Myosin-Vc特异性片段的原核表达及其抗血清的制备

Prokaryotic expression,purification and polyclonal antibody preparation of human myosin-Vc

目的原核表达Myosin-Vc(Myo5c)蛋白并纯化,制备小鼠、家兔多克隆抗体,为探索Myo5c在病毒感染中的作用提供研究工具。方法采用RT-PCR方法从人胃粘膜组织中克隆编码Myo5c特异性蛋白的基因片段,构建该片段与6×His标签的融合蛋白表达质粒pQE-31/Myo5c,原核表达与蛋白纯化后,分别免疫BALB/c小鼠和新西兰兔,制备Myo5c抗血清。采用ELISA检测抗体效价,Westernblot和间接免疫荧光染色检验抗体特异性。结果获得Myo5c特异性片段约42×103的纯化蛋白。ELISA检测小鼠和家兔抗血清效价分别为1∶12800、1∶6400。Westernblot和间接免疫荧光染色显示所制备的抗体能特异性识别Myo5c。结论获得Myo5c特异性蛋白,并成功地制备了Myo5c特异性的小鼠和家兔抗血清。

Objective To obtain purified myosin-Vc （Myo5c） protein and prepare its polyclonal antibodies of mouse and rabbit. Methods The fragment encoding Myo5c specific protein （297 amino acids） was amplified with RT-PCR from human gastric mucosa. The product and the prokaryotic expression plasmid pQE-31 containing 6×His were used to construct pQE-31/Myo5c. The recombinant was transformed into E. coli JM109 and induced to express with IPTG. The objective product was purified. BALB/c mice and rabbits were immunized with the purified Myo5c protein and the antiserum was obtained. Titer and specificity of the polyclonal antibodies were determined by ELISA and Western blotting. Results pQE-31/Myo5c was successfully constructed. The fusion protein of Myo5c with molecular weight 42 000 was obtained and purified. The recombined protein showed immunogenicity. Mouse or rabbit antiserum with high level of specific antibodies for Myo5c was obtained. Indirect immunostaining and Western blotting analysis demonstrated that the antibodies could recognize native Myo5c protein. Conclusion Our results suggest that the prepared antibodies might be useful in studying the function of Myo5c in intracellular trafficking of endocytic vesicle, such as viruses.