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Construction and application of attenuated Salmonella choleraesuis for expressing mycoplasma hyopneumoniae p65 protein

A technology of Mycoplasma hyopneumoniae and Salmonella, which is applied in the direction of application, medical preparations containing active ingredients, bacteria, etc., can solve biological safety problems and other problems, and achieve good biological safety and good immune protection effects

Inactive Publication Date: 2012-10-17
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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  • Construction and application of attenuated Salmonella choleraesuis for expressing mycoplasma hyopneumoniae p65 protein
  • Construction and application of attenuated Salmonella choleraesuis for expressing mycoplasma hyopneumoniae p65 protein
  • Construction and application of attenuated Salmonella choleraesuis for expressing mycoplasma hyopneumoniae p65 protein

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1 Preparation of gene fragments capable of prokaryotic expression of the Mycoplasma hyopneumoniae p65 protein

[0066] For the cloning method of the p65 protein gene fragment of Mycoplasma hyopneumoniae, see figure 2 shown.

[0067] 1. The primers required for preparing the p65 gene fragment capable of prokaryotic expression are shown in Table 1.

[0068] Table 1 prepares the primers required for the p65 gene fragment capable of prokaryotic expression

[0069]

[0070] Note in Table 1: The underlined part of the primer is the enzyme cutting site.

[0071] 2. Preparation of p65 gene fragment capable of prokaryotic expression

[0072] Genomic DNA extracted from Mycoplasma hyopneumoniae live vaccine (purchased from Nanjing Tianbang Biotechnology Co., Ltd., which contains Mycoplasma hyopneumoniae Mhp168 strain and adjuvant) (extraction method is in accordance with the bacterial genome of Tiangen Biochemical Technology (Beijing) Co., Ltd. DNA Extraction Kit I...

Embodiment 2

[0076] Construction and identification of embodiment 2 recombinant plasmid pYA-65

[0077] 1. Construction of recombinant plasmid Primers required for construction of recombinant plasmid pYA-65

[0078] Table 2 constructs the primers required for recombinant plasmid pYA-65

[0079]

[0080] Note in Table 2: The underlined part of the primer is the restriction site.

[0081] 2. Construction and identification of recombinant plasmid pYA-65

[0082] Depend on Figure 8 As shown, the plasmid pGEX-KG-65 capable of prokaryotic expression of the Mycoplasma hyopneumoniae p65 gene after the site-directed mutation is used as a template, and the primers p65(SaII) / p65(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 the ligated product was electrotransformed into Escherichia col...

Embodiment 3

[0083] Example 3 Construction and identification of Salmonella choleraesuis C500 (pYA-65) expressing Cpro-65 fusion protein

[0084] The construction method of Salmonella choleraesuis C500 (pYA-46) is as follows: Figure 8 shown. The correct recombinant plasmid pYA-65 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-65) is as follows: Figure 8 shown. Pick a single bacterium colony on the DAP negative plate and cultivate, carry...

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Abstract

The invention belongs to the field of animal bacteria engineering technology, and specially relates to a construction of a resistance marker-free recombinant attenuated Salmonella choleraesuis for expressing a main immunogenic membrane protein of mycoplasma hyopneumoniae, a vaccine preparation and an application thereof. The resistance marker-free attenuated Salmonella choleraesuis C500(pYA-65) for expressing mycoplasma hyopneumoniae p65 protein is obtained, and the collection number is CCTCC NO: M2011107. An asd gene necessary for the growth of Salmonella choleraesuis is deleted in the attenuated strain, and the attenuated strain contains a plasmid capable of expressing the asd gene and the p65 gene of mycoplasma hyopneumoniae. The invention further discloses a Salmonella choleraesuis and mycoplasma hyopneumoniae vaccine prepared from the attenuated strain, a preparation method and an application thereof. The bivalent vaccine of the invention can stimulate a swine to generate a protective immune response resisting the Salmonella choleraesuis and mycoplasma hyopneumoniae, and effectively prevent infection of the Salmonella choleraesuis and mycoplasma hyopneumoniae.

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 p65 gene of Mycoplasma hyopneumoniae without a resistance marker and its 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] Salmonella as a va...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/31A61K39/116A61K39/112A61K39/02A61P31/04C12R1/42C12R1/35
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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