Construction of pMSV-Slfn5-GFP plasmid and analysis of gene structure in mice / 重庆医学

ABSTRACT

Objective To perform mouse pMSV-Slfn5-GFP gene recombinant expression plasmid construction and gene structure analysis.Methods Total RNA was extracted from mouse liver and turned into cDNA by reverse transcription.Mouse Slfn5 coding sequence (CDS) fragment was amplified by PCR and connected with the pGEM-T Easy vector.The connected product was transferred the E.coli DH5a.The positive clones were selected for extracting plasmid,which was identified by double enzyme of restriction endonuclease Hpa Ⅰ and Xho Ⅰ.Then correct plasmid identified by enzyme digestion was sequenced by Macrogen USA.Then correct plasmid by sequencing was connected with pMSV-GFP by HindⅢ and Xbo Ⅰ,which was named as pMSV-Slfn5-GF.UCSC (http://genome.ucsc.Edu/) was used to analyze mouse Slfn5 and its family genomic structure.Slfn5 protein structural domain was determined by NCBI.Results Slfn5 full-length gene sequence was cloned into the expression vector pMSV-GFP,the fragment size was about 2.65 kb by enzyme digestion identification.The conservatism of AAA_4 protein domain in Slfn5 protein family was determined by phylogeny.fr.Conclusion Mouse full-length gene pMSV-Slfn5-GFP expression vector is successfully constructed.