it reaches about 2.0, the number of viable bacteria is about 6×10 9 CFU / mL, at this time, mix the bacterial solution and 20% skim milk (sterilized) in the ultra-clean workbench at a ratio of 1:1, pack in 2 mL / bottle, seal with sterilized absorbent cotton, and place in- After freezing in a refrigerator at 20°C for 24 hours, freeze and vacuum-dry them in a freeze dryer at -50°C, then take them out, cover them, and store them at -20°C. At the same time, the finished product inspection of the vaccine is c...

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Abstract

The invention discloses construction and identification of a medicine resistance marker-free serotype 5 swine actinobacillus pleuropneumoniae (APP) Apx I C / Apx II C double gene deleted vaccine candidate strain SW1 delta I C delta II C. The strain with a collection number CCTCC NO:M2011343 was collected in the China Center for Type Culture Collection (CCTCC) on 11th October, 2011. According to the gene deleted strain, activating gens C(Apx I C / Apx II C) of hemolysis exotoxin Apx I and Apx II of a serotype 5 APP segregating strain (SW1) which is used as a parent strain are deleted by a genetic engineering technology, and the magnitude of deleted fragments is 475bp and 451bp. The constructed gene deleted strain does not have a medicine resistance marker, meets the biological safety requirement, is high in genetic stability and is not abnormal and can be used as the vaccine candidate strain.

Description

technical field [0001] The invention relates to a serotype 5 Actinobacillus pleuropneumoniae Apx I C / Apx II C double-gene deletion vaccine candidate strain without a drug resistance marker, which belongs to the field of animal bacterial genetic engineering and veterinary biological products. 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...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12Q1/68C12R1/04
Inventor 曹三杰文心田刘琼黄小波龚雨恒文翼平赵勤马晓平张宇周家强
Owner SICHUAN AGRI UNIV
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