论文部分内容阅读
目的 构建含幽门螺杆菌 (Hp)Mr 2 6 0 0 0外膜蛋白编码基因的重组载体 ,并在E .coliBL2 1中表达。方法 用PCR从Hp染色体中 ,扩增Mr 2 6 0 0 0外膜蛋白编码基因片段。将目的基因与pET32a(+)同时经BamHI、HindⅢ双酶切、纯化、连接后 ,构建含有目的基因的重组载体。以含目的基因片段的重组载体转化大肠杆菌BL2 1(DE30 )并表达。表达产物经纯化后 ,用ELISA法检测其抗原性。结果 经酶切、测序分析表明 ,插入的基因片段为HpMr 2 6 0 0 0的外膜蛋白编码基因 ,与Tomb等的报道相比较 ,有 1.1%的bp发生变异 ,1.5 1%的氨基酸残基改变。经SDS PAGE分析发现 ,融合基因表达的蛋白Mr为 4 6× 10 3 ,可溶性表达产物占细菌总蛋白的 38.96 %。重组蛋白经Ni NTA琼脂糖树脂纯化后 ,其纯度达 95 %以上。ELISA法检测显示 ,该重组蛋白可被Hp阳性患者的血清所识别 ,具有良好的抗原性。结论成功地克隆并表达Mr为 2 6 0 0 0的Hp外膜蛋白编码基因 ,为Hp蛋白疫苗的研制和快速诊断试剂盒的研究打下了基础
Objective To construct a recombinant vector encoding the outer membrane protein of Helicobacter pylori (Hp) Mr 26 0 0 0 and express it in E.coli BL21. Methods PCR amplification of Mr 2 0 0 0 outer membrane protein encoding gene fragment from Hp chromosome was performed. The target gene and pET32a (+) at the same time by BamHI, Hind Ⅲ double digestion, purification, ligation, construct a recombinant vector containing the gene of interest. Escherichia coli BL21 (DE30) was transformed with the recombinant vector containing the gene fragment and expressed. The expressed product was purified and its antigenicity was tested by ELISA. Results After digestion and sequencing analysis, the inserted gene fragment was encoded by the outer membrane protein of HpMr260o. Compared with the reports by Tomb et al., 1.1% bp was mutated and 1.5% of the amino acid residues change. SDS PAGE analysis showed that the fusion protein expressed Mr 4 6 × 10 3 and the soluble protein accounted for 38.96% of the total bacterial protein. The purity of recombinant protein was above 95% after purified by Ni NTA agarose resin. ELISA test showed that the recombinant protein can be recognized by the serum of Hp positive patients and has good antigenicity. Conclusion The successful cloning and expression of Hp outer membrane protein gene Mr = 2600 laid the foundation for the development of Hp protein vaccine and the rapid diagnostic kit