Prokaryotic expression,purification and polyclonal antibody preparation of human myosin-Vc / 第三军医大学学报

ABSTRACT

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.