摘要
目的 :克隆表达人血管内皮生长因子 16 5 (humanvascularendothelialgrowthfactor 16 5 ,hVEGF16 5 )基因 ,制备VEGF免疫学检测工作标准品。方法 :用RT -PCR从肝癌组织扩增目的基因VEGF16 5 ,插入到pUCm -T质粒中并测序鉴定。构建原核表达重组质粒 pET32a -hVEGF16 5 ,转化入大肠杆菌 ,经IPTG诱导 ,固化Ni2 + 吸收色谱纯化得到重组人VEGF16 5 (recombinanthumanVEGF16 5 ,rhVEGF16 5 )。用WsternBlot检测rhVEGF16 5免疫原性 ,并通过HUVEC增殖试验检测rhVEGF16 5生物学活性。按标准品的要求制备VEGF检测工作标准品 ,最后用ELASA试剂盒标定。结果 :PCR产物为 6 0 0bp ,测序结果表明其序列正确 ,人VEGF16 5可在大肠杆菌内可溶性表达 ,rhVEGF16 5蛋白相对分子质量为 38kD ,具有生物学活性。工作标准品浓度标定为 5 0 5pmol/L。结论 :成功地克隆了有生物学活性的VEGF16 5基因 ,并制备出VEGF免疫学检测工作标准品。
Objective To clone, express and determine activity of hVEGF165 in E.coli and prepare a working standards for immunoassay. Methods Using RNA of human liver cancer tissues as templates, VEGF gene was amplified with RT-PCR. The PCR product was interpolated into pUCm-T plasmid and the sequenc verified. A prokaryotic expression plasmid pET32a harbouring VEGF165 was constructed ,ten transformed into BL21trxB(DE3) cells. IPTG was used to induce the expression of VEGF165 in E.coli. The product was purified with his bind resin affinity chromatography. Western blotting were performed to demonstrate the antigenicity. Furthermore the bioactivity of VEGF165 was preliminarily detected with HUVEC proliferation test. The working standard for immunoassay was detected with ELASA kit for VEGF. Results Sequence of 600bp VEGF165 cDNA was proved to be correct by dequence analysis. The expression of soluble VEGF165 was identified by SDS-PGAE, Western bolotting indicated that the molecular weight of VEGF165 protein was 38KD with good antigenicity and high specificity. rhVEGF165 had biological activities of hVEGF165. The concentration of working standard was 5.05pmol/L. Conclusion VEGF165 gene has been successfully cloned and expressed in this laboratory. The working standard for immunoassay was prepared as well.
出处
《放射免疫学杂志》
CAS
2004年第5期395-398,共4页
Journal of Radioimmanology
基金
上海市科委重点基金资助 0 2DJ0 413 7
