T7噬菌体衣壳蛋白IOA的表达、纯化及多克隆抗体的制备

目的表达纯化T7噬菌体衣壳蛋白10A，并免疫家兔得到抗10．4多克隆抗体．为研究噬菌体展示抗体技术，进而筛选致病微生物蛋白抗体奠定基础。方法37℃培养含有17噬菌体10A蛋白质粒的大肠杆菌BLT5615，用异丙基-β-D-硫代半乳糖苷直接诱导表达该蛋白；SDS．PAGE鉴定表达状态，盐酸胍变性一尿素复性纯化以包涵体形式存在的蛋白并免疫家兔：辛酸一硫酸铵沉淀法纯化多克隆抗体，间接ELISA测定效价，WesternBlot鉴定特异性。结果成功表达了T7噬菌体衣壳蛋白10A。SDS—PAGE显示所表达的蛋白相对分子质量约为37kDa。主要以包涵体形式存在：复性后SDS—PAGE分析获得单一条带蛋白。利用该蛋白免疫家兔，6次免疫后抗血清的效价均达到1：160003。纯化后的多克隆抗体可以和17噬菌体衣壳蛋白10A特异结合。结论成功表达、纯化了17噬菌体衣壳蛋白10A，并制备了其多克隆抗体。

EXPRESSION,PURIFICATION AND POLYCLONAL ANTIBODY PREPARATION OF T7 BACTERIOPHAGE CAPSID PROTEIN 10A

Objective To express and purify T7 bacteriophage capsid protein 10A and produce its rabbit polyclonal antibody, so as to provide a technical support for T7 phage display and screening pathological microorgan- ism pyotein antibodies. Methods E. coli （ BLT5615 ） containing T7 bateriophage capsid protein 10A plasmid were cultured at 37℃. Then the expression of 10A capsid protein was induced by iso-propyl-β-D-thiogalactoside （IPTG）. The expressed protein was identified with SDS-PAGE, and the inclusion bodies was denatured with guani- dine hydrochloride and renatured with urea. Two rabbits were Immunized with the purified protein to obtain poly- clonal antibody. The final polyclonal antibody was purified by caprylic acid-ammonium sulfate precipitation meth- od;its titers were mesured by indirect ELISA, and specificity was identified by Western blot. Results The molecular weight of the expressed 1031 protein was approximately 37 kDa according to SDS-PAGE analysis. The expression product was existed as inclusion bodies. Single strap of the renatured protein was obviously observed in the SDS- PAGE analysis. Titers of the polyelonal antibody reached dilution of 1 ： 160 000 after the 6th immunization. The purified polyclonal antibody could bind to 10A protein specifically. Conclusion The expression, purification and polyclonal prepartion of 10A protein were successful.