基因片段Myo5a原核表达、纯化及多克隆抗体制备  

Prokaryotic expression of Myo 5 a gene fragment,purification and preparation of polyclonal antibody

作　　者：游荷花 唐锐敏 YOU Hehua;TANG Ruimin(Department of Immunology and Laboratory Immunology,Fenyang College of Shanxi Medical University,Fenyang 032200,China)

机构地区：[1]山西医科大学汾阳学院免疫学与免疫学检验教研室,汾阳032200 [2]山西农业大学生命科学学院,太谷030801

出　　处：《中国免疫学杂志》2023年第10期2227-2231,共5页Chinese Journal of Immunology

基　　金：国家自然科学基金青年基金项目(31900450)。

摘　　要：目的:制备了PGEX-4T-3/Myo5a原核表达载体,并表达与纯化融合蛋白,制备其多克隆抗体。方法:通过PCR扩增Myo5a末端一个基因小片段,Bam HⅠ和XhoⅠ双酶切后,连接到原核载体pGEX-4T-3,制备原核表达载体PGEX-4T-3/Myo5a。鉴定后,在BL21菌中诱导表达蛋白并对重组蛋白进行纯化,重组蛋白PGEX-4T-3/Myo5a采用聚丙烯酰胺凝胶电泳和免疫印迹进行鉴定。采用融合蛋白免疫家兔,制备其抗血清,测定抗血清效价和特异性。结果:经鉴定后原核表达载体PGEX-4T-3/Myo5a制备成功,表达的重组蛋白相对分子量约为30 kD。免疫家兔后抗血清滴度为1∶128 000,免疫印迹和间接免疫荧光证明其特异性。结论:实验成功制备了PGEX-4T-3/Myo5a原核表达载体,融合蛋白具备了较高的免疫反应性,并得到了其多克隆抗体,为进一步探讨Myo5a的生物学意义做了铺垫。Objective:The prokaryotic expression vector pGEX-4T-3/Myo5a was prepared,the fusion protein was expressed and purified,and polyclonal antibody was prepared.Methods:One small fragment of Myo5a was amplified by PCR.After BamHⅠand XhoⅠdouble digestion,the fragments were inserted into prokaryotic expression vector pGEX-4T-3 to construct one prokaryotic expression vectors pGEX-4T-3/Myo5a.After correct identification,BL21 prokaryotic expression bacteria were transferred to IPTG induction.The recombinant protein pGEX-4T-3/Myo5a was purified and the recombinant protein was identified by SDS-PAGE electrophoresis and Western blot.The rabbit was immunized with fusion protein,whose antiserum was prepared and its titer and specificity were determined.Results:After identification,the prokaryotic expression vector pGEX-4T-3/Myo5a was prepared correctly,and the relative molecular weight of the expressed recombinant protein was about 30 kD.After immunizing rabbits,the antiserum titer was 1∶128000,and the specificity was proved by Western blot and indirect immunofluorescence.Conclusion:The PGEX-4T-3/Myo5a prokaryotic expression vector is successfully prepared,the fusion protein had high immune reactivity,and its polyclonal antibody is obtain,which paved the way for further exploration of the biological significance of Myo5a.

关 键 词：PGEX-4T-3/Myo5a 原核表达 蛋白表达与纯化 多克隆抗体 

分 类 号：R730.2[医药卫生—肿瘤]