%0 Journal Article
%T 幽门螺杆菌lpp20基因在食品级乳酸菌中的表达及免疫活性鉴定<br>Expression of Helicobacter pylori lpp20 gene in food-grade Lactococcus lactis and its immunoreactivity
%A 孙
%A 楠
%A 张荣光
%A 段广才
%A 陈帅印
%A 彭晓燕
%A 范清堂
%A 郗园林
%J 郑州大学学报（医学版）
%D 2016
%X 目的:构建分泌表达幽门螺杆菌lpp20基因的重组乳酸乳球菌菌株,并鉴定其免疫活性,为研究安全高效的幽门螺杆菌口服疫苗奠定基础。方法:采用PCR法从幽门螺杆菌基因组DNA中扩增lpp20基因,将lpp20先与T载体(pMD19-T)连接,进而经双酶切与表达载体pNZ8149-SPusp45连接,用电穿孔法将重组质粒转入食品级乳酸乳球菌菌株NZ3900中,并用Nisin诱导Lpp20表达,表达产物通过Western blot法鉴定免疫活性。结果:lpp20基因的PCR扩增产物大小约为540 bp,重组质粒的酶切、PCR和测序鉴定结果符合预期; 在菌体和培养基中均检出表达的Lpp20蛋白,重组表达的Lpp20蛋白相对分子质量约为20 000,且具有与小鼠抗幽门螺杆菌血清反应的免疫活性。结论:成功构建了能够分泌表达幽门螺杆菌Lpp20抗原的乳酸乳球菌菌株,该重组菌株在口服疫苗研究中具有应用潜力。<br>Aim: To construct a food-grade expression system of Helicobacter pylori(H. pylori) lpp20 gene in Lactococcus lactis(L.lactis) and evaluate its immunoreactivity with aim to establish basis for development of safe and efficient orally administrable vaccine against H. pylori.Methods: The lpp20 gene was obtained by PCR amplification from the genomic DNA of H. pylori, and ligated to plasmid pMD19-T and then to the expression vector pNZ8149-SPusp45. The recombinant pNZ8149-SPusp45-Lpp20 was electrotransformed into L.lactis NZ3900. The resultant recombinant strains were induced to express the exogenous gene with Nisin, and the expression products were assessed by Western blot.Results: The length of the amplified lpp20 gene by PCR was estimated 540 bp. The gene fragments were correctly inserted into the vector as identified by enzyme digestion, PCR and gene sequencing. The expression product of lpp20 gene was detected both in the bacterium digestion and the culture media samples, and with a relative molecular weight of 20 000,which was immunoreactive to anti-H. pylori mouse sera.Conclusion: The recombinant strain of L.lactis capable of secretary expression of H. pylori antigen Lpp20 has been successfully constructed and, shows potential for being an anti-H. pylori vaccine candidate
%K 乳酸菌
%K lpp20基因
%K 幽门螺杆菌
%K 分泌表达
%K 食品级&lt
%K br&gt
%K Lactococcus lactis
%K lpp20 gene
%K Helicobacter pylori
%K secretary expression
%K food-grade
%U http://jms.zzu.edu.cn/oa/darticle.aspx?type=view&id=201604011