[Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].
[Construction and expression of recombinant adenovirus vector carrying human endostatin, K5 and endostatin-K5 gene].
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发表时间：
2009-07
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通讯作者：
Hui-jin Li;Weifeng Zhang;Dong-yang Wang;Rong-hua Li;Zong-li Qi;Jun-li Zhao;Hai-bin Xia
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作者：
Hui-jin Li;Weifeng Zhang;Dong-yang Wang;Rong-hua Li;Zong-li Qi;Jun-li Zhao;Hai-bin Xia
目的构建分别表达人内皮抑素、K5和内皮抑素-K5基因的重组腺病毒载体，并研究其体外生物活性。方法采用PCR扩增人内皮抑素、K5和内皮抑素-K5基因，分别通过酶切和连接法将其亚克隆到穿梭载体pAd5-CMV-H1H2-MCS-6His中。用磷酸钙沉淀法将Pac I线性化的阳性重组质粒与Pac I线性化的腺病毒主干质粒共转染HEK 293细胞。重组病毒采用CsCl密度梯度离心纯化。Western blot检测不同时间点（24 h、48 h、72 h）的蛋白表达情况。MTT法检测该蛋白对ECV-304生长的抑制作用。结果在HEK293细胞中成功构建了表达人内皮抑素、K5和内皮抑素-K5基因的重组腺病毒载体。Western blot检测蛋白表达。随着感染的HeLa细胞孵育时间的延长，蛋白表达水平升高。重组腺病毒载体表达的3种蛋白对ECV-304细胞生长均有明显的抑制作用。结论携带内皮抑素、K5和内皮抑素-K5基因的腺病毒载体表达的蛋白对ECV-304细胞的生长有明显的抑制作用。
AIM To construct the recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene respectively, and study their bioactivity in vitro. METHODS Human endostatin, K5 and endostatin-K5 gene were amplified by PCR, which were then subcloned into shuttle vector pAd5-CMV-H1H2-MCS-6His by enzyme and ligation respectively. The positive recombinant plasmids linearized by Pac I were cotransfected into HEK 293 cells with the Pac I linearized adenoviral backbone plasmid using calcium phosphate precipitation method. The recombinant viruses were purified by CsCl density gradient centrifugation. The protein expression at different time points (24 h, 48 h and 72 h) was determined by Western blot. The inhibitory effect of the protein on ECV-304 growth was detected by MTT. RESULTS The recombinant adenoviral vectors expressing human endostatin, K5 and endostatin-K5 gene were successfully constructed in HEK293 cells. Protein expression was detected by Western blot. The level of protein expression was increased with the prolonged incubation of the infected HeLa cells. Three kinds of the protein expressed by the recombinant adenoviral vectors showed obvious inhibitory effect on ECV-304 cell growth. CONCLUSION The protein expressed by adenoviral vectors carrying endostatin, K5 and endostatin-K5 gene has an obvious inhibitory effect on ECV-304 cell growth.