【摘要】 目的构建N-TIMP3重组腺病毒载体,为深入研究组织金属蛋白酶抑制因子-3(tissue inhibitor of metalloproteinases-3,TIMP-3)N端结构域对胰腺癌的作用奠定基础。方法PCR扩增编码人TIMP3信号肽及其N端结构域的DNA序列,定向克隆至空载穿梭质粒pShuttle-CMV,构建重组穿梭质粒pShuttle-N-TIMP3,并测序鉴定。将重组pShuttle-N-TIMP3转化BJ5183感受态细菌,与其内的腺病毒骨架质粒pAdEasy-1进行同源重组,构建重组腺病毒载体pAd-N-TIMP3,并经酶切与PCR鉴定。将线性化的重组载体转染QBI-293A细胞包装病毒,TCID50法检测病毒滴度,用Western印迹检测目的蛋白的表达。结果重组腺病毒载体pAd-N-TIMP3经鉴定正确,病毒滴度为5×108PFU/ml,并检测到目的蛋白的表达。结论成功构建了重组腺病毒载体pAd-N-TIMP3。更多还原

【Abstract】 Objective To construct and identify recombinant adenoviral plasmid expressing N-TIMP3, for further investigating the effect of N-TIMP3 on pancreatic cancer.Methods Construction of recombinant shuttle plasmid pShuttle-N-TIMP3 was carried out by inserting Signal Peptide encoding DNA sequencing and TIMP-3 N-terminal domain into pShuttle-CMV.pShuttle-N-TIMP3 was identified by sequencing.Subsequently, the recombinant adenoviral plasmid pAd-N-TIMP3 was obtained by homologous recombination between pShuttle-N-TIMP3 and the adenoviral backbone plasmid pAdEasy-1.The recombinant Ad-N-TIMP3 adenovirus particles were produced by transfection of QBI-293A cells with Pac I linearized pAd-N-TIMP3.Virus titer was determined by TCID50, and N-TIMP3 expression was tested by Western blot.Results The recombinant pAd-N-TIMP3 was successfully constructed and identified.The recombinant Ad-N-TIMP3 was titered at 5×108 PFU/ml, and the expression of N-TIMP3 was confirmed.Conclusion The recombinant pAd-N-TIMP3 was successfully constructed and identified.更多还原