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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SL32 6 1,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% (397/40 2 ) ,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15 0 0 0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口服疫苗奠定了基础。
Objective: To establish an attenuated Salmonella typhimurium vaccine strain expressing Helicobacter pylori neutrophil activating protein (Hp NAP). Methods: The Hp NAP gene was amplified by PCR and cloned into prokaryotic expression plasmid pTrc99A. The gene sequence was determined by PCR and restriction enzyme digestion, and compared with the homology of related sequences in GenBank. The recombinant plasmid pTrc99A NAP was transformed into attenuated Salmonella typhimurium SL32 6 1. After screening, the positive colonies were screened and the plasmids were extracted for PCR and restriction enzyme digestion. The expressed Hp NAP protein was identified by SDS PAGE and the protein content was analyzed by thin layer scanning. Results: The nucleotide sequence analysis and homology analysis confirmed that the homology of the cloned Hp NAP gene with the related sequences in GenBank was 98% (397/40 2) and the amino acid sequence homology was 98% (131 / 133). The attenuated Salmonella typhimurium transformed with the recombinant plasmid pTrc99A NAP expressed Hp NAP protein of Mr about 1500, accounting for 37.5% of the bacterial protein content. Conclusion: An attenuated Salmonella typhimurium vaccine strain that expresses Hp NAP gene was established, which laid the foundation for further development of oral vaccine against Hp.