Construction And Identfication Of Recombinant Adenovirus Vectors And Adeno-Associated Virus Vectors Of Dmrt7

Dmrt7 gene can express protein Dmrt7, which is important to mammalian reproductive system and contains DM domain. The main biological functions of Dmrt7 are the regulation of spermatogenesis and sex differentiation. There are rich values and broad prospects in the application of Dmrt7 protein in the treatment of low sperm production and no sperm production of male animals. While the natural Dmrt7 protein is scarce, and can not meet the needs of clinic and scientific research, the production of Dmrt7 protein using transgenic technology are becoming the main focus of current research.In our study, total RNA was firstly separated and extracted from mouse testis and then the cDNA of Dmrt7 was obtained by RT-PCR. After being amplified by PCR, Dmrt7 was recombined with Adenovirus vector and adeno-associated virus vector.The results of this study included as follows:(1) The 1.0% agarose gel electrophoresis results of RT-PCR showed the 1100bp gene fragment was obtained from mouse testis total RNA and amplification with a pair of special primers designed at first. The sequence of Dmrt7 gene obtained was consistent with the reported, and had no mutation.(2) Construction of Dmrt7 recombinant adenovirus vector using Homologous recombination in bacteria. After being digested and identified by restrictive endonuclease, pShuttle-Dmrt7 -IRES-GFP was linearized by PmeⅠand subsequently co-transfected into BJ 5183 with adenoviral backbone plasmid pAdEasy-1. The results of 1.0% agarose gel electrophoresis of recombinant Ad-Dmrt7 digested by restrictive endonuclease PacⅠshowed a special band of 2.9kb. It confirmed that the Ad-Dmrt7 was constructed successfully. The homologous recombination in bacteria was a convenient and efficient way to construction of recombinant adenoviral vectors.(3) Recombinant Ad-Dmrt7 vector was transfected into HEK293 cell by liposome. After fluorescence was observed in 293 cells which were infected by 24h, the viral supernatant was collected and identified by PCR. The results indicated that there is a distinct band at 1100bp; the titer of recombinant Ad-Dmrt7 which was determined by half fluorescent cell method was 0.95×10~8PFU/ml.(4) Construction of Dmrt7 recombinant AAV vector using Homologous recombination in HEK 293 cells. The Dmrt7 gene was subcloned into pAAV-MCS,then the obtained recombinant pAAV-Dmrt7 plasmid was digested and identified by restrictive endonuclease. After being purifed, the pAAV-Dmrt7 plasmid was co-transfected into HEK293 cells with pRC plasmid and pHelper plasmid and packaged into recombinant AAV-Dmrt7. The fluorescence can be observed after being transfected by 24h. The titer of recombinant AAV-Dmrt7 which was determined by half fluorescent cell method was 1.5×10~9PFU/ml.