摘要
目的 :克隆大肠杆菌不耐热肠毒素 B亚单位 (L TB)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.coli4 4 815株与 V.cholerae东 74株基因组 DNA中扩增 L TB与 CTB基因 ,T- A克隆后测定核苷酸序列。分别构建 p ET32 a的 LTB及 CTB表达载体 ,在 E.coli BL2 1DE3宿主菌中用不同浓度的 IPTG诱导表达。采用 SDS- PAGE及 GM1- EL ISA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 98.5 4 %～ 99.4 2 % ,氨基酸序列同源性分别高达 97.5 8%～99.19%和 96.77%～ 99.19%。p ET32 a- L TB- BL2 1DE3和 p ET32 a- CTB- BL2 1DE3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- EL ISA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 L TB与 CTB融合蛋白具有粘膜免疫佐剂活性。
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.
出处
《浙江大学学报（医学版）》
CAS
CSCD
2003年第1期17-20,共4页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金 (39970 6 78)
国家教育部优秀年轻教师基金
浙江省科技厅项目 (0 0 1110 438)
