大肠杆菌LTKA63突变体的构建及重组LTKA63免疫佐剂活性机理的研究

目的构建大肠杆菌不耐热肠毒素A亚单位(heat-labile enterotoxin subunit A,LTA)基因减毒突变体LTKA63及其原核表达系统,探讨重组LTKA63(rLTKA63)粘膜免疫佐剂活性的机制。方法采用高保真PCR从大肠杆菌44815株基因组DNA中扩增LTA基因,利用定位突变技术构建LTKA63突变体,T-A克隆后测序。采用无His-tag的原核表达载体pET-15b,构建LTKA63原核表达系统,通过SDS-PAGE鉴定表达产物。采用三色流式细胞术确定rLTKA63激活T细胞途径。结果从大肠杆菌44815株DNA模板中扩增获得预期大小的LTA基因条带。与报道的LTA序列比较,所克隆的LTA基因核苷酸和氨基酸序列相似性分别为99.03%~99.61%和98.33%~99.22%。LTA基因定位突变后获得序列正确的LT-KA63突变体。rLTKA63表达量占细菌总蛋白的15%左右。rLTKA63作用后Th1和Th2细胞较阴性对照组分别增加21.9%和36.2%。结论本文成功地构建了LTKA63原核表达系统,所表达的rLTKA能同时激活Th1和Th2细胞。

Construction of Escherichia coli LTKA63 mutant and possible mechanism of immunoadjuvant activity of its recombinant

To construct detoxified mutant LTKA63 of the heat-labile enterotoxin subunit A(LTA) gene of Escherichia coli and its prokaryotic expression system,and to study the mechanism of mucosal immunoadjuvant activity of the recombinant LTKA63(rLTKA63).The LTA gene from E.coli strain 44815 genome DNA was amplified by high fidelity PCR.Site mutation technique was applied to construct the mutant LTKA63.Two cloned genes were sequenced after T-A cloning.Then the prokaryotic expression system of LTKA63 gene was constructed by using pET-15b without His-tag and its expressed product was identified with SDS-PAGE.The pathways activating T lymphocytes of rLTKA63 were determined with Three Color Flow Cytometry.And amplification fragment of LTA gene with the expected size was obtained from E.coli strain 44815 genome DNA template.In comparison with the reported LTA gene sequences, the nucleotide and amino acid sequences of the cloned LTA gene showed the similarities of 99.03%～99.61% and 98.33%～99.22%,respectively.The mutant LTKA63 with correct sequence was obtained through the site mutation of the cloned LTA gene and expression output of rLTKA63 was approximate 10% of the total bacterial proteins.After co-incubation with LTKA63,the Th1 and Th2 lymphocytes were increased with 21.9% and 36.2%,respectively,compared to those of the negative controls.In conclusion,the prokaryotic expression system of LTKA63 gene was successfully established in this study and the expressed rLTKA63 can activate both Th1 and Th2 lymphocytes.