大眼鰤鲈鱼真皮肿瘤病毒(WDSV)辅助基因orfA与orfC重组腺病毒的制备及应用  

Preparation and preliminary application of recombinant adenoviruses based on accessory genes orfA and orfC of Walleye dermal sarcoma virus(WDSV)

作　　者：张优优[1] 徐坤[1] 王亮亮[1] 张婷婷[1] 辛颖[1] 任充华[1] 闫强[1] 张智英[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2012年第12期34-40,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30870119)

摘　　要：【目的】扩增大眼鰤鲈鱼真皮肿瘤病毒(WDSV)辅助基因orfA和orfC,制备orfA和orfC重组腺病毒,并感染Hela229和SPC-A-1细胞,以检测orfA和orfC对肿瘤细胞的影响。【方法】从pWDSV全长表达载体中扩增WDSV辅助基因orfA和orfC,构建orfA和orfC基因的重组腺病毒载体以及对照空载体,采用脂质体介导法转染HEK293细胞进行包装和扩增,用绿色荧光蛋白法测定重组腺病毒的滴度;将重组腺病毒转染肿瘤细胞Hela229和SPC-A-1,采用Weastern Blot方法检测基因orfA和orfC的表达情况,并用MTT法检测其对肿瘤细胞Hela229和SPC-A-1的影响。【结果】PCR扩增获得了891bp的orfA基因和360bp的orfC基因,成功构建了对照空载体pAdEasy-l-CK和含有orfA和orfC基因的腺病毒载体pAdEasy-l-orfA和pAdEasy-l-orfC,转染HEK293细胞并进行包装后获得了重组腺病毒Ad-CK、Ad-orfA和Ad-orfC,用Hela229细胞测得其滴度分别为5.67×109,2.00×109和7.33×108 GFU/mL。重组腺病毒对肿瘤细胞Hela229和SPC-A-1的生长具有抑制作用。【结论】初步证明了orfA和orfC基因对人类肿瘤细胞Hela229和SPC-A-1的生长具有抑制作用,为进一步研究WDSV辅助基因对人类肿瘤的影响以及肿瘤萎缩的分子机理奠定了基础。【Objective】 This study aimed to amplify the accessory genes orfA and orfC of Walleye Dermal Sarcoma Virus（WDSV）,and prepare the recombinant adenovirus based on amplified genes to detect their effects on Hela229 and SPC-A-1 cells.【Method】 WDSV accessory genes orfA and orfC from pWDSV were amplified by PCR technology before being inserted into adenovirus vector（pAdTrack） to generate pAdTrack-orfA and pAdTrack-orfC.Then they were recombined with pAdEasy in BJ5183 to obtain the pAdEasy-orfA and pAdEasy-orfC,and the control pAdEasy-CK was also generated.Then pAdEasy-orfA,pAdEasy-orfC,and the control pAdEasy-CK were transfected into HEK293 with Lipofectamine2000 to get the recombinant adenovirus particles Ad-orfA,Ad-orfC and Ad-CK.The titers of the recombinant adenovirus were validated through the green fluorescent protein methods.Ad-orfA,Ad-orfC and Ad-CK were transfected to human tumor cells Hela229 and SPC-A-1,respectively.The expressions of the orfA and orfC genes were detected through western blot and effects of Ad-orfA and Ad-orfC on Hela229 and SPC-A-1 were measured by MTT assay.【Result】 We amplified the 891 bp orfA and 360 bp orfC by PCR and got the shuttle vectors pAdtrack-orfA and pAdtrack-orfC.Recombination of these shuttle vectors in BJ5183 with pAd-easy produced pAdEasy-orfA and pAdEasy-orfC.pAdEasy-orfA,pAdEasy-orfC and control vector were packaged in HEK293 to get Ad-orfA,Ad-orfC and Ad-CK.Titers of adenovirus measured in Hela229 cells were 5.67×109,2.00×109 and 7.33×108 GFU/mL,respectively.Ad-orfA,Ad-orfC and Ad-CK infected tumor cells Hela229 and SPC-A-1.The expression of orfA and orfC showed inhibiting effects on tumor cell growth.【Conclusion】 The results showed that orfA and orfC genes inhibited the growth of human tumor cells Hela229 and SPC-A-1.This work is a good start for further studies on the WDSV auxiliary genes in human tumors and the molecular mechanism of tumor shrinkage.

关 键 词：大眼鰤鲈鱼真皮肿瘤病毒 orfA基因 orfC基因 重组腺病毒 肿瘤 

分 类 号：Q740[生物学—分子生物学] R730.2[医药卫生—肿瘤]