牛支原体EF-Tu基因的克隆表达、亚细胞定位及间接ELISA方法建立

doi:10.11843/j.issn.0366-6964.2019.01.016

Abstract

Abstract:

The purpose of the present study was to establish an indirect enzyme linked immunosorbent assay (ELISA) detection method via obtaining prokaryotic expression products of elongation factor Tu (EF-Tu) referring Mycoplasma bovis (M. bovis) and analyze its subcellular localization in M. bovis, which could provide theoretical basis for studying the biological function of EF-Tu in M. bovis and lays the essential foundation for establishing effective serological diagnosis method and construction of subunit vaccine against M. bovis. In this study, specific primers were designed referred to gene sequence of EF-Tu from M. bovis PG45 reported in Uniprot. And Overlap PCR-amplified products of EF-Tu of M. bovis was inserted into the pMD19-T vector. With right sequences, prokaryotic expression vector of pET32-EF-Tu was constructed and converted into Escherichia coli (BL21) competent cells. Then, the expression of target protein was induced with 1 mmol·L^-1 of IPTG. After that, the recombinant protein of EF-Tu was purified via Ni-NTA affinity chromatography, with which New Zealand rabbits were immunized to prepare polyclonal antibodies, and the titer of antibody was measured by ELISA. Subsequently, subcellular localization of EF-Tu was analyzed using Western blot and ELISA. The indirect ELISA detection method was established by recombinant protein of EF-Tu. Results were as follows:Recombinant protein of EF-Tu showed an expected molecular mass of 66 kD and was expressed in soluble form. The antibody titer of rabbit serum was 1:6 400. The results of ELISA and Western blot showed that the recombinant protein of EF-Tu showed good immunogenicity, and elongation factor was detected in both cytoplasm and cell membrane of M. bovis with similar expression amounts. The indirect ELISA method for the detection of M. bovis established with EF-Tu showed good specificity and sensitivity. The recombinant protein of EF-Tu was successfully obtained, and elongation factor was expressed in both cytoplasm and cell membrane of M. bovis showing similar expression amounts. The indirect ELISA method showed great potential for serological diagnosis of M. bovis.

Key words: Mycoplasma bovis, prokaryotic expression, polyclonal antibody, subcellular localization, ELISA

CLC Number:

S852.62

WANG Yanfang, ZHOU Yaping, GUO Ting, ZHANG Xinzhu, WU Qian, CHEN Xindi, HAO Yongqing. Cloning, Expression, Subcellular Localization and Indirect ELISA Method of EF-Tu Gene in Mycoplasma bovis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 134-142.

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Cloning, Expression, Subcellular Localization and Indirect ELISA Method of EF-Tu Gene in Mycoplasma bovis

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