Construction And Identfication Of Recombinant Adenovirus Vectors Of FOXL2 And Par-4

FOXL2 gene can express FOXL2 protein, which is important to animal embryonic development, the formation of animal eyelid and ovary. FOXL2 gene only express in the animal eyelid and ovary. Animals will suffer BPES and premature ovarian failure, if FOXL2 gene have been mutation.FOXL2 gene also play a key role in the animal sex control.Par-4 gene can express Par-4 protein,which can induce apoptosis.It has broad prospects in cancer treatment.While the natural Par-4 protein is scarce, and can not meet the needs of clinic and scientific research, the production of Par-4 protein using transgenic technology are becoming the main focus of current research.In our study, total RNA was firstly separated and extracted from mouse ovary and testis, then the cDNA of FOXL2 and Par-4 was obtained by PCR. After being amplified by PCR, FOXL2 and Par-4 was recombined respectively with adenovirus vector.The results of this study included as follows:(1) The 1.0% agarose gel electrophoresis results of PCR showed the 1250bp gene fragment was obtained from mouse ovary total RNA and amplification with a pair of special primers designed at first. The sequence of FOCL2 gene obtained was consistent with the reported, and had no mutation.(2) Construction of FOXL2 recombinant adenovirus vector using Homologous recombination in bacteria. After being digested and identified by restrictive endonuclease, pShuttle-FOXL2-IRES-GFP was linearized by PmeⅠa nd subsequently co-transfected into BJ 5183 with adenoviral backbone plasmid pAdEasy-1. The results of 1.0% agarose gel electrophoresis of recombinant Ad-FOXL2 digested by restrictive endonuclease PacⅠshowed a special band of 4.5kb. It confirmed that the Ad-FOXL2 was constructed successfully. The fluorescence can be observed after being transfected by 24 h.And the virus titer of Ad-FOXL2 is TCID50=10^-8.61/0.1 ml.(3) The 1.0% agarose gel electrophoresis results of PCR showed the 1050bp gene fragment was obtained from mouse testis total RNA and amplification with a pair of special primers designed at first. The sequence of Par-4 gene obtained was consistent with the reported, and had no mutation.(4) Construction of Par-4 recombinant adenovirus vector using Homologous recombination in bacteria. After being digested and identified by restrictive endonuclease, pShuttle-Par4-IRES-GFP was linearized by PmeⅠa nd subsequently co-transfected into BJ 5183 with adenoviral backbone plasmid pAdEasy-1. The results of 1.0% agarose gel electrophoresis of recombinant Ad-Par4 digested by restrictive endonuclease PacⅠshowed a special band of 4.5kb. It confirmed that the Ad-Par4 was constructed successfully. The fluorescence can be observed after being transfected by 24 h.And the virus titer of Ad-Par4 is TCID50=10^-8.72/0.1 ml.