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Recombinant Salmonella choleraesuis expressing Mycoplasma hyopneumoniae p46 protein and its preparation method and application

A technology of mycoplasma hyopneumoniae and salmonella, which is applied in the field of genetic engineering of animal bacteria, can solve problems such as biological safety issues, and achieve good biological safety and good immune protection effects

Inactive Publication Date: 2017-03-15
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many defects in the traditional method of constructing recombinant Salmonella strains; for example, expression plasmids carrying resistance genes were commonly used in the past, which were not accepted by people due to biosafety problems

Method used

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  • Recombinant Salmonella choleraesuis expressing Mycoplasma hyopneumoniae p46 protein and its preparation method and application
  • Recombinant Salmonella choleraesuis expressing Mycoplasma hyopneumoniae p46 protein and its preparation method and application
  • Recombinant Salmonella choleraesuis expressing Mycoplasma hyopneumoniae p46 protein and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Cloning of the gene fragment of embodiment 1 Mycoplasma hyopneumoniae p46 protein

[0070] For the cloning method of the p46 protein gene fragment of Mycoplasma hyopneumoniae, see figure 2 shown.

[0071] 1. The primers required for preparing the p46 gene fragment capable of prokaryotic expression are shown in Table 1.

[0072] Table 1 prepares the primers required for the p46 gene fragment capable of prokaryotic expression

[0073]

[0074] Note in Table 1: The underlined part of the primer is the enzyme cutting site.

[0075] 2. Preparation of prokaryotic expressed p46 gene fragments

[0076] Genomic DNA extracted from the live vaccine strain Mhp168 of Mycoplasma hyopneumoniae (purchased from Nanjing Tianbang Biotechnology Co., Ltd. Beijing) Co., Ltd. Bacterial Genomic DNA Extraction Kit Instructions Operation) as a template, utilize the primer p46(BamH I) / p46(HindIII) PCR amplification p46 gene fragment as shown in Table 1, the amplification system is as foll...

Embodiment 2

[0079] Construction and identification of embodiment 2 recombinant plasmid pYA-46

[0080] For the construction method of recombinant plasmid pYA-46, see Figure 8 shown.

[0081] 1. The primers required to construct the recombinant plasmid pYA-46 are shown in Table 2.

[0082] Table 2 Primers required for recombinant plasmid pYA-46

[0083]

[0084] Note in Table 2: The underlined part of the primer is the restriction site.

[0085] 2. Construction and identification of recombinant plasmid pYA-46

[0086] Depend on Figure 8 As shown, the plasmid pGEX-KG-46 capable of prokaryotic expression of the Mycoplasma hyopneumoniae p46 gene after the site-directed mutation is used as a template, and the primers p46(SalI) / p46(HindIII) shown in Table 2 are used for PCR amplification and recovery. The product and the shuttle plasmid pYA3493 (gifted by Dr. Roy Curtiss III, University of Washington, USA) were digested with Sal I and HindIII respectively, ligated after recovery, and ...

Embodiment 3

[0087] Example 3 Construction and identification of Salmonella choleraesuis C500 (pYA-46) expressing Cpro-46 fusion protein

[0088] The construction method of Salmonella choleraesuis C500 (pYA-46) is as follows: Figure 8 shown. The recombinant plasmid pYA-46 identified above was electrotransformed (parameters: voltage 2.0KV, time 4ms, capacitance 25μF and pulse resistance 200Ω) to C500 competent cells of the asd gene deletion strain (see literature: Xu Yindi et al., Salmonella choleraesuis C500 Construction and identification of a balanced lethal vector system for strain ΔcrpΔasd deletion strain. Acta Biological Engineering, 2006, 5(3): 366-371. Attached is a commitment certificate for distributing attenuated Salmonella choleraesuis vaccine strain C500ΔcrpΔasd deletion strain to the public). The construction process of Salmonella choleraesuis C500 (pYA-46) is as follows: Figure 8 shown. Pick a single bacterium colony on the DAP negative plate and cultivate it, carry out ...

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Abstract

The invention belongs to the genetic engineering technology field of animal bacteria, especially relates to construction, vaccine preparation and application of recombinant salmonella choleraesuis expressing a main immunogenic membrane protein of mycoplasma hyopneumoniae and the expression contains no resistance markers. A strain of salmonella choleraesuis C500 (pYA-46) expressing mycoplasma hyopneumoniae p46 protein from the expression which contains no resistance markers is provided by the invention, and the preservation number is: CCTCC NO: M2011106. The recombinant strain lacks of an asd gene which is essential for the growth of samonella choleraesuis, and contains plasmid which can express asd in the strain and mycoplasma hyopneumoniae p46 protein. The invention also discloses the preparation method and the application of salmonella choleraesuis and pig mycoplasma pneumonia vaccine prepared by the recombination stain. The bivalent vaccine prepared in the invention can stimulate porcine to produce an immune reaction to protect porcine from the salmonella choleraesuis and the porcine pneumonia mycoplasma and can effectively prevent from being infected by the salmonella choleraesuis and the porcine pneumonia mycoplasma.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of animal bacteria, and in particular relates to a recombinant Salmonella choleraesuis live vaccine strain expressing the membrane protein p46 gene of Mycoplasma hyopneumoniae without a resistance marker, its preparation method and application. Background technique [0002] As a live vaccine carrier, Salmonella can carry the immunogenic genes of various bacteria, viruses or parasites, thus becoming a multivalent recombinant genetically engineered live vaccine for salmonellosis and other diseases. In recent decades, the study of using attenuated Salmonella as the expression vector of live vaccines has received extensive attention. Among them, Salmonella strains weakened by genetic engineering methods are used as live bacterial vectors to express foreign genes, which have been widely used in vaccine research for tumors, viral diseases, bacterial diseases, and parasitic diseases. [0003...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12R1/42C12R1/35
Inventor 何启盖马丰英邹浩勇陈焕春郭爱珍徐高原吴斌张灏
Owner HUAZHONG AGRI UNIV
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