表达幽门螺杆菌NAP基因的减毒沙门菌疫苗株的建立

目的：建立表达幽门螺杆菌(H．pylori)中性粒细胞活化蛋白(Hp—NAP)的减毒沙门菌疫苗株。方法：用PCR扩增Hp—NAP基因，并克隆至原核表达质粒pTrc99A中，经PCR及酶切鉴定后测定其基因序列，并与GenBank中相关序列的同源性进行比较。以重组质粒pTrc99A—NAP转化减毒伤寒沙门菌SL3261，培养后筛选阳性菌落，抽提质粒进行PCR及酶切鉴定。表达的Hp—NAP蛋白用SDS—PAGE进行鉴定，用薄层扫描分析蛋白含量。结果：核苷酸序列测定及同源性分析证实，克隆的Hp—NAP基因与GenBank中相关序列的同源性为98％(397／402)，氨基酸序列的同源性为98％(131／133)。以重组质粒pTrc99A—NAP转化的减毒沙门菌，可表达Mr约15000的Hp—NAP蛋白，表达量约占菌体蛋白量的37．5％。结论：建立了可表达Hp—NAP基因的减毒鼠伤寒沙门疫苗株，为进一步研制Hp口眼疫苗奠定了基础。

Establishment of an attenuated salmonella typhimurium vaccine strain SL3261 expressing Hp-NAP gene

AIM: To develop an attenuated salmonella typhimurium vaccine strain SL3261 expressing Helicobacter pylori neutrophile activation protein( Hp NAP). METHODS: The Hp NAP gene was amplified by PCR and was cloned into prokaryotic expression plasmid pTrc99A to construct recombinant plasmid pTrc99A NAP. After the pTrc99A NAP was performed successively by PCR, enzyme digestion analysis and sequencing. The homology was compared between the cloned Hp NAP gene and related genes in GenBank. The vaccine strain SL3261 was transformed by pTrc99A NAP. After cultivation, positive colonies were picked out and the plasmid was amplified by PCR and enzyme digestion analysis again. The expression of Hp NAP protein in the SL3261 bacteria was proved by SDS PAGE and thin layer scanning. RESULTS: Sequencing and homologous analysis showed that the homology between the cloned Hp NAP gene and related genes in GenBank reached 98% for their nucleotide and deduced amino acid sequences. The expression of Hp NAP protein could be detected in the culture fluid of SL3261 bacteria. Relative molecular mass ( M r) of expression product was about 15 000, and expression amount accounted for 37.5% of total bacterial protein. CONCLUSION: An attenuated salmonella typhimurium vaccine strain SL3261 has been established successfully, which lays the foundation for further developing oral HP vaccine.