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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。
OBJECTIVE: To construct the expression plasmid of 5 ’end of human Toll-like receptor-9 (hTLR-9) gene in Escherichia coli and immunize mice with expressed hTLR-9-N antibody. Methods: The thioredoxin expression plasmid pET32a-hTLR-9-N at the 5 ’end of hTLR-9 gene (707 ~ 1167 bp) was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) and expressed under the induction of 0.5 mmol / L IPTG. The expression product was identified by SDS-PAGE. Antigen of hTLR-9-N was prepared from immunized Kunming mice by using 8 mol / L urea dissolved inclusion bodies as immunogen. Results: The fusion expression plasmid pET32a-hTLR-9-N was expressed in E.coli BL21 (DE3) as a inclusion body with a molecular weight of 31000. Western blot analysis showed that hTLR-9-N was correctly expressed in E. coli . The purity of the expressed protein was above 90% after purification. The titer of the antiserum prepared with hTLR-9-N as antigen was 1:25600. The antiserum reacted with HEK293 stably expressing full-length hTLR-9 Cells produce a binding reaction. CONCLUSION: The hTLR-9 N-terminal protein was successfully expressed in E. coli and the prepared anti-hTLR-9-N antibody specifically binds to eukaryotic cells expressing full-length hTLR-9.