Xia Xiao-Ping, Yan Jie, Zhao Shou-Feng
Department of Medical Microbiology and Parasitology, College of Medical Sciences, Zhejiang University, Hangzhou 310031, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2003 Feb;32(1):17-20. doi: 10.3785/j.issn.1008-9292.2003.01.005.
To clone the LTB gene of E.coli and the CTB gene of V.cholerae, and to construct expression vectors of these genes.
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.
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% approximate, equals 99.71% and 98.54% approximate, equals 99.42%, while their putative amino acid sequence homologies were as high as 97.58% approximate, equals 99.19% and 96.77% approximate, equals 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.
The expression systems of LTB and CTB genes have been successfully established. Both the expressed LTB and CTB fusion proteins possess mucosal adjuvant immunoactivity.
克隆大肠杆菌的LTB基因和霍乱弧菌的CTB基因，并构建这些基因的表达载体。
通过高保真PCR扩增大肠杆菌44815株的LTB基因和霍乱弧菌东方74株的CTB基因。T-A克隆后对两个目标DNA扩增片段的核苷酸序列进行测序。构建插入LTB和CTB基因的pET32a表达载体。用不同剂量的IPTG诱导LTB和CTB融合蛋白在大肠杆菌BL21DE3中表达。通过SDS-PAGE和G(M1)-ELISA对两种表达产物进行鉴定。
与报道的LTB和CTB序列相比，克隆的LTB基因和CTB基因的核苷酸序列同源性分别约为99.12%（等于99.71%）和98.54%（等于99.42%），而其推测的氨基酸序列同源性高达约97.58%（等于99.19%）和96.77%（等于99.19%）。pET32a-LTB-BL21DE3和pET32a-CTB-BL21DE3系统中LTB和CTB融合蛋白的表达量分别约占总细菌蛋白的30%和10%。ELISA证实LTB和CTB融合蛋白能够与牛G(M1)结合。
成功建立了LTB和CTB基因的表达系统。表达的LTB和CTB融合蛋白均具有黏膜佐剂免疫活性。
