携带HPV16 E7病毒癌基因的重组腺病毒穿梭质粒的构建及鉴定

①目的构建携带人乳头瘤病毒16型（HPV16）E7病毒癌基因的复制缺陷型重组腺病毒穿梭质粒pAdTrack-CMV-E7。②方法应用PCR技术从宫颈癌组织中扩增E7病毒癌基因全长片段，并导入T载体进行测序，与Nucleotide中HPV16标准毒株序列比较鉴定。目的基因及穿梭质粒载体pAdTrack-CMV分别经Bgl Ⅱ和Hind　Ⅲ双酶切后连接，转化宿主菌E．coli DH5α，应用PCR法及限制性内切酶消化法双重鉴定重组质粒，并测序。③结果PCR扩增产物与Nucleotide中HPV16E7序列一致，重组穿梭质粒经Bgl　Ⅱ和Hind　Ⅲ双酶切后电泳，可观察到9220bp大小的载体条带和313bp大小的目的基因条带；将经PCR和双酶切鉴定的重组阳性克隆测序，结果证实克隆成功。④结论成功地构建了携带HPV16E7基因的重组腺病毒穿梭质粒。

CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT ADENOVIRUS PADTRACK-CMV-E7 SHUTTLE VECTOR CARRYING THE HUMAN PAPILLOMAVIRUS TYPE 16 E7 VIRUS ONCOGENE

Objective To construct recombinant adenovirus pAdTrack-CMV-E7 shuttle vector carrying the human papillomavirus type 16 E7 virus oncogene. Methods PCR was performed to get HPV 16 E7 gene from human cervical carcinoma tissues. The PCR product and the pAdTrack-CMV shuttle vector were cut by the two same restriction endonucleases, and then the E7 gene was subcloned into the pAdTrack-CMV shuttle vector by T4 DNA ligase enzyme. Results It was confirmed by sequencing that the PCR product was the gene of HPV16 E7 virus oncogene. Restriction endonuclease analysis confirmed the successful cloning of the E7 gene into the pAdTrack-CMV. Conclusion The recombinant adenovirus pAdTrack-CMV shuttle vector carrying the human papillomavirus type 16 E7 is constructed successfully.