功能未知基因C17orf77重组蛋白及多克隆抗体的制备

Recombinant protein induction and polyclonal antibody preparation of novel gene C17orf77

目的体外克隆表达C17orf77重组蛋白,制备其多克隆抗体,初步探索该蛋白的生物学作用,观察其细胞系分布特征。方法以HepG2细胞cDNA为模板,PCR扩增C17orf77目的基因片段,构建pET-32a(+)-C17orf77原核表达载体。转化大肠埃希菌,IPTG诱导C17orf77重组蛋白表达并进行Western blot鉴定。以重组蛋白免疫大耳白兔,获得兔抗C17orf77蛋白多克隆抗体,并分析其特异性及检测效价。MTT法观察不同浓度重组蛋白对HepG2细胞增殖的影响。利用Western blot观察C17orf77蛋白在HepG2、L02、LX2和Jurkat细胞系的表达情况。结果 PCR扩增获得C17orf77基因片段,成功诱导表达了C17orf77重组蛋白。制备了多克隆抗-C17orf77,ELISA检测抗体效价>1∶1280000。不同浓度C17orf77重组蛋白对HepG2细胞无明显增殖促进或抑制活性(P>0.05)。C17orf77蛋白在HepG2、L02、LX2和Jurkat细胞系均有分布。结论利用体外克隆表达的C17orf77重组蛋白可制备效价和特异性均较高的多克隆抗体。HBV感染相关基因C17orf77在体外肝脏的间质和实质细胞,以及CD4+T细胞系(Jurkat细胞)均有不同程度的表达。提示该基因可能与HBV感染的致病过程相关。

Objective To clone, express and purify C17orf77 recombinant protein for preparation of specific polyclonal antibody and to determine the expression of C17orf77 protein in different cell lines, and further elucidate its biological function. Methods PCR was applied to amplify the gene C17orf77 in vitro, pET-32a （＋）-C17orf77, the recombinant protein prokaryotic expression vector, was transformed into E. coli. Then IPTG was taken as the inductive agent to obtain C17or77 recombinant protein, which was analyzed by the recombinant protein with sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS- PAGE） and Western blot. Specific polyclonal antibody was derived from rabbits immunized by recombinant protein. ELISA and Western blot were applied to test its titer and specificity, respectively. MTT cell proliferation experiment was carried out to observe the effects of different concentration of recombinant protein on proliferation of HepG2 cells. Western blot was used to observe the expression of C 17orf77 protein in HepG2, L02, LX2 and Jurkat cells, respectively. Results The C17orf77 recombinant protein was expressed in a large quantity. Data of ELISA indicated that the titer of polyclonal antibody was higher than 1 ： 1 280 000. And the antibody also had a good specificity, which was confirmed by Western blot. C 17orf77 recombinant protein had no impact on the proliferation of HepG2 cells （P ;〉 0.05）. C 17orf77 protein could be observed in all HepG2, L02, LX2 and Jurkat cell lines. Conclusions Through C 17orf77 recombinant protein, we can prepare the polyclonal antibody with great titer and good specificity. Human novel gene C17orf77 expresses in HepG2, L02, LX2 and Jurkat cells at different level, suggesting that it may participate in the pathogenic processes of HBV infection related diseases.