鸡RNF165蛋白多克隆抗体制备及其生物信息学分析

doi:10.16431/j.cnki.1671-7236.2022.11.006

Abstract

Abstract: 【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.

Key words: ring finger protein 165 (RNF165); polyclonal antibody; bioinformatics analysis

CLC Number:

LIU Chao, WANG Houkun, ZHU Lilin, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Polyclonal Antibody Preparation and Bioinformatics Analysis of Chicken RNF165[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4159-4167.

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Polyclonal Antibody Preparation and Bioinformatics Analysis of Chicken RNF165

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