人VEGF基因杆状病毒表达载体的构建及生物学活性鉴定

首都医科大学学报 ›› 2006, Vol. 27 ›› Issue (5): 623-626.

人VEGF基因杆状病毒表达载体的构建及生物学活性鉴定

孙丽翠¹, 王雅梅¹, 闫豫东², 张静宜², 司杨², 祁雅慧²

1. 首都医科大学生物化学与分子生物学系;2. 首都医科大学医学实验与测试中心

收稿日期:2006-01-05 修回日期:1900-01-01 出版日期:2006-10-24 发布日期:2006-10-24
通讯作者: 祁雅慧

Construction of Baculovirus Expression System of Human VEGF Gene and Assessment of Biological Activity

Sun Licui¹, Wang Yamei¹, Yan Yudong², Zhang Jingyi², Si Yang², Qi Yahui²

1. Faculty of Biochemistry and Molecular Biology, Capital University of Medical Sciences;2. Medical Experiment and Test Center, Capital University of Medical Sciences

Received:2006-01-05 Revised:1900-01-01 Online:2006-10-24 Published:2006-10-24

摘要/Abstract

摘要： 目的构建含人血管内皮细胞生长因子(VEGF)基因的杆状病毒表达载体,并对其表达产物的生物学活性进行测定.方法用Hind Ⅲ对pcDNA3.1/VEGF进行酶切,回收575 bp的VEGF片段;pFast a载体用Hind Ⅲ酶切回收,与VEGF片段连接.NcoI酶切鉴定方向,得到正向连接重组载体pFast/VEGF;将其转化DH10BAC感受态细胞,经卡那霉素、四环霉素、庆大霉素及蓝白斑筛选得到重组杆状病毒载体pBacmid-Fa-VEGF,PCR扩增鉴定得到单一VEGF条带.将该病毒载体转染sf9细胞,用噬斑筛选及SDS-PAGE和Western blot检测VEGF表达,并通过MTT法检测VEGF促内皮细胞生长活性.结果病毒液在稀释到10-7时出现噬斑,Western blot检测出现单一条带,MTT检测促内皮细胞生长率为19.44%.结论成功构建了含VEGF基因的杆状病毒表达载体,且表达产物具有显著的促内皮细胞生长作用.

关键词: 人血管内皮细胞生长因子, 重组杆状病毒载体, Western blot, sf9细胞, MTT

Abstract: Objective To construct recombinant baculovirus vector containing VEGF gene and study its biological activity.Methods Using Hind Ⅲ enzyme to digest pcDNA3.1/VEGF,the VEGF fragment(575 bp) was obtained.Then recombinant vector pFast/VEGF including VEGF gene was constructed.It was transformed into DH10BAC competent cell and cultured in LB plate which contained Kan,Ter,Get and X-gal/IPTG.After transfected into sf9 cells the virus plaque was screened.Western blot and MTT methods were used to determine the expression and biological activity of VEGF gene. Results The virus plaque was observed successfully in diluting to 10~(-7) concentration.Western blot showed only one band was identified.Cell growth promotion rate was 19.44% by MTT method.Conclusion The vector pBacmid-Fa-VEGF was successfully constructed and showed significant biological activity to stimulate endothelial cells proliferation.

Key words: human VEGF, recombinant baculovirus vector, Western blot, sf9 cell, MTT

中图分类号:

孙丽翠;王雅梅;闫豫东;张静宜;司杨;祁雅慧. 人VEGF基因杆状病毒表达载体的构建及生物学活性鉴定[J]. 首都医科大学学报, 2006, 27(5): 623-626.

Sun Licui;Wang Yamei;Yan Yudong;Zhang Jingyi;Si Yang;Qi Yahui. Construction of Baculovirus Expression System of Human VEGF Gene and Assessment of Biological Activity[J]. Journal of Capital Medical University, 2006, 27(5): 623-626.

[1]	纪晓方, 李丽英, 常娜. 高内涵分析及Western blotting法在蛋白质核质分布研究中的应用及比较研究[J]. 首都医科大学学报, 2016, 37(5): 616-620.
[2]	崔世娟;杨静;顾园;魏骏飞;王少华;杨雅平;诸欣平. 不同培养条件对旋毛虫肌幼虫产生排泄分泌抗原的影响[J]. 首都医科大学学报, 2010, 31(5): 591-595.
[3]	黄雯;李晓璐;陈燊;张红;杨敏. 内源性H₂S对糖尿病肾病细胞外基质积聚的作用[J]. 首都医科大学学报, 2009, 30(2): 133-136.
[4]	孙丽翠;王雅梅;祁雅慧;闫豫东;张静宜. 人VEGF基因昆虫杆状病毒表达载体的构建[J]. 首都医科大学学报, 2004, 25(2): 159-161.
[5]	谢文杰;刘欣;王喆;王娜;付桂芳;张瑾峰. 莪棱舌草Ⅰ号对人肝癌细胞抑制作用的研究[J]. 首都医科大学学报, 2003, 24(2): 100-103.
[6]	吕婴;张海燕;刘晓勇;李卫红. EGF对人牙周膜细胞增生与ALP活性表达的影响[J]. 首都医科大学学报, 2002, 23(2): 171-173.
[7]	董苍转;吕国蔚. 缺氧预适应小鼠脑匀浆提取液对PC12细胞的影响[J]. 首都医科大学学报, 2001, 22(2): 97-99.
[8]	左大鹏;付蓁;潘颖;黄如彬. 重组人肿瘤坏死因子对白血病细胞生长抑制的研究[J]. 首都医科大学学报, 1995, 16(2): 139-143.
[9]	石梦辉;杨连雪;盛树力. 用SDS—PAGE、Western blot和ELISA方法检测猪囊虫囊液中有效抗原的研究[J]. 首都医科大学学报, 1989, 10(1): 9-11.