人Toll 样受体-9胞外区N端蛋白的表达及其抗体的制备

目的:构建人Toll样受体-9(hTLR-9)基因5′端序列的表达质粒,在大肠杆菌中表达,并以表达的hTLR-9-N免疫小鼠制备抗体。方法:构建hTLR-9基因5′端(707~1167bp)的硫氧还蛋白(thioredoxin)融合表达质粒pET32a-hTLR-9-N。将重组质粒转入大肠杆菌BL21(DE3)中,在0.5mmol/LIPTG诱导下表达。表达产物用SDS-PAGE进行鉴定。以8mol/L尿素溶解的包涵体作为免疫原,免疫昆明种小鼠制备抗hTLR-9-N的抗体。结果:融合表达质粒pET32a-hTLR-9-N在E.coliBL21(DE3)中以相对分子质量(Mr)为31000的包涵体形式表达;Westernblot分析证明hTLR-9-N在大肠杆菌中得到正确表达。表达的目的蛋白初步纯化后的纯度达90%以上;以hTLR-9-N为抗原制备的抗血清效价为1∶25600;该抗血清可以与转染后稳定表达全长hTLR-9的HEK293细胞产生结合反应。结论:成功地在大肠杆菌中表达hTLR-9N末端蛋白,制备的抗hTLR-9-N抗体可特异性结合表达全长hTLR-9的真核细胞。

Expression of human Toll-like receptor-9 in E.coli and the preparation of antibody

AIM: To express N terminal fragment of human Toll-like receptor-9 (hTLR-9) in E.coli and prepare its antiserum. METHODS: The thioredoxin fusion expression plasmid of terminal fragment of hTLR-9 gene (707-1 167 bp) was constructed and then transformed into E.coli BL21 (DE3). The fusion protein was expressed in E.coli BL21 (DE3) under induction of 0.5 mmol/L IPTG. Inclusion body dissolved in urea was used as immunogen to prepare mouse anti-hTLR-9 antibody. RESULTS: The fusion protein was expressed in the form of inclusion body with molecular mass of about 31kD. The purity of fusion protein reached to 90%; The result of immunohistochemical staining showed that the antisera against recombinant fusion protein could react specifically to HEK293 cells transfected with hTLR-9. CONCLUSION: The fusion protein of hTLR-9 N-terminal fragment was expressed in E.coli BL21 (DE3). The prepared anti-hTLR-9 antibody can react specifically to HEK293 cells transfected with full length hTLR-9.