摘要
目的 构建表达幽门螺杆菌的组成成分热休克蛋白A亚单位 (HspA)和尿素酶B亚单位 (UreB)的重组蛋白质的侯选菌株。方法 用PCR方法从幽门螺杆菌的染色体DNA上分别扩增出HspA和UreB基因片段 ,将它们融合插入原核表达载体pET 2 2b(+)中 ,并在BL2 1(DE3)大肠杆菌表达。结果 经测序 ,HspA UreB(HU)融合基因片段由 2 0 6 1bp组成 ,为编码 6 87个氨基酸残基的多肽。SDS PAGE和免疫印迹分析检测发现 ,融合基因表达的蛋白相对分子质量约为 78× 10 3,并证实该重组蛋白可以被幽门螺杆菌感染阳性患者的血清所识别 ,其免疫小鼠所得血清抗体可与纯化的HspA和UreB重组蛋白单体或融合体发生特异性的结合反应。同时观察到HspA的存在可使融合蛋白的尿素酶活性增加 2倍以上。结论 HspA UreB融合蛋白有可能作为一种有效的蛋白质疫苗用于幽门螺杆菌感染的预防和治疗。
Objective Both heat shock protein A subunit (HspA) and urease B (UreB) subunit as effective immunogen may stimulate the immunoresponse protecting human body against challenge of H.pylori. A recombinant strain which expresses bivalent antigen of HspA and UreB subunit was constructed. Methods Gene encoding the structural A/B subunit of H.pylori heat shock protein/ urease was amplified from H.pylori chromosomal DNA by PCR techniques. The genes were inserted into the prokaryotic expression vector pET 22b(+), and then was transformed into the BL 21(DE3) E.coli strain, expressed which HspA UreB recombinant protein. Results HspA UreB gene was found to be 2061 base pairs ,the recombinant fusion protein encoded polypeptides of 687 amino acid residues, corresponding to calculated molecular masses of 78kD. Western blot analysis of HspA UreB recombinant protein confirmed that it could be specifically recognized by the serum from H.pylori infected patients, and could also be recognized by the serum from BALB/c mice immunized with HspA UreB itself. The urease activity of E.coli cells containing the UreB gene plus the HspA gene was two times greater than that of UreB lonely. Conclusion This result suggested that HspA UreB recombinant protein may be an potential vaccine for control and treatment of H.pylori infection.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2000年第3期232-235,共4页
Chinese Journal of Microbiology and Immunology
关键词
幽门螺杆菌
热休克蛋白A
尿素酶B
双价疫苗
Helicobacter pylori
Heat shock protein A
Urease B
Bivalent genetic vaccine
