摘要
目的构建乙肝表面抗原-人微小病毒B19 VP2融合蛋白的原核表达系统,并检测其表达蛋白的抗原反应性。方法用PCR扩增乙肝表面抗原(HBsAg)基因及B19病毒VP2基因,分别重组入pGEM-T,构建出pGEM-T-HBs及pGEM-T-VP2并测序分析;测序证实序列正确后,将pGEM-T-HBs中的HBs基因克隆入pQE30表达载体中,得到pQE30-HBs;再将pGEM-T-VP2中的VP2基因克隆入pQE30-HBs中,得到pQE30-HBs-VP2后转化至BL21(DE3)宿主菌中,并以IPTG诱导表达融合蛋白,以Western-blot鉴定。结果pQE30-HBs-VP2可在大肠杆菌BL21(DE3)中高效表达,Western-blot检测结果显示所表达融合蛋白均可与抗-HBs和抗-VP2结合反应。结论成功构建pQE30-HBs-VP2原核表达载体,且其表达的重组蛋白具有良好的HBsAg和VP2蛋白的双重抗原的反应原性。从而建立了表达B19和HBV的联合抗原表达系统,同时给HBV和B19病毒重组联合疫苗的研制和复合诊断试剂提供了抗原参考。
Objective To construct efficient expression vector of HBsAg and B19-VP2 gene, and to detect the antigenicity of the expression products. Methods HBsAg gene and B19 VP2 gene were amplified by PCR. After being cloned with pGEM-T vector and sequencing, the HBsAg gene was inserted into the prokaryotic expression vector pQE30. After the recombinant pQE30-HBs had been formed, the VP2 gene was inserted into pQE30-HBs from pGEM-T-VP2 to construct pQE30-HBs-VP2. Then the pQE30-HBs-VP2 was transformed into E. coli BL21（DE3）, and induced to express the fusion proteins by IPTG. Finally,the methods of SDS-PAGE and Western-blot were used to detect the level and the antigenicity of the fusion proteins. Results pQE30-HBs-VP2 recombinant vector could express efficiently in BL21. And the result of Western-blot indicated that the recombinant fusion protein had the better antigenicity, as it could be recognized by the antibody of anti-HBs and anti-VP2. Conclusion The pQE30-HBs-VP2 expression vector has been constructed successfully, its expression production has both the antigenic characterization of HBsAg and B19 VP2 protein. And the expression system of HBs and B19 VP2 is constructed successfully. It may provide some information for developing B19 and HBsAg recombinant combined vaccine, and also provide potential antigen for multi-diagnostic reagents.
出处
《河南科技大学学报（医学版）》
2005年第4期241-244,共4页
Journal of Henan University of Science & Technology:Medical Science
基金
河南省科技攻关基金资助项目(01140512)
