关键词: 条件复制型腺病毒；穿梭载体；E1A基因

Abstract:

Abstract:Objective To construct and identify a novel single targeted CRAd shuttle vector pshuttle-E1A-E1Bp-E1B55K selectively delated CR2 region of E1A gene,and lay the foundation for the research of gene therapy of cancer. Methods The   E1B55K and E1A-E1Bp genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from human embryonic kidney cells (293T);E1A gene deleted CR2 region was amplified by overlap-PCR.The shuttle vector of pshuttle-E1A-E1Bp-E1B55K was 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 CR2 region were consistent with that published on GenBank,indicating that the E1B55K and E1A-E1Bp deleted CR2 region genes were cloned successfully. Furthermore,the recombinant plasmid pshuttle-E1A-E1Bp-E1B55K was identified by endonuclease digestion and PCR,which could be digested into two fragments of XbaⅠ or KpnⅠ，one length of 817 base pairs for XbaⅠ and the length of 1 244 base pairs for KpnⅠ,and the lengthes of  amplified fragments by PCR were 250 base pairs and 1 148 base 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.

Key words: conditionally replicative , adenovirus；shuttle vector；E1A gene