鸡RNF165蛋白多克隆抗体制备及其生物信息学分析

【目的】制备鸡环指蛋白165(RNF165)多克隆抗体并对RNF165蛋白进行生物信息学分析，以期为研究鸡RNF165的生物学功能提供参考。【方法】以鸡胚成纤维细胞(DF-1)为试验材料，提取总RNA,反转录获得cDNA,通过PCR扩增RNF165基因，将其连接至pET-28a(+)质粒构建重组表达质粒pET-28a-RNF165。将测序正确的重组表达质粒转化大肠杆菌BL21感受态细胞进行诱导表达，将表达的融合蛋白纯化后按照制定的免疫程序免疫7周龄BALB/c雌鼠。第3次免疫7 d后，分离小鼠血清，用Western blotting检测鼠抗RNF165蛋白多克隆抗体的特异性，间接ELISA法测定其效价。使用在线生物信息学软件对RNF165蛋白理化性质、信号肽、磷酸化位点、亚细胞定位、跨膜结构、二级结构和保守结构域进行分析。【结果】成功扩增得到大小为1 068 bp的RNF165基因。成功构建原核表达载体pET-28a-RNF165,并诱导表达出约43 ku的RNF165蛋白。Western blotting结果显示，制备的鼠抗RNF165蛋白多克隆抗体能特异性识别DF-1细胞中过表达的...

Polyclonal Antibody Preparation and Bioinformatics Analysis of Chicken RNF165

【Objective】 The aim of this study was to prepare a polyclonal antibody against chicken ring finger protein 165 (RNF165) and perform bioinformatics analysis on RNF165 protein, in order to provide references for studying the biological function of chicken RNF165.【Method】 Using chicken embryo fibroblasts (DF-1) as the test material, total RNA was extracted from DF-1 and reverse transcribed to obtain cDNA. The RNF165 gene was amplified by PCR, and it was connected to the pET-28a (+) plasmid to construct the recombinant expression plasmid pET-28a-RNF165. The correctly sequenced recombinant expression plasmid was transformed into E. coli BL21 competent cells for induced expression, and the expressed fusion protein was purified and immunized 7-week-old BALB/c female mice according to the established immunization program. Seven days after the third immunization, the mouse serum was separated, and the specificity of mouse anti-RNF165 protein polyclonal antibody was detected by Western blotting, and the titer was determined by indirect ELISA. The physicochemical properties, signal peptide, phosphorylation site, subcellular localization, transmembrane structure, secondary structure and conserved domain of RNF165 protein were analyzed using online bioinformatics softwares.【Result】 The RNF165 gene with a size of 1 068 bp was successfully amplified. The prokaryotic expression vector pET-28a-RNF165 was successfully constructed, and the RNF165 protein of about 43 ku was induced. Western blotting results showed that the prepared mouse anti-RNF165 protein polyclonal antibody could specifically recognize the overexpressed RNF165 protein in DF-1 cells with a titer of 1:32 000. The results of bioinformatics analysis showed that RNF165 protein was composed of 350 amino acids, with a molecular weight of 39.88 ku, an isoelectric point of 7.13, and an instability index of 69.87. RNF165 protein was a hydrophilic protein without signal peptide and transmembrane domain, and it contained 29 phosphorylation sites. The secondary structures of RNF165 protein was mainly composed of random coil, alpha helix, extended chain and beta turn. The highest probability of RNF165 appeared in the nucleus was 47.8%. The amino acid residues 296-341 of the C-terminal of RNF165 protein were conserved domains of the RING-Ubox superfamily.【Conclusion】 The mouse anti-RNF165 polyclonal antibody that was prepared in this study had high reactivity and specificity. RNF165 protein was a hydrophilic protein and was mainly expressed in the nucleus. The results could provide materials for exploring the biological function of RNF165 protein.