人VEGF基因杆状病毒表达载体的构建及生物学活性鉴定

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

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HindⅢ对pcDNA3.1/VEGF进行酶切,回收575 bp的VEGF片段;pFast a载体用HindⅢ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。

Objective To construct recombinant baculovirus vector containing VEGF gene and study its biological activity. Methods Using Hind HI 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.