【摘要】 目的获得大量具有良好IgE结合活性的Bla g2重组变应原,以用于变态反应性疾病特异性诊断及免疫治疗的研究。方法提取德国小蠊总RNA,采用RT-PCR的方法扩增Bla g2片段,产物连接进入pMD18-T载体。挑取重组子,用NdeⅠ和NotⅡ双酶切后将目的片段亚克隆入pET24a(+),经IPTG诱导表达后,通过Ni2+亲和层析柱对重组变应原进行纯化,并采用Western-blot检测其免疫学结合活性。结果扩增出编码328个氨基酸的长度为984个碱基对的核苷酸片段,序列分析结果和GenBank上的基因序列(U28863)完全一致。重组变应原在E.coliBL21 star中得到高效表达,具有良好的反应原性。结论成功构建了德国小蠊变应原Bla g2的原核表达载体,并纯化出具有免疫原特性的重组蛋白,为进一步开展Bla g2蛋白和重组疫苗研究奠定了基础。更多还原

【Abstract】 To obtain large amount of the recombinant allergen Bla g2 of the major cockroach（Blattella germanica） for the specific diagnosis and vaccine development of the allergic disorders,the Bla g2 gene fragment amplified by RT-PCR was cloned into vector pMD18-T and the recombinant plasmid pET24a（+）-Bla g2 was constructed by ligating the Bla g2 gene to the expression vector pET24a（+） and then was transformed to E.coli BL21 Star.After induction with IPTG,the expressed Bla g2 protein was purified through metal(Ni²⁺) chelating affinity chromatography.Western blotting was used to determine the immunological binding activity of the recombinant allergen.It was found that a 984 bp cDNA fragment encoding 328 amino acids was amplified,and as demonstrated by sequence analysis,the homology with the published gene sequence of GenBank U28863 was 100%.The recombinant allergen of Bla g2 protein was overexpressed in E.coli,and it could be highly purified through one-step affinity chromatography.As demonstrated by Western blotting analysis,it was shown to have high IgE-binding potency when sera from patients allergic to cockroach were used as the corresponding antibody.We have successfully constructed the prokaryotic expression vector for Bla g2 gene encoding the cockroach allergen protein and have purified the recombinant protein with highly monogenetic capacity,thus providing the basis for the further studies on the recombinant vaccine with Blab g2 protein.更多还原