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Attenuated vaccine of salmonella choleraesuis capable of expressing surface antigen of haemophilus parasuis

A technology of Haemophilus suis and Salmonella, applied in the direction of bacterial antigen components, bacteria, antibacterial drugs, etc., can solve the problems of bacteriolysis and death, and achieve the effect of convenient operation, broad application prospects, and good immune protection

Active Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The asd gene deletion strain cannot form a complete cell wall in the absence of exogenous DAP, resulting in lytic death

Method used

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  • Attenuated vaccine of salmonella choleraesuis capable of expressing surface antigen of haemophilus parasuis
  • Attenuated vaccine of salmonella choleraesuis capable of expressing surface antigen of haemophilus parasuis
  • Attenuated vaccine of salmonella choleraesuis capable of expressing surface antigen of haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Salmonella choleraesuis asd gene deletion strain asd - Construction of the C500

[0036] 1. Main experimental materials

[0037] Salmonella choleraesuis attenuated vaccine strain C500 was purchased from China Veterinary Drug Control Institute. pBluescriptSK(+) carrier plasmid (see attached Figure 13 ) was purchased from Stratagene, USA. Suicide plasmid pRE112 (see attached Figure 12 ), Escherichia coli χ7213 were kindly donated by Professor Dr.Roy Curtiss III, University of Washington, USA.

[0038] PCR-related reagents such as Taq enzyme, endonucleases such as BamH I and Hind III and related Buffer, T4 ligase and Buffer, DH5α competent cells, etc. are all products of Treasure Bioengineering (Dalian) Co., Ltd. Bacterial Genomic DNA Extraction Kit was purchased from Tianjin Tiangen Biochemical Technology (Beijing) Co., Ltd. UNIQ-10 Column DNA Gel Recovery Kit was purchased from Shanghai Sangon Bioengineering Technology Co., Ltd. DNA Marker (2K or 2K Pl...

Embodiment 2

[0051] Embodiment 2: Cloning of the outer membrane protein gene HbpB of Haemophilus parasuis

[0052] 1. Main experimental materials

[0053] NAD (nicotinamide adenine dinucleotide) and DAB chromogenic diagnostic kits are all products of Sigma Company, and newborn bovine serum is a product of Hangzhou Sijiqing Biological Products Co., Ltd. TSB, TSA for Difco TM product. Refer to Example 1 for the rest of the main experimental materials.

[0054] 2. Target gene analysis and primer design

[0055] The sequence registration number of the target antigen HbpB involved in the present invention and its application in Haemophilus parasuis SH0165 strain (Cai Xuwang, Research on the isolation and identification of Haemophilus parasuis and its diagnostic method and inactivated vaccine, June 2006, Huazhong Agricultural University Doctoral dissertation, National Library of China, National Digital Library of China (http: / / res4.nlc.gov.cn / home / search.trs?method=showDetail&channelid=3&id=...

Embodiment 3

[0065] Embodiment 3: Construction of recombinant Salmonella choleraesuis asd-C500 / pYA-HbpB strain

[0066] 1. Main experimental materials

[0067] Escherichia coli χ6097 (ara Δ (lac-pro)rpslΔasdA4Δ[zhf-2.∵Tn10]thiΦ80d / lacZΔM15) was kindly provided by Dr. Roy CurtissIII, University of Washington, USA. CaCl 2 , Glycerin and other reagents are products of Shanghai Sinopharm Chemical Reagent Co., Ltd. The other main experimental reagents are shown in Example 1.

[0068] 2. Preparation of competent state of χ6097 (CaCl 2 Law)

[0069] Using CaCl 2 Escherichia coli χ6097(ara Δ (lac-pro)rpslΔasdA4Δ[zhf-2.∵Tn10] thiΦ80d / lacZΔM15) competent cells, the specific steps are as follows: take out Escherichia coli χ6097 from -80℃ refrigerator Handed over to China.Wuhan.Wuhan University China Type Culture Collection Center for preservation, its preservation number is CCTCCNO: M2012344) freeze-dried powder, after thawing, put a line on the LB plate, and incubate at a constant temperatur...

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Abstract

The invention belong to the field of gene engineering vaccines for animal bacterial diseases, and particularly relates to construction, vaccine preparation and application of an attenuated vaccine strain of recombined salmonella choleraesuis capable of expressing a surface antigen gene HbpB of haemophilus parasuis without resistance markers. The recombined salmonella choleraesuis asd-C500 / pYA-HbpB capable of the expressing surface antigen gene HbpB of hemophilus parasuis without resistance markers is obtained (the preservation number is CCTCC No: M2013052); the asd gene on the genome of the salmonella choleraesuis is lost by the recombined strain, so that the asd gene and the recombinant plasmid pYA-HbpB of an outer membrane antigen of haemophilus parasuis can both be expressed in the strain. The invention further discloses a construction method of the recombination strain asd-C500 / pYA-HbpB and a corresponding manufacturing method of the attenuated vaccine of the recombination strain asd-C500 / pYA-HbpB, and application to the preparation of vaccines of salmonella choleraesuis and haemophilus parasuis.

Description

technical field [0001] The invention belongs to the field of genetic engineering vaccines for animal bacterial diseases, and in particular relates to the construction, vaccine preparation and application of a recombinant Salmonella choleraesuis attenuated vaccine strain expressing the surface antigen gene of Haemophilus parasuis without a resistance marker. Background technique [0002] Haemophilus parasuis (HPS) is a common bacterium in the upper respiratory tract of conventionally reared pigs, but under certain conditions it can invade the body and cause severe systemic diseases, such as fibrinous polyserositis , arthritis and meningitis as the main features ( , 1910). In recent years, in view of the continuous adjustment of feeding technology and the continuous occurrence of new respiratory syndrome, Haemophilus parasuis has become more and more popular, which has caused great harm to the pig industry at home and abroad. The main means of prevention and control of Haem...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/116A61K39/112A61K39/102A61P31/04C12R1/42C12R1/21
Inventor 金梅林赵建平周明光宋岱松张强张安定石建康超徐高原陈焕春
Owner HUAZHONG AGRI UNIV
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