人血管内皮抑制素vasostatin120-180在大肠杆菌中的表达纯化及初步的抗血管生成活性研究-东南大学学报医学版

人血管内皮抑制素vasostatin120-180在大肠杆菌中的表达纯化及初步的抗血管生成活性研究

单位：南京大学,医药生物技术国家重点实验室,江苏,南京,210093

分类号：Q78

出版年·卷·期（页码）：2008·27·第二期（75-80）

摘要：

目的:在大肠杆菌中克隆和表达人源血管内皮抑制素vasostatin120-180(VAS)基因,并进行分离纯化及抑制新生血管生成活性鉴定.方法:根据大肠杆菌偏爱密码子,应用化学合成和分子克隆方法得到人源血管内皮抑制素120-180片段(即VAS)的编码基因.通过PCR和限制性内切酶消解,将VAS编码基因克隆入大肠杆菌表达载体pET28a中,并在BL21(DE3)菌株中经IPTG诱导、表达带有His融合标签的His-VAS重组蛋白.VAS蛋白的表达水平大约占细菌总蛋白的45%,大多数目的蛋白以包涵体形式存在,经过体外包涵体变性、复性及纯化,采用SDS-PAGE分析鉴定,用鸡胚尿囊膜血管生成抑制试验进行重组蛋白活性的生物活性鉴定.结果:经DNA序列分析鉴定,重组质粒pET28a-VAS构建成功,IPTG诱导后的融合蛋白在大肠杆菌中得到高效表达,Western blotting分析并鉴定了VAS的分子属性.通过鸡胚尿囊膜试验验证,重组VAS蛋白能够很好地抑制微血管生成.结论:获得高效表达的、高纯度的VAS,为VAS进一步的功能分析和抑制血管生成活性研究奠定了基础.

Objective To clone,highly express the gene encoding human vasostatin120-180(VAS) in E.coli and to purify and validate its anti-angiogenic activity.Methods According to codon usage preference for E.coli,the optimized coding sequence of human VAS was obtained by chemical synthesis and molecular cloning methods.Using PCR and enzyme digestion,the full encoding sequence for VAS was cloned into the E.coli expression vector pET28a and overexpressed as a N-terminal His-tagged fusion protein.Between His-tag and VAS,an enterokinase recognition site was introduced to release the intact VAS.Most of the target protein existed in inclusion body,which was analysed by SDS-PAGE.Anti-angiogenic activity of recombinant VAS protein was displayed by chick embryo chorioallantoic membrane assay(CAM).Results The recombinant plasmid was identified and confirmed by DNA sequencing.VAS was expressed at high level and purified successfully and its anti-angiogenic activity was confirmed by CAM.Conclusion A novel and highly efficient method for producing bioactive VAS by inclusion body refolding strategy is reported by us,which demonstrates a promising prospect of the further in-depth study and potential clinic application of recombinant VAS protein.

参考文献：

[1] PANDYA N M, DHALLA N S, SANTANI D D. Santani Angiogenesis-a new target for future therapy. 2006(5). doi:10.1016/j.vph.2006.01.005
[2] FOLKMAN J, BRAUWALD E. Harrison's textbook of internal medicine, 2001
[3] HANAHAN D, FOLKMAN J. The hallmarks of cancer. 2000. doi:10.1016/S0092-8674(0)81683-9
[4] O'REILLY M S, BOEHM T, SHING Y. An endogenous inhibitor of angiogenesis and tumor growth. 1997. doi:10.1016/S0092-8674(0)81848-6
[5] O'REILLY M S, HOLMGREN L, SHING Y. Angiostatin:a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. 1994. doi:10.1016/0092-8674(94)90200-3
[6] PIKE S E, YAO L, JONES K D. Vasostatin,a calreticulin fragment,inhibits angiogenesis and suppresses tumor growth. 1998. doi:10.1084/jem.188.12.2349
[7] MAESHIMA Y, SUDHAKAR A, LIVELY J C. Tumstatin,an endothelial cell-specific inhibitor of protein synthesis. 2002. doi:10.1126/science.1065298
[8] PIKE S E, YAO L, SETSUDA J. Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth. 1999
[9] PERSANO L, CRESCENZI M, INDRACCOLO S. Anti-angiogenic gene therapy of cancer:Current status and future prospects. 2007(1). doi:10.1016/j.mam.2006.12.005
[10] GROSJEAN F, FIERS W. Preferential codon usage in prokaryotic genes:the optimal codon anticodon interaction energy and the selective codon usage in efficiently expressed genes. 1982. doi:10.1016/0378-1119(82)90157-3
[11] FANG L, SUN Q M, HUA Z C. Expression of recombinant Chinese bovine enterkinase catalytic subunit in P.pastoris and its purification and characterization. 2004(7). doi:10.1093/abbs/36.7.513
[12] HEHIR K M, BAGUISI A, PENNINGTON S E. A potential antitumor peptide therapeutic derived from antineoplastic urinary protein. 2004(4). doi:10.1016/j.peptides.2004.02.003

服务与反馈：

【文章下载】【发表评论】【查看评论】【加入收藏】

提示：您还未登录，请登录！点此登录