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A kind of attenuated Salmonella vaccine of Haemophilus parasuis

A technology of Haemophilus suis and Salmonella, which is applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., can solve the problems of antibiotics threatening human health and public health safety, economic losses of pig industry, etc., and achieve good immune protection, Good humoral immunity and easy operation

Active Publication Date: 2015-10-28
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The infection rate of Haemophilus parasuis in large-scale breeding pigs reaches 50%-70%, and the fatality rate exceeds 10%, which has caused huge economic losses to the pig industry
At present, the prevention and control of Haemophilus parasuis in the market mainly relies on inactivated vaccines and antibacterial drugs, but the development and utilization of traditional vaccines has become increasingly difficult to pass the safety and quality control requirements put forward by the health management department, and drug resistance caused by antibiotics The problem is also increasingly threatening human health and public health security

Method used

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  • A kind of attenuated Salmonella vaccine of Haemophilus parasuis
  • A kind of attenuated Salmonella vaccine of Haemophilus parasuis
  • A kind of attenuated Salmonella vaccine of Haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cloning of Haemophilus parasuis outer membrane antigen gene hps06257 and construction of recombinant plasmid pYA-06257

[0043] 1. Main experimental materials

[0044] Escherichia coli χ6097 (ara Δ (lac-pro)rpslΔasdA4Δ[xhf2::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. PCR-related reagents such as Taq enzyme, endonucleases such as EcoR I and BamH I and related Buffers, T4 ligase and corresponding Buffers, DH5α competent cells, etc. are all products of Treasure Bioengineering (Dalian) Co., Ltd. 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. Bacterial Genomic DNA Extraction Kit was purchased from Tianjin Tiangen Biochemical Tec...

Embodiment 2

[0063] Example 2 Construction of Salmonella choleraesuis asd gene deletion mutant strain asd-C500

[0064] 1. Main experimental materials

[0065] Attenuated commercial vaccine strain C500 of Salmonella choleraesuis was purchased from China Veterinary Drug Control Institute. pBluescriptSK(+) carrier plasmid (see attached for the physical map of the plasmid Figure 4 ) was purchased from Stratagene, USA. Suicide plasmid pRE112 (see attached Figure 5 ), Escherichia coli χ7213 strain was kindly donated by Professor Dr. Roy Curtiss III, University of Washington, USA.

[0066] PCR-related reagents such as Taq enzyme, endonucleases such as Xba I and BamHI, related Buffer, T4 ligase and Buffer, DH5a 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...

Embodiment 3

[0079] Embodiment 3: recombinant Salmonella choleraesuis asd - C500 / pYA-06257 strain construction

[0080] 1. Salmonella choleraesuis asd - Preparation of C500 Competent Cells

[0081] Removal of Salmonella choleraesuis asd from -80°C freezer - C500 freeze-dried powder, directly dipped with sterile platinum wire and streaked on the surface of LB plates containing 50 μg / mL DAP, cultured upside down at 37°C for 16-18 hours; pick a single colony and inoculate into 4 mL LB containing 50 μg / mL DAP In the culture medium, cultivate overnight at 37°C and 180r / min; transfer the culture to 50mL LB culture medium (containing 50μg / mL DAP) at a volume ratio of 1:100, and culture with shaking at 37°C and 230r / min for 2-3h; take out 100-200 μL culture to detect OD 600 , when OD 600 When the temperature is around 0.8, cool the bacteria solution to 0°C in an ice bath; then centrifuge at 5000r / min for 10 minutes at 4°C to collect the bacteria, discard the supernatant; wash the bacteria ...

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Abstract

The invention belongs to the technical field of animal bacteriosis gene engineering vaccines, particularly relates to strain construction, vaccine preparation and application of a recombined haemophilus parasuis attenuated salmonella vaccine strain without resistance makers and used for expressing a surface antigen hps 06257 of haemophilus parasuis, and aims to obtain a recombinant salmonella choleraesuis asd-C500 / pYA-06257 (the preservation number is CCTCC NO: M2013054) without resistance makers and used for expressing the surface antigen HPS 06257 of haemophilus parasuis. The recombinant bacteria is lack of an asd gene on a salmonella choleraesuis genome, and contains a recombinant plasmid pYA-06257 which can express the asd gene and an outer membrane antigen hps 06257 gene of haemophilus parasuis on the strain. The invention further discloses a construction method of the recombinant strain asd-C500 / pYA-06257 and a corresponding preparation method of the haemophilus parasuis attenuated salmonella vaccine, as well as application to the preparation of the haemophilus parasuis attenuated salmonella vaccine.

Description

technical field [0001] The invention belongs to the field of genetic engineering vaccines for animal bacterial diseases, and in particular relates to strain construction, vaccine preparation and vaccine application of a novel Haemophilus parasuis attenuated Salmonella vaccine capable of expressing Haemophilus parasuis antigen genes without resistance markers. Background technique [0002] Salmonella Choleraesuis (Salmonella Choleraesuis) is the main pathogen that causes paratyphoid fever in piglets aged 2-4 months. Economic losses. Currently widely used piglet paratyphoid commercial vaccine was developed by Fang Xiaowen of the China Veterinary Drug Administration in the 1960s. The vaccine strain Salmonella choleraesuis C500 used in the vaccine has good immunogenicity, but has a certain residual virulence, and the genetic background is not clear , and there is a risk of virulence reversion. With today's strict and standardized safety approval standards, it is very difficult...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/63C12N15/31A61K39/116A61K39/112A61K39/102A61P31/04C12R1/42
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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