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.
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)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
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)。
