大肠杆菌LTB与霍乱弧菌CTB基因的克隆和表达及鉴定

目的：克隆大肠杆菌不耐热肠毒素B亚单位（LTB）及霍乱弧菌肠毒素B亚单位（CTB）基因，构建其表达载体。方法：采用高保真PCR分别从E.coli44815株与V.cholerae东74株基因组DNA中扩增LTB与CTB基因，T-A克隆后测定核苷酸序列。分别构建pET32a的LTB及CTB表达载体，在E.coliBL21DE3宿主菌中用不同浓度的IPTG诱导表达。采用SDS-PAGE及GM1-ELISA鉴定表达产物。结果：所克隆的LTB和CTB基因与报道的相应核苷酸序列同源性分别为99.12％-99.71％和98.54％-99.42％，氨基酸序列同源性分别高达97.58％-99.19％和96.77％-99.19％。pET32a-LTB-BL21DE3和pET32a-CTB-BL21DE3系统表达的融合蛋白产量分别占细菌总蛋白的30％和10％左右。GM1-ELISA结果证实LTB及CTB融合蛋白均能与牛GM1结合。结论：本研究成功地构建了LTB与CTB表达系统，所表达的LTB与CTB融合蛋白具有粘膜免疫佐剂活性。

Cloning,expression and identification of Escherichia coli LTB gene and Vibrio cholerae CTB gene

Objective: To clone the LTB gene of E.coli and the CTB gene of V.cholerae, and to construct expression vectors of these genes. Methods: The LTB gene from E.coli strain 44815 and the CTB gene from V.cholerae strain eastern-74 were amplified by high fidelity PCR. The nucleotide sequences of the two target DNA amplification fragments were sequenced after T-A cloning. pET32a expression vectors with inserted LTB and CTB genes were constructed. The LTB and CTB fusion proteins were expressed in E.coli strain BL21DE3 inducted by IPTG at different dosages. The two expression products were identified by SDS-PAGE and G M1-ELISA. Results: In comparison with the reported LTB and CTB sequences, the nucleotide sequence homologies of the cloned LTB gene and CTB gene were from 99.12%~99.71% and 98.54%~99.42%, while their putative amino acid sequence homologies were as high as 97.58%~99.19% and 96.77%~99.19%. The expression outputs of LTB and CTB fusion proteins in pET32a-LTB-BL21DE3 and pET32a-CTB-BL21DE3 systems were approximately 30% and 10% of the total bacterial proteins, respectively. The LTB and CTB fusion proteins were able to combine with bovine G M1 confirmed by ELISA. Conclusion: The expression systems of LTB and CTB genes have been successfully established. Both the expressed LTB and CTB fusion proteins possess mucosal adjuvant immunoactivity.