目的 获取人基因C16orf68,构建原核表达载体,诱导重组蛋白的表达,制备兔抗C16orf68蛋白多克隆抗体,观察C16orf68在各细胞系的表达特征.方法 用RT-PCR技术,从肝星状细胞系LX2的总RNA中获得编码C16orf68基因功能片段的cDNA,构建原核表达质粒pET-32a(+)-C16orf68,并导入大肠埃希菌BL21中,IPTG诱导表达重组的重组蛋白.利用Western blot技术、生物质谱技术对表达的C16orf68进行确认.利用纯化的C16orf68重组蛋白免疫新西兰大白兔,获得抗C16orf68蛋白的多克隆抗体,利用Western blot和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测.利用Western blot技术观察C16orf68在多个细胞系的表达特征.结果 扩增获得C16orf68基因片段,测序结果与GenBank已公开的基因序列相一致,成功表达了C16orf68重组蛋白,经Western blot鉴定、生物质谱Q-TOF分析均显示表达正确.利用纯化后的重组蛋白,制备了兔抗人C16orf68多克隆抗体,酶联免疫吸附法检测证实多克隆抗体效价＞1:320 000,Western blot检测证明多克隆抗体的特异性良好,Western blot结果显示各细胞系该蛋白主要表达于肝实质细胞.结论 C16orf68在肝脏主要表达于肝实质细胞,可能与肝细胞损伤相关.%Objective To establish the prokaryotic expression vector of human gene C16orf68, and get the C16orf68 recombinant protein. To prepare the C16orf68 specific rabbit polyclonal antibody, and observe the expression of C16orf68 in different cell lines. Methods C16off68 cDNA was gotten from LX2 cells and coloned into the prokaryotic expressive vector pET-32a( + ), and the recombinant plasmid was transformed into E. coil BL21. The expression of C16off68 protein was induced by IPTG and analyzed by SDS-PAGE and Western blot. The recombinant protein was also confirmed with mass spectrometry. Then the pET-32a( + )-C16orf68 recombinant protein was used to immunize New Zealand rabbits to obtain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Westem blot and ELISA. The expression of C16off68 in different cell lines was observed by Western blot. Results The C16orf68 recombinant protein was highly expressed. The recombinant C16orf68 had same DNA sequence with GenBank and had the same molecular weight with prediction accessed by Western blot. The protein sequence was confirmed by Mass spectrometry. ELISA analysis indicated the titer of polyclonal antibody ＞ 1: 320 000. The high specificity was confirmed by Western blot. The expression of C16orf68 in HepG2 cells was higher than that in LX2 cells, but lower than that in HepG2.2.15 cells. Conclusion The recombinant C16off68 fusion protein was correctly constructed and the specific antibody was obtained, which could provide valuable tools for the investigation on the biological function of C16orf68. HBV replication may up-regulate the expression of C16orf68 in hepatocytes.
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