牛支原体新疆分离株P48基因的克隆、原核表达及重组蛋白免疫原性研究

doi:10.16431/j.cnki.1671-7236.2018.07.028

Abstract

Abstract:

In order to determine the immunogenicity of Mycoplasma bovis P48 gene and lay the foundation for further screening of the immunoprotective gene of Mycoplasma bovis.In this study,Mycoplasma bovis Xinjiang isolate as the research object,Overlap PCR method was used to amplify P48 gene point mutation of Mycoplasma bovis Xinjiang isolate and construct the prokaryotic expression vector pET-32a(+)-P48,which was transformed into Escherichia coli BL21 (DE3),the recombinant protein P48 was obtained under the induction of ITPG.BALB/c mice was immunized by purified recombinant P48 protein to produce polyclonal antibody,the reactivity and immunogenicity of the recombinant protein were tested using Western blotting and ELISA methods.The results showed that the prokaryotic expression vector pET-32a(+)-P48 was successfully constructed,and the recombinant protein P48 was 66 ku.The purified recombinant P48 protein of Mycoplasma bovis immunized in mice could produce good immune response,and serum antibody titers reached a higher level (D_{450 nm}=1.126).Western blotting results showed that there was obvious antibody response of anti-Mycoplasma bovis P48 recombinant protein of mouse serum,Mycoplasma bovis P48 recombinant protein and Mycoplasma bovis total protein antigen,P48 recombinant protein had good immunogenicity and reactogenicity,which could be used as a candidate gene of Mycoplasma bovis vaccine.The homology of P48 gene was high between Mycoplasma bovis Xinjiang isolate and 5 domestic Mycoplasma bovis,and the genetic relationship was closer.

Key words: Mycoplasma bovis Xinjiang strain; P48 gene; clone; prokaryotic expression; immunogenicity

CLC Number:

S852.62

ZHANG Rui, YANG Mingwei, REN Jingjing, ZHU Ling, LIU Xuwei, YAN Genqiang. Study on Cloning, Prokaryotic Expression and Immunogenicity of P48 Gene of Mycoplasma bovis Xinjiang Isolate[J]. , 2018, 45(7): 1949-1957.

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Study on Cloning, Prokaryotic Expression and Immunogenicity of P48 Gene of Mycoplasma bovis Xinjiang Isolate

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