摘要
目的构建canstatin原核表达载体,表达重组人canstatin蛋白,并验证其生物学活性。方法利用BamH Ⅰ和HindⅢ从重组质粒pUCm-T／canstatin上切下canstatin基因,插入表达质粒 pET-22b(+)相应位点,转化E.coli BL21。异丙基硫代-β-D半乳糖苷(IPTG)诱导重组蛋白表达,SDS- PAGE电泳分析蛋白表达情况。超声破碎菌体,Ni-NTA柱亲和层析纯化重组蛋白,PBS对其透析复性。鸡胚尿囊膜(CAM)实验验证目的蛋白抗血管生成活性。结果重组质粒pUCm-T／canstatin在 BamHⅠ和HindⅢ双酶切后,电泳证实切下目的基因canstatin cDNA,将其插入质粒pET-22b(+)相应位点,转化E.coli BL21后,琼脂板上生长出多个白色菌落。挑选7个白色菌落做BamH Ⅰ和Hind Ⅲ双酶切,所有菌落均切出分别对应原质粒与目的基因的2条特异性条带,证实均为阳性克隆。IPTG 诱导其中1个克隆表达蛋白,SDS-PAGE电泳分析表明在相对分子质量24 000左右出现新的蛋白表达条带,诱导后1、2、3、4 h表达蛋白占菌体总蛋白百分比分别为18．2％、18．8％、23．0％和23．4％。Ni- NTA柱亲和层析后,在125、250mmol／L洗脱液中纯化出目的蛋白。CAM实验显示重组人canstatin 蛋白能抑制新生血管形成,且与剂量呈正相关。结论成功构建了canstatin原核表达载体,表达并纯化出有活性的重组人canstatin蛋白,为进一步研究其临床应用打下基础。
Objective To express the recombinant human canstatin protein, and to examine its biological activity. Methods Canstatin cDNA was cut off from the plasmid pUCm-T/canstatin with restriction enzymes BamHⅠ and Hind Ⅲ. The cDNA fragment was then ligated into the correspondence sites of plasmid pET-22b（＋） by T4 DNA ligation enzyme and transformed into E. coli BL21 which was induced to express proteins with isopropyl-1-thio-b-dgalactopyranoside （IPTG）. The expressed proteins were analyzed by SDS-PAGE and purified through Ni NTA column affinity chromatography. Chick chorioallantoic membrane （CAM） assay was performed to determine the activity of the recombinant protein. Results Canstatin cDNA from pUCmT showed one clear objective DNA band with electrophoresis. Seven of positive colonies were selected and identified by restriction enzyme analysis with BamH Ⅰ and Hind Ⅲ. Electrophoresis revealed that all selected colonies had two specific bands, one near the location of primary plamid, the other near that of objective gene fragment. After IPTG induction, there was a new protein band about 24 000 on SDS-PAGE. The induced product over total bacterial proteins in 1, 2, 3, and 4 hours after induction was 18. 2%, 18. 8%, 23. 0% and 23. 4%, respectively, by densitornetry examination. CAM assay demonstrated that the recombinant canstatin protein significantly inhibited the embryonic neovascularization in a dose-dependent manner. Conclusion The prokaryotic expression vector of human canstatin gene has been successfully constructed, laying the foundation for further clinical study.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2006年第2期92-95,共4页
Chinese Journal of Digestion
基金
上海市自然科学基金资助项目(03JcI4007)
关键词
血管生成
抑制剂
鸡胚尿囊膜实验
Agiogenesis
Inhibitor
Chick chorioallantoic membrane assay
