奶牛S100A12基因克隆、原核表达及体外抗菌活性分析

Cloning and expression of cDNA of S100A12 from Holstein cow and its antibacterial activity

克隆奶牛S100A12基因并在大肠杆菌中高效表达。采用RT-PCR扩增奶牛外周血白细胞中S100A12基因,构建原核表达载体pCold TF-S100A12,在大肠杆菌BL21(DE3)中IPTG诱导表达并纯化。SDS-PAGE和Westernblotting分析显示S100A12基因以融合蛋白形式表达,表达量占菌体总蛋白的46.7%,纯化的重组蛋白(80mg/L)。琼脂扩散法测定重组蛋白对乳腺炎主要致病菌大肠杆菌和金黄色葡萄球菌的抗菌活性。结果显示:重组蛋白对大肠杆菌有抗菌活性,而对金黄色葡萄球菌无抗菌活性。本研究为S100A12蛋白抗体制备及基因功能研究奠定了基础。

S100A12 cDNA from bovine peripheral blood was cloned by RT-PCR,then purified PCR products was connected with prokaryotic expression vector pCold TF（TaKaRa） to construct pro-karyotic expression plasmid pCold TF-S100A12. Subsequently, the plasmid was expressed in E. coli BL21 （DE3） cell induced by IPTG. Result of SDS-PAGE and western blotting indicated that S100A12 gene was expressed in form of fusion protein, accounting for 46.7% of total protein of the strain. Agar hole diffusion-inhibition zone method determined the purified protein S100A12, with 80 mg/L. The antibacterial activity was assayed by agar hole diffusion-inhibition zone method using the purified recombination protein against E. coli and Sta. aureu. The recombinate protein had antibacterial activity against Escherichia coll. In contrast,it was negative against Staphylococ- cus aureus. The results of this study provide a foundation for the further understanding expression characteristic and function of bovine S100A12 gene.