志贺毒素B(ShT-B)在E.coli中的融合表达及特异性抗体制备

Fusion Expression of Shiga Toxin B Subunit of Shigella dysenteriae Type I in E, coli and its Generation of Specific Antibody

用KpnⅠ、Hind Ⅲ酶切克隆载体pGEM-ShTB3，然后亚克隆入带有二氢叶酸还原酶(DHFR)的融合表达栽体pQE40，构建重组质粒pQE40-ShTB,转化到E。coli M15中，经IPTG诱导，实现了ShTB-DHFR融合蛋白的高表达，表达量约占菌体总蛋白的21．9％，为包涵体形式，在变性条件下，包涵体蛋白经螯合镍离子的次氨基三乙酸(Ni-NTA)亲和柱一步纯化，得到了纯度为90．5％的重组ShT-B．以纯化的shTB-DHFR融合蛋白免疫昆明鼠，并结合腹腔注射S180细胞，成功制备了抗ShTB腹水多克隆抗体，效价达1：1×10^6间接ELISA和Westem印迹结果表明，抗ShT-B多抗与重组蛋白和天然ShT均有特异性结合,本试验结果为毒素检测方法的研究奠定了基础。

pGEM-ShTB3 cloning vector and prokaryotic pQE-40 expression vector were digested respectively with the endonucleases Kpn Ⅰ and Hind Ⅲ , and the recombinant vector named as pQE40-ShTB was obtained. The resulting expression vector pQE40-ShTB was transformed into E. coli M15 competent cells, and highly expressed an ShTB-DHFR fusion protein induced by IPTG. The target fusion protein occupied 21.9 % of total cell protein and in inclusion body form. The fusion protein was used for immunizing Kunming mice, and ascitic polyclonal antibodies raised against recombinant ShT-B were generated by using sarcoma 180 cells and the antibody liter of ascites was up to 1: 1×106 by the indirect ELISA. Western blot analysis showed that the recombinant ShT-B had a specific affinity for polyclonal antibody against ShT-B. The results laid the foundation for the detection of toxin and genetic vaccine development.