大肠杆菌肠毒素基因突变体在乳酸菌中的表达与免疫学鉴定

Expression of E. coli enterotoxin mutant gene mlt63 in lactic acid bacterium and immunological identification of the expression products

目的构建表达大肠杆菌肠毒素基因突变体mlt63的乳酸菌工程菌株,为黏膜免疫佐剂研究建立重要基础。方法从前期构建的质粒p BV-mlt63中经PCR扩增mlt63基因,经双酶切将mlt63与质粒载体p NZ8110连接。用连接产物转化大肠杆菌MC1061菌株,从重组菌株中提取p NZ8110-mlt63,用于电转化Lactococcus lactis NZ3900菌株;经质粒双酶切和测序,鉴定重组菌株L.lactis NZ3900/p NZ8110-mlt63。采用nisin诱导重组乳酸菌表达m LT63,对表达产物进行SDS-PAGE和Western blot分析。结果 mlt63 PCR扩增产物长度与预期相符;重组菌株鉴定结果显示L.lactis NZ3900/p NZ8110-mlt63构建正确;Western blot分析在重组菌株中检出2条mlt63表达的蛋白带,分子量分别为10 k D和40 k D。结论本研究首次将mlt63基因克隆至乳酸乳球菌中,并表达出具有抗原活性的蛋白,鉴于目前亟需安全有效的黏膜免疫佐剂,本研究发现将对该领域研究产生重要影响,对胃肠感染性疾病口服疫苗研究具有促进作用。

Objective To construct an engineered strain of lactic acid bacterium expressing Escherichia coli enterotoxin mutant gene mlt63 with aim to lay the crucial basis for researches on mucosal immune adjuvant. Methods mlt63 gene was amplified by PCR from the plasmid p BV-mlt63 built previously,undergone restriction enzyme digestion,ligated to the plasmid vector p NZ8110,and then used to transform E. coli MC1061. The recombinant plasmid p NZ8110-mlt63 was separated from the recombinants,and introduced into Lactococcus lactis NZ3900. The recombinant L. lactis NZ3900/p NZ8110-mlt63 was identified by using dual enzyme digestion and gene sequencing. The engineered L. lactis strain was induced with nisin to express m LT63,and the expression products were assayed by using SDS-PAGE and Western blot. Results mlt63 PCR product had the length as expected. As demonstrated by the gene sequencing,L. lactis NZ3900/p NZ8110-mlt63 had been constructed correctly. Western blot assays showed the expression products of mlt63 in the engineered L. lactis consisted of two protein bands,with molecular weights of 10 k D and 40 k D,respectively. Conclusion This study successfully performed cloning the mlt63 gene to L. lactis,obtained expression products with antigenicity. Considering the situation that the safe and efficient mucosal immune adjuvants are in urgent need,these findings of this study will have considerable effects in this research area,and accelerate the advancement of oral vaccine development against gastrointestinal infectious diseases.