表达幽门螺杆菌尿素酶B亚单位基因减毒鼠伤寒沙门疫苗菌的构建和鉴定

目的构建表达幽门螺杆菌(Helicobacter

Construction and identification of recombinant attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori urease subunit B

Objective To construct a recombinant live attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori urease subunit B(ureB). Methods By genetic engineering methods, ureB gene was amplified by PCR and cloned into a prokaryotic expression plasmid pTrc99A, and the identified recombinant plasmid was then used to transform an attenuated Salmonella typhimurium vaccine strain SL3261, and the positive clones were screened by PCR and restriction enzyme digestion. The ureB expressed in the recombinant vaccine strain was analyzed by SDS PAGE and optical density scanning. Two and 10 days after recombinant strain intragastric immunization, the C57BL/6 mice were sacrificed, and the spleens and terminal ileums were cultured for recombinant strain. Results The ureB gene could be amplified from the recombinant prokaryotic expression plasmid pTrc99A ureB and the plasmids extracted from transformed SL3261 strain by PCR, and also the ureB gene flagment could be produced from these plasmids after restriction enzyme digestion. SDS PAGE and optical density scanning indicated that ureB was expressed in the recombinant vaccine strain SL3261 (pTrc99A ureB) as a protein with 66 kD of molecular weight and was 26% of the total bacterial protein. Recombinant strain was found in both spleen and terminal ileum of each mouse two and ten days after intragastric immunization. Conclusions A recombinant liver attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori ureB was constructed and identified, and this study will help to develop an oral recombinant live vaccine against Helicobacter pylori infection.