Construction And Identification Of Recombinant Adenovirus Genome AD-E7 Vector Carrying The Human Papillomavirus Type 16 E7 Virus Oncogene

Objective To construct recombinant adenovirus genome Ad-E7 vector carrying the human papillomavirus type 16 E7 virus oncogene for further being transfected into 293 cells to be packed into an Ad to study the role of HPV E7 in the regulation of cell cycle and the development of human cervical carcinoma.Methods The HPV16 E7 gene was obtained by PCR from the genomic DNA of cervical carcinoma tissue which was already proved to be HPV 16 E7(+).Then it was inserted into pMD18-T Vector in order to test whether its sequence was correct. The sequenced E7 gene and pAdTrack-CMV plasmid were cut by the two same restriction endonucleases, after that they were retrieved and ligated into a pAdTrack-CMV-E7 plasmid. The plasmid pAdEasy-1 was transformed into Bacillus Coli RecA~+BJ5183 to obtain a new kind of host bacillus. pAdTrack-CMV-E7 cut by Pme I was co-transfected into the new host Bacillus Coli RecA+BJ5183(with pAdEasy-1) so as to form a recombinant and amplified it. The recombinant was identified by the methods of PCR cutting by Pac I and Bam H I.Results (1)The size of PCR product was 313bp; (2)Enzyme digestion and PCR reaction showed that the PCR product was inserted into pMD18-T Vector; (3)The PCR product was identified by sequencing to be HPV 16 E7 gene; (4)The successful construction of recombinant plasmid of pAdTrack-CMV-E7 was identified by PCR reaction and enzymes digestion; (5)The plasmid obtained from the new host bacillus was cut by Bam H I into two part ,which were the same with pAdEasy-l.(6)The recombinant plasmid identified by PCR reaction and enzymes digestion were constructed successfully. Conclusion (1)The HPV 16 E7 gene can be obtained by PCR from the genomic DNA of cervical carcinoma tissue which was already proved to be HPV16 E7(+). (2)The recombinant plasmid pAdTrack-CMV-E7 can be constructed successfully by genetic engineering technology. (3)The new host Bacillus Coli RecA+ BJ5183 (containing pAdEasy-1) can be obtained by transforming plasmid pAdEasy-1 with the effective 1 xTSS method. (4)The recombinant adenovirus genome Ad-E7 carrying HPV 16 E7 gene can be constructed by AdEasy? System as well as the homologous recombination...