Midkine启动子调控的HSV-TK基因重组腺病毒的构建

目的构建以人midkine（MK）启动子调控的TK基因的重组复制缺陷型腺病毒。方法以Adeno-XTM表达试剂盒为基础，应用分子克隆技术，将穿梭质粒pShuttle的CMV启动子替换为MK启动子，并将由pHSV-106获取HSV-TK基因的编码序列亚克隆至其下游，酶切鉴定阳性重组穿梭质粒pShuttle-MK-TK。通过I-CeuⅠ和PI-SceⅠ两个稀有酶切位点．将目的基因与腺病毒质粒DNA（pAdeno-X）进行体外连接，获得含目的基因的重组腺病毒质粒DNA，后者经限制性内切酶PacⅠ切割后，两端露出反向末端重复序列（ITR），利用脂质体转染293细胞，获得含有目的基因重组腺病毒上清，PCR检测。结果酶切结果显示．MK启动子与TK基因均正向插入pShuttle中．TK位于MK启动子的下游。PCR检测显示重组腺病毒中含有MK启动子及TK基因片段。结论体外连接法可成功构建以人MK启动子调控的TK基因的重组腺病毒。

Construction of replication recombinant adenovirus containing human mindkine promoter and HSV-TK

Objective: To construct the replication-deficient recombinant adenovirus containing human mindkine promoter and thymidine kinase gene of Herpes simplex virus(HSV-TK).Methods: Based on Adeno-X~(TM) expression system,CMV promoter of pShuttle was replaced by MK promoter.The SHV TK gene,which was obtained from plasmid p HSV-106,was subcloned into p Shuttle vector under MK promoter.The proper p Shuttle-MK-TK was identified by special restriction endonuclease analysis.Both of the plasmid p Shuttle-MK-TK and Adeno-X DNA were digested with restriction endonuclease PI-SceI and I-CeuI,the target fragment(MK+TK) was subcloned into the Adeno-X DNA with ligation in vitro and the recombinant Adeno-MK-TK was obtained.After linearized by digesting with restriction endonucleases PacI,the plasmid Adeno-MK-TK was transfected into HEK 293 to produce recombinant virus stocks.Using PCR method,the virus stocks was identified whether it contained the interest gene MK and TK.Results: The restriction endonuclease analysis showed that the MK promoter and TK gene were inserted into the pShuttle successfully.And,the TK gene was located on the downstream of the MK promoter.PCR products were in coincidence with expectedness.Conclusion: By ligation in vitro,the recombinant HSV-TK gene under MK promoter mediated by adenovirus vector was constructed successfully.