Fusion gene of mycoplasma hyopneumoniae and preparation method thereof

A technology of Mycoplasma hyopneumoniae and gene, applied in the field of genetic engineering, can solve problems such as lack, achieve high protection rate, easy purification and preparation, and obvious effects of lung tissue

Inactive Publication Date: 2018-10-02
HEBEI NORMAL UNIV
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  • Claims
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Problems solved by technology

Due to the lack of effective vaccines to prevent Mycoplasma hyopneumoniae infection in my country, in order to more effectively control and reduce the harm of Mhp to animal husbandry, this experiment focuses on the immunogenicity of the fusion protein P65-DnaKc of Mycoplasma hyopneumoniae

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  • Fusion gene of mycoplasma hyopneumoniae and preparation method thereof
  • Fusion gene of mycoplasma hyopneumoniae and preparation method thereof
  • Fusion gene of mycoplasma hyopneumoniae and preparation method thereof

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Embodiment Construction

[0032] The fusion gene of the present invention is obtained through the following steps: firstly, according to the P46 and P65 genes published on Genebank, primers are designed and PCR amplification is performed. The PCR product of the P46 gene and the prokaryotic expression plasmid pET-28a were digested with restriction endonucleases BamHI and SalI at 37°C for 3 h, and ligated overnight with T4 DNA ligase. The recombinant expression vector pET28a-P46 was constructed. The PCR product of the P65 gene and the recombinant expression vector pET28a-P46 were respectively digested with restriction endonucleases SalI and HindIII at 37°C for 3 hours, and ligated overnight with T4 DNA ligase to construct a recombinant expression vector pET28a containing the fusion gene -P46-P65, and through the in vitro mutation technique, three TGA codons in the P46 gene are converted into TGG codons, thereby constructing a recombinant expression vector that can be expressed in prokaryotic cells. The ...

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Abstract

The invention discloses a fusion gene of mycoplasma hyopneumoniae and a preparation method of the fusion gene, and belongs to the field of genetic engineering technology. P46 and P65 genes adopted in the invention can directly design specific primers containing corresponding restriction sites according to a coding region sequence released in NCBI GeneBank. A PCR product of the P46 gene is subject to double enzyme digestion and connection with a prokaryotic expression vector pET-28a, so as to construct a recombinant expression vector pET-28a-P46; and the recombinant expression vector pET-28a-P46 is subject to double enzyme digestion and connection with a PCR product of the P65 gene, so as to directly construct a recombinant expression vector pET-28a-P46-P65. The fusion gene and the preparation method have the following advantages: instead of Linker connection, the P46 and P65 genes are directly subject to double enzyme digestion connection so as to prepare the recombinant expression vector pET-28a-P46-P65; and compared with a process of connecting genes by virtue of Linker and then connecting to a prokaryotic expression vector, the recombinant vector construction process is simple and is easy to prepare the recombinant expression vector, and consumed time is significantly shortened.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the research on gene vaccines for animal bacterial infectious diseases, in particular to a fusion gene of mycoplasma hyopneumoniae and a preparation method thereof. Background technique [0002] Mycoplasma hyopneumoniae (Mhp) can cause bronchopneumonia in pigs, commonly known as swine asthma. The harm of the disease is that Mhp can specifically adhere to the cilia of porcine respiratory epithelial cells, thereby destroying the respiratory mucosa-cilia barrier of the animal body, causing infection of other pathogens, exacerbating respiratory diseases, and then causing porcine respiratory disease syndrome (Porcine respiratory disease syndrome). disease complex, PRDC). [0003] Mhp has high requirements on culture conditions and is difficult to cultivate. So far, only the genes from 4 pathogenic types of mycoplasma have been reported, which are 168 strains, J strains, 7448...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/70
Inventor 赵宝华刘思远彭丽萍朱晓萍陈珍姚昌茹
Owner HEBEI NORMAL UNIV
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