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

摘要/Abstract

摘要：

旨在获得牛支原体延伸因子EF-Tu蛋白，分析其在牛支原体菌体内的分布情况，建立EF-Tu间接ELISA法，为进一步研究牛支原体EF-Tu的生物学功能提供理论依据，也为建立牛支原体有效的血清学诊断方法和亚单位疫苗的研制奠定基础。参照Uniprot数据库中M.bovis PG45株EF-Tu基因序列，应用Overlap PCR扩增获得EF-Tu基因并将其克隆至pMD19-T载体，测序正确后，构建pET32a-EF-Tu原核表达载体，转化大肠杆菌BL21表达菌，IPTG诱导表达，纯化后的表达产物免疫新西兰兔（New Zealand rabbit）制备多克隆抗体，利用间接ELISA测定高免血清抗体效价，用Western blot和间接ELISA法初步定位EF-Tu在菌体内的分布，通过优化反应条件，建立间接ELISA检测法。结果表明：重组蛋白EF-Tu约为66 ku，主要以可溶性形式表达；采用间接ELISA测定的多克隆抗体效价为1∶6 400； Western blot和ELISA表明该蛋白在牛支原体细胞质和细胞膜中均有表达，且含量相当。利用EF-Tu建立的ELISA法具有很好的特异性和敏感性。通过原核表达系统成功得到牛支原体延伸因子的重组蛋白，该蛋白分布于菌体细胞质和膜表面，且含量基本相当。本研究所建立的EF-Tu ELISA法适合大规模的血清学诊断检测。

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.

中图分类号:

S852.62

王艳芳, 周雅坪, 郭婷, 张新竹, 吴倩, 陈昕迪, 郝永清. 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[J]. 畜牧兽医学报, 2019, 50(1): 134-142.

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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