摘要
目的构建人巨细胞病毒(HCMV)gB基因,并对pET28a/gB重组质粒进行诱导表达,鉴定表达的目的蛋白。方法根据Genebank中HCMV核苷酸序列设计引物,并在引物的5′、3′端分别加入BamHⅠ、EcoRⅠ限制性内切酶位点。特异性扩增gB编码基因片段,同时构建含HCMVgB编码基因的原核表达载体,对重组质粒进行诱导表达,用间接酶联免疫吸附测定(ELISA)法对纯化的Ni/gB融合蛋白的灵敏度和特异度等生物活性进行鉴定,灵敏度可达95%,特异度达96.7%。结果目的蛋白经诱导后表达于上清液中,经鉴定,目的蛋白保留了天然蛋白原有的生物活性。结论 Ni/gB融合蛋白的获得,为研制以重组蛋白代替传统全病毒作为检测抗原的新型HCMV特异性免疫学检测试剂盒奠定了基础。
Objective To construct and identify the procaryotic expression vector of gB. Trying to induce the pET28a/gB and i- dentify the expressed protein. Methods According to the nucleotide sequence of HCMV in Genbank,a pair of primers with BarnH I and EcoR I sites at 5r-terminal 3＇-terminal were designed and synthesized respectively and a HCMV gB DNA fragment was am- pli/ied by PCR. The amplified gB gene fragment was subcloned into pET28a vector by a series of molecular biological methods in- cluding restriction digest and ligation, and transfection. The recombinant plasmid was induced under optimum conditions, then the expressed production was identified by SDS-PAGE. The recombinant strain was lyzed by ultrasonic and harvested by centrifugation. The sensitivity（95 %） and specificity（96.7%） of pET28a/gB protein were identified by indirect ELISA. Results The interest pro- tein was found in the supernatant,and its original biological activity is retained when compared with native protein. Conclusion The pET28a/gB protein was successfully expressed and it is a solid foundation for developing new type-specific serological detection of HCMV.
出处
《国际检验医学杂志》
CAS
2014年第4期390-391,393,共3页
International Journal of Laboratory Medicine
