ETEC粘附素蛋白亚基Fae G在乳酸乳球菌中的表达及免疫反应性分析

Cloning and expression of the Fae G gene of enterotoxigenic Escherichia coli K88 fimbrial subunit in Lactococcus lactis

为探索研制预防仔猪腹泻口服基因工程疫苗,本研究根据GenBank中登录的产肠毒素大肠杆菌(ETEC)Fae G基因序列设计一对引物,以ETEC C83907菌株为模板,通过PCR方法扩增出K88菌毛粘附素主要蛋白亚基Fae G的基因,将其定向克隆到乳酸乳球菌(L.lactis)分泌表达载体pNZ8112中,构建了分泌表达载体pNZ8112-fae G,并转化到L.lactis NZ9000中。重组菌株经5ng/mL nisin诱导3h后,仅在L.lactis菌体内检测到Fae G的表达,上清液中未检测到Fae G蛋白。SDS-PAGE电泳检测结果显示,表达的目的蛋白分子量大小约为27ku,表达产物的量达到了菌体总蛋白的13.56%。Western blot分析结果表明,表达蛋白具有免疫反应性。本实验为进一步研究FaeG在L.lactis中分泌表达的影响因素奠定了基础。

The Fae G gene of K88 main fimbrial subunit was amplified by PCR from ETEC C83907 using primers designed according to the published enterotoxigenic Escherichia coil （ETEC） Fae G gene sequence. The PCR product was inserted into pNZ8112 and transformed into L. lactis NZ9000. Fae G was expressed after induced with 0.5 ng/mL nisin. SDS-PAGE showed that expressed Fae G was about 27 ku which accounted for up to 13.56 % of the total cellular protein. The immuno-reactivity of the protein was confirmed by western blot. The production of protein could provide foundations for further study on heterologous protein secretion in L. lactis, preventing diarrhea of piglets and promoting problotic properties of L. lactis.