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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 and express the recombinant protein C17orf77 in vitro and prepare its polyclonal antibody. The biological function of the protein was explored preliminarily and the cell distribution characteristics were observed. Methods The gene fragment of C17orf77 gene was amplified by PCR using HepG2 cDNA as a template. The prokaryotic expression vector pET-32a (+) - C17orf77 was constructed. The recombinant plasmid was transformed into Escherichia coli. IPTG induced the expression of C17orf77 recombinant protein and identified by Western blot. Rabbit anti-C17orf77 protein polyclonal antibody was obtained by immunizing rabbits with recombinant protein, and its specificity and detection titer were analyzed. MTT assay of different concentrations of recombinant proteins on HepG2 cell proliferation. Western blot was used to observe the expression of C17orf77 protein in HepG2, L02, LX2 and Jurkat cell lines. Results C17orf77 gene fragment was amplified by PCR and the recombinant protein C17orf77 was successfully induced. Polyclonal anti-C17orf77 was prepared. Antibody titers> 1: 1280000 by ELISA. Different concentrations of C17orf77 recombinant protein on HepG2 cells did not significantly promote proliferation or inhibition (P> 0.05). The C17orf77 protein is distributed in HepG2, L02, LX2 and Jurkat cell lines. Conclusion The polyclonal antibody with high titer and specificity can be prepared by using C17orf77 recombinant protein cloned in vitro. The HBV infection-related gene C17orf77 expressed in different degree in vitro in interstitial and parenchymal cells of liver and CD4 + T cell line (Jurkat cell). Suggesting that the gene may be associated with the pathogenesis of HBV infection.