大肠杆菌不耐热肠毒素基因克隆和无毒突变体mLT63的构建及序列分析

Clonging gene for E.coli heat-labile enterotoxin and constructing its site-directed mutant mLT63 and analysis of their nucleotide sequences

目的:大肠杆菌不耐热肠毒素(LT)是很强的免疫原,也具有很强的粘膜免疫佐剂作用,但LT的毒性限制了它在人类疫苗中使用。本研究希望通过定点诱变构建无毒或减毒并保持LT免疫学特性的突变体。方法:以人源肠毒素大肠杆菌(ETEC)菌株E.coli44815为研究对象,应用本研究室改进的SDS裂解法小量制备含LT基因的野生大质粒,PCR扩增LT基因,构建重组质粒pNEB193_elt;采用重叠延伸法PCR对LT第63位氨基酸进行体外定点诱变,构建LT突变基因的重组克隆载体,测序并进行序列分析鉴定。结果:采用本研究室改进的SDS裂解法,可成功地小量提取满足扩增模板要求的大质粒;所克隆的LT基因与GenBank公布的一致;定点诱变正确,无非特异性突变。结论:克隆的LT野生基因和定点诱变基因可用于构建重组表达载体,表达重组蛋白,对其粘膜免疫佐剂性或其他相关特性进一步研究。

Objective: Escherichia coli heat-labile enterotoxin is a strong immunogen and a powerful mucosal adjuvant as well.However,the high toxicity of LT renders it unsuitable for practical human use.We hope to reduce the toxicity by site-directed mutagenesis and to obtain a mutant of LT retaining the immunological properties.Methods: The big wild type plasmid containing the gene for LT was extracted by a modified method of SDS lysis from the LT+ strain E.coli 44815 isolated from human.The wild type gene of LT was amplified by PCR.The site-directed mutant gene for mLT63 was obtained by over-lap extension PCR.The two genes were identified by nucleotide sequencing and were inserted into plasmid pNEB193 respectively to construct recombinant plasmids.Results: The modified SDS lysis methods for extracting the big plasmids were effective.The gene for wild type LT we cloned is identical to that published in GenBank.The site-directed mutagenesis of mLT63 gene was performed correctly.Conclusion: With the genes obtained,we can construct recombinant expression plasmids and take more associated researches.