幽门螺杆菌抗原基因ureB在乳酸菌中的克隆表达与免疫反应性研究  

Expression of Helicobacter pylori ureB gene in Lactococcus lactis NZ3900 and its immunoreactivity

作　　者：张小娟[1] 张荣光[2] 段广才[2] 范青堂[2] 

机构地区：[1]河南科技大学病原生物学教研室,洛阳471003 [2]郑州大学河南省分子医学重点学科开放实验室,郑州450001

出　　处：《中国人兽共患病学报》2011年第10期925-928,932,共5页Chinese Journal of Zoonoses

摘　　要：目的为了开发幽门螺杆菌(Helicobacter pylori,Hp)口服疫苗,将Hp尿素酶B(UreB)在食品级乳酸乳球菌NZ3900菌株中进行表达,并研究其免疫反应性。方法 PCR扩增Hp MEL-HP27菌株ureB基因(基因号:FJ436980),将其克隆入大肠杆菌-乳酸乳球菌穿梭质粒pNZ8110中并转化乳酸乳球菌NZ3900;采用正交试验确定目的蛋白表达的适宜条件;应用western-blot鉴定其免疫反应性。结果成功扩增了Hp MEL-HP27菌株ureB基因,构建了ureB基因的乳酸菌NICE(Nisin-controlled expression)原核表达系统;UreB蛋白适宜的表达条件为:在ureB重组菌生长至对数生长前期(OD600≈0.3～0.4)加入终浓度为40ng/mL的nisin,诱导表达5h,可溶性UreB蛋白表达量最高,可达27.26μg/mL培养基。可溶性UreB蛋白的表达占上清蛋白的比例最高可达20.19%;Western-blot结果显示乳酸乳球菌表达的UreB抗原蛋白具有良好的免疫反应性。结论结果提示应用乳酸乳球菌构建幽门螺杆菌食品级疫苗可能具有较好前景。To lay a foundation for the further study of Hp live oral vaccine,Hp urease subunit B（UreB） was expressed in a food-grade delivery vehicle,and Lactococcus lactis NZ3900 and its immunoreactivity was studied.The UreB gene was amplified from genome DNA of H.pylori MEL-Hp27（GenBank accession no.FJ436980） and then cloned into the E.coli-L.lactis shuttle vector pNZ8110 and transformed into E.coli MC1061.The positive recombinant plasmid was electro-transformed into L.lactis NZ3900.The conditions of UreB expression in the L.lactis transformant were optimized by orthogonal experiment.Western blot was adopted to confirm whether the UreB expressed by L.lactis transformant had immunoreactivity.As a result,the recombinant engineering bacteria has been successfully constructed through DNA recombinant technique,which were identified by specific PCR and restriction enzyme digestion of recombinant plasmids.The 40ng/mL nisin was added into the medium and induced for 5h when the recombinant engineering bacteria grew to logarithmic growth phase（OD600≈0.3-0.4）.The maximum yield of UreB was 27.26μg/ml of medium,and the maximum percentage of UreB in cell extracts of the L.lactis transformant reached its peak at 20.19%.Western blot analysis showed that the UreB protein expressed by L.lactis transformant has favorable immunoreactivity.All these results make an appealing case for construction of the food-grade vaccine for Hp.

关 键 词：幽门螺杆菌 乳酸乳球菌 NICE UREB 

分 类 号：R378[医药卫生—病原生物学]