弹性蛋白酶保护因子--Elafin结构基因腺病毒载体穿梭质粒的构建、鉴定及表达

构建能表达弹性蛋白酶保护因子———elafin基因腺病毒表达载体的穿梭质粒,为进一步包装能高效表达结构蛋白的腺病毒载体做准备.以含有elafin基因的真核质粒pEGFP-N1-Elafin为模板,PCR扩增elafin基因,PCR产物以SalⅠ、EcoRⅤ双酶切,定向插入到腺病毒穿梭质粒pAdTrack-CMV中CMV启动子下游SalⅠ与EcoRⅤ位点之间,获得重组腺病毒穿梭质粒pAdTrack-CMV-Elafin,通过SalⅠ及EcoRⅤ双酶切、PCR及插入片段序列测定对该质粒进行鉴定,将pAdTrack-CMV-Elafin转染293细胞,以RT-PCR及Western-Blot检测其在293细胞中的瞬时表达.结果表明:双酶切、PCR及测序鉴定证实,pAdTrack-CMV-Elafin穿梭质粒的插入片段为elafin基因,用pAdTrack-CMV-Elafin穿梭质粒转染293细胞后可见绿色荧光蛋白的表达,RT-PCR和Western-blotting表明其可在293细胞中瞬时表达.

Construction, Identification and Expression of a Recombinant Adenovirus Shuttle Plasmid Containing Antiproteinase Protector Elafin Structure Gene

Objective To construct a recombinant adenovirus shuttle plasmid containing antiproteinase protector elafin structure gene in order to construct the adenovirus vector.Methods The elafin gene was amplified by PCR according to the pEGFP-N1-elafin,and inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of the adenovirus shuttle plasmid(pAdTrack-CMV),then the recombinant plasmids(pAdTrack-CMV-elafin)were obtained.The recombinant plasmid was identified by endonuclease,PCR and sequencing.Antiproteinase protector elafin gene was expressed transiently with Lipofectamine 2000 coated in 293 cells which was confirmed by RT-PCR and Western-Blotting.Results: Insert DNA of the recobinant plasmid was confirmed to be elafin gene by endonuclease,PCR and sequencing.The recombinant plasmid can express elafin gene transiently in 293 cells which was confirmed by RTPCR and Western-Blotting.Conclusions: The recombinant adenovirus shuttle plasmid containing antiproteinase protector elafin gene was constructed successfully.This should be useful to construct adenovirus vector which can express antiproteinase protector elafin gene.