长爪沙鼠CST3序列同源性分析及蛋白体外表达

目的 分析长爪沙鼠半胱氨酸蛋白酶抑制剂C (Cystatin C，CST3)cDNA序列同源性，并建立CST3蛋白原核表达体系，为长爪沙鼠CST3抗体制备和后续基因功能研究奠定基础。方法 对长爪沙鼠Cst3 cDNA序列进行克隆、同源性分析及密码子优化；将优化后序列酶切连接到pET28a载体，完成重组CST3蛋白表达载体构建；将该载体转化到感受态细胞中，通过异丙基硫代半乳糖苷(Isopropyl β-D-Thiogalactoside，IPTG)诱导实现CST3蛋白的原核表达，并用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)验证。结果 长爪沙鼠Cst3基因与人和小鼠Cst3基因的序列同源性较低。密码子优化后的长爪沙鼠Cst3表达序列插入到pET28a质粒中，获得了CST3蛋白表达载体。经1 mmol/L IPTG 37 ℃诱导12 h，可获得大量CST3蛋白。结论 成功地构建了长爪沙鼠CST3蛋白的体外表达体系。

Homology Analysis and Prokaryotic Expression of Mongolian Gerbil CST3

Objective To analyze the sequence homology of CST3 and establish a prokaryotic expression system of CST3 protein in vitro for producing anti-Mongolian gerbil Cystatin C (CST3) antibodies and perform function studies in future. Method After cloning and homology analysis, the condon optimization of Cst3 cDNA sequences of Mongolian gerbils were conducted. The recombinant CST3 protein expression vector was constructed by inserting the optimized sequences into pET28a vectors, which were then transformed into competent cells. The prokaryotic expression of CST3 protein was induced by Isopropyl β-D-Thiogalactoside (IPTG) and verified by SDS-PAGE gel electrophoresis and Western blotting. Result Both nucleotide and amino acid sequences of CST3 in Mongolian gerbils displayed low identity with human or mice CST3. After codon optimization, the Cst3 expression sequences of Mongolian gerbils were inserted into the pET28a plasmids, and a large amount of CST3 protein was obtained after induced expression with 1 mmol/L IPTG at 37 ℃ for 12 h as an optimized condition. Conclusion We successfully established a prokaryotic expression system of CST3 protein in vitro.