人膜联蛋白Ⅴ的表达及核素标记

Expression of Human Annexin Ⅴ and Its Nuclide Labeling

目的构建人膜联蛋白Ⅴ(AnnexinⅤ)表达载体并诱导其表达,应用双功能螯合剂联肼尼克酰胺(HYNIC)偶联后应用核素99mTc标记。方法PCR扩增含AnnexinⅤ编码基因序列,将扩增产物克隆至His融合表达载体pET-28a(+),以异丙基硫代-β-D-半乳糖苷(IPTG)诱导His融合AnnexinⅤ的表达,应用Ni-NTA Super-flow纯化并经测序、SDS-PAGE及Western blot确证。表达的蛋白通过HYNIC偶联后进行99mTc标记,考察标记物的特性。结果琼脂糖凝胶电泳示PCR扩增产物碱基数量与目的片段大小一致。对重组质粒的分析表明,插入片段的序列与发表的AnnexinⅤ基因编码序列一致。在IPTG诱导下,经Western blot及SDS-PAGE证实,重组大肠埃希菌DH5α高效表达分子量约36 kD的目的产物9。9mTc-HYNIC-AnnexinⅤ标记率及放化纯均达到90%以上,稳定性在6 h以上,可以满足核素显像的要求。结论人AnnexinⅤ编码序列已被克隆至His融合表达载体pET-28a(+)上,并在大肠埃希菌DH5α中高效、大量表达。经HYNIC偶联后99mTc标记,可以得到较高的标记率和放化纯9。9mTc-HYNIC-AnnexinⅤ有望成为一种有良好临床应用前景的凋亡显像剂。

Objective To construct the expression plasmid of human Annexin Ⅴ , express it in E. coli and label Annexin Ⅴ with ^99mTc using hydrazino nieotinamido （HYNIC）. Methods The coding sequence of human Anncxin Ⅴ gene was amplified by PCR. The PCR product was cloned into His fusion expression vector pET-28a （＋）. The fusion protein was induced by IPTG and purified by Ni-NTA Superflow. SDS-PAGE and Western blot were used to identify the protein. The expressed protein was labeled with ^99mTc using HYNIC, and the characteristic of ^99mTc-HYNIC-Annexin Ⅴ was studied. Results A specific band of 980 bp from PCR amplification was seen in gel electrophoresis. The sequence of thc insert in the plasmid was identical to the published coding-region sequence of human Annexin Ⅴ gcne. A protein product with a molecular weight of about 36 kD could be induced by IPTG and confirmed by Western blot and SDS-PAGE, The radiochemical purity and radiolabeling yield of ^99mTc-HYNIC-Annexin Ⅴ were more than 90%. The stability could last 6 h. Conclusion The coding sequence of human Annexin Ⅴ gcne was successfully cloned into the His fusion expression vector pET-28a （＋） and highly expressed in E. coli DH5α. High radiochemical purity and radiolabcling yield could be gained when labeled with ^99mTc using HYNIC. It was suggested that ^99mTc-HYNIC-Annexin Ⅴ may be a promising agent for apoptosis imaging and clinical application.