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. requirem

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
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Example Embodiment

[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 the China Veterinary Drug Administration.

[0031] Escherichia coli is cultured in LB liquid or solid medium, and ampicillin (Amp) or kanamycin (Kan) with a final concentration of 100μg / ml is added according to different needs; infectious pleuropneumonia rods are cultured in TSB liquid Cultivate in solid medium or TSA, and add NAD at a final concentration of 10μg / ml.

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

[0033] The plasmid small amount extraction kit and DNA gel recovery kit were purchased from OMEGA. Taq DNA polymerase and DNA Marker were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Various restriction enzymes, DNA Liga...

Example Embodiment

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

[0111] (1) Growth characteristics test

[0112] Inoculate single colonies of the double gene deletion strain (SW1ΔI C / ΔII C) and parental strain (SW1) in TSB liquid medium and culture overnight, and then take 50μL of the above bacterial liquid to inoculate 50mL TSB liquid medium and cultivate, wait for OD 600 Start measuring when the value is 0.13, take samples every 1h, and read the OD with a nucleic acid protein analyzer 600 Value through each time point OD 600 The value size compares their growth rate. According to OD 600 Draw the growth curve of the parental strain and the gene-deficient strain to compare the growth characteristics of the two.

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

Example Embodiment

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

[0121] The gene-deficient strain was inoculated into a test tube containing 5mL TSB culture solution and recovered for 6-8 hours, then streaked on a TSA plate and incubated overnight at 37°C. On the next day, a single colony with good growth was picked and inoculated into 100mL TSB culture. Expand the culture on the basis, 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 bacteria liquid and 20% skimmed milk (sterilized) in the ultra-clean workbench at a ratio of 1:1, and divide them into 2mL / bottle, seal with sterilized absorbent cotton, and place- After being frozen and stored in a refrigerator at 20°C for 24 hours, it was freeze-vacuum dried in a freeze dryer at -50°C, then taken out, covered, and stored at -20°C. At the same time, the finished vaccine is inspected. The inspection 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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