摘要
目的在原核系统中表达全长HIV-1 p24抗原,对其抗原性进行鉴定,探讨其对HIV早期诊断的价值。方法利用PCR技术从含有HIV-1 gag基因质粒PTM-1中扩增p24蛋白基因,通过酶切消化后连接到表达载体pET28a上,转化大肠埃希菌BL21(DE3),经IPTG诱导,表达p24抗原。通过ELISA和Western blotting(WB)法检测表达产物的免疫反应活性及特异性。结果PCR产物和构建的重组质粒pET28a-p24经双酶切均显示约690 bpDNA片段,与预期p24抗原全基因片段大小一致。SDS-PAGE电泳可见纯化蛋白为1条相对分子量约26×103Mr的外源表达蛋白带,与预期大小一致。ELISA及WB实验证实表达的p24抗原具有较好的活性及特异性。时间分辨荧光免疫双抗体夹心法显示与市售p24抗原线性一致。结论构建了HIV-1 p24/pET28a原核高效表达系统,表达产物与市售的p24抗原具有相似的抗原性。
Objective To express recombinant HIV-1 core antigenicity and clinical application of HIV early diagnosis. p24 antigen in prokaryotic system, and investigate its Methods HIV-1 p24 gene amplified by PCR from plasmid （PTM-1-HIV-I-gas） was subcloned into vector pET28a （＋） after enzymatic digestion. The recombinant plasmid was transformed into E.coli BI21, and expressed after IPTG induction. Double enzyme digestion was used to confirm the correct insert in the recombinant plasmid. Western blotting and ELISA were used to analyze the purity and antigenicity of expressed p24 protein. The difference between recombinant p24 antigen and commercial p24 antigen were compared by time-resolved fluoroimmunoassay （TRFIA）. Results PCR product and exogenous gene section from the recombinant plasmid pET28a-p24 showed the same size of 690 bp which is equal to p24 gene sequences. An exogenous expressed protein band of Mt 26x103 Mr was obtained after purified by SDS-PAGE electrophoresis. The activity and specificity of p24 protein were tested by ELISA and WB. There was no significant difference between recombinant p24 and commercial p24 antigen. Conclusion The recombinant p24 antigen constructed and expressed in E.coli had a good antigenicity same as the commercial p24 antigen.
出处
《热带医学杂志》
CAS
2013年第4期424-427,436,共5页
Journal of Tropical Medicine
基金
广东省"211工程"项目
