单重靶向条件复制型腺病毒穿梭载体pshuttle-E1A-E1Bp-E1B55K的构建及鉴定  

Construction and verification of a novel single targeted CRAd shuttle vector pshuttle-E1A-E1Bp-E1B55K

作　　者：王宏芳[1] 李金华[1] 刘扬[1] 李艳博[1] 王志成[1] 刘威武[1] 龚守良[1] 

机构地区：[1]吉林大学公共卫生学院卫生部放射生物学重点实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2010年第5期821-824,共4页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(30870747;30970681)

摘　　要：目的:构建并鉴定一种新型E1A基因CR2区选择性缺失的腺病毒穿梭载体pshuttle-E1A-E1Bp-E1B55K,为包装单重靶向条件复制型腺病毒进行肿瘤基因治疗奠定基础。方法:采用RT-PCR法从人胚肾细胞(293T)中扩增条件复制型腺病毒必需的E1区基因,包括E1A、E1Bp及E1B55K基因片段;重叠PCR法扩增CR2区缺失的E1A基因;采用基因重组技术构建pshuttle-E1A-E1Bp-E1B55K重组质粒,并进行PCR及酶切鉴定。结果:经测序证实获得的E1B55K及CR2区缺失的E1A基因与GenBank公布的完全一致,E1A基因CR2区缺失的pshuttle-E1A-E1Bp-E1B55K质粒经分别经XbaⅠ与KpnⅠ单酶切,得到大小约为817和1244bp的酶切片段,PCR鉴定得到约250及1148bp的基因片段,鉴定结果与预期结果完全一致。结论:E1A基因CR2区选择性缺失的单重靶向条件复制型腺穿梭载体pshuttle-E1A-E1Bp-E1B55K构建成功。Objective To construct and identify a novel single targeted CRAd shuttle vector pshuttle-E1A-E1Bp-E1B55Kselectively delated CR2region of E1Agene,and lay the foundation for the research of gene therapy of cancer.Methods The E1B55Kand E1A-E1Bp genes were amplified by reverse transcriptase polymerase chain reaction（RT-PCR）from human embryonic kidney cells（293T）;E1Agene deleted CR2region was amplified by overlap-PCR.The shuttle vector of pshuttle-E1A-E1Bp-E1B55Kwas constructed by gene recombinant technique,PCR and enzyme analysis were used to identify the shuttle vector.Results These sequences of amplified E1B55K and E1A-E1Bp deleted CR2region were consistent with that published on GenBank,indicating that the E1B55Kand E1A-E1Bp deleted CR2region genes were cloned successfully.Furthermore,the recombinant plasmid pshuttle-E1A-E1Bp-E1B55Kwas identified by endonuclease digestion and PCR,which could be digested into two fragments of XbaⅠor KpnⅠ,one length of 817base pairs for XbaⅠand the length of 1 244base pairs for KpnⅠ,and the lengthes of amplified fragments by PCR were 250base pairs and 1 148base pairs.These were coincident with the anticipated result.Conclusion A novel recombinant CRAd shuttle vector pshuttle-E1A-E1Bp-E1B55K is constructed successfully which is useful for cancer gene therapy.

关 键 词：条件复制型腺病毒 穿梭载体 E1A基因 

分 类 号：Q78[生物学—分子生物学]