摘要
目的构建表达人凝血因子Ⅶ(FⅦ)的重组腺病毒载体,利用腺病毒载体介导哺乳动物细胞表达重组人凝血因子Ⅶ(rh FⅦ)。方法利用Bam HⅠ和EcoRⅤ双酶切pc DNA 3.1-FⅦ载体得到FⅦ基因片段,补平后,插入经SalⅠ单酶切、补平并去磷酸化处理的加强型绿色荧光蛋白(GFP)表达盒的p Ad Track-CMV腺病毒穿梭载体,构建p Ad Track-CMV-FⅦ,继而进行酶切和测序鉴定;经PmeⅠ单酶切线性化后,p Ad Track-CMV-FⅦ在BJ5183感受态菌内与p Ad Easy-1腺病毒骨架发生同源重组,构建重组表达FⅦ的腺病毒质粒p Ad-FⅦ;经PacⅠ酶切后的p AdFⅦ,使用PEI试剂转染293A细胞,包装表达FⅦ的重组腺病毒Ad-FⅦ;利用Western印迹检测重组腺病毒Ad-FⅦ介导293A细胞表达FⅦ的蛋白水平;通过观察EGFP的表达来判断质粒转染及病毒感染细胞的效率。结果经酶切和测序鉴定,FⅦ克隆到p Ad Track-CMV腺病毒穿梭载体中,获得重组质粒p Ad Track-CMV-FⅦ;将其与p Ad Easy-1在细菌内发生同源重组,成功获得FⅦ重组腺病毒质粒p Ad-FⅦ;在293A细胞内包装重组腺病毒,Western印迹检测到FⅦ蛋白的表达。结论成功构建重组腺病毒Ad-FⅦ,并实现了rh FⅦ在哺乳动物细胞中的表达,为利用哺乳动物细胞表达rh FⅦ的研究奠定了基础。
Objective To construct a recombinant adenovirus expressing human coagulation factor Ⅶ （FⅫ）. Methods The FⅫ fragment obtained by double restriction enzymes （BamH I and EcoR V ） digested from the vector pcDNA3.1-FⅫ and blunted by T4 DNA polymerase was cloned into the shuttle vector pAdTrack-CMV a cassette that expresses enhanced green fluorescence protein（EGFP）. The resuhant recombinant plasmid pAdTrack-CMV-FⅫ was identified by restriction en- zyme digestion and DNA sequencing. Then, the plasmid was linearized by digesting with restriction endonuclease Pme I and subsequently transformed into competent BJ5183 cells together with the adenoviral backbone plasmid pAdEasy-1. Recombinants were selected for kanamycin resistance and recombination was confirmed by restriction endonuclease analy- sis. In order to obtain the recombinant adenovirus Ad-FⅦ, the confirmed recombinant adenovirus plasmid pAd-FⅦ was digested with Pac I to liberate both inverted terminal repeats （ITRs） and transfected into 293A cells. The level of protein FⅦ expression in 293A cells infected with the recombinant adenovirus Ad-FⅦ was detected by Western blot. Transfection efficiency and infection efficiency were determined by the observation of EGFP expression in 293A cells. Results The FⅦ recombinant adenovirus was packaged successfully, and the level of protein FⅦ expression in 293A cells infected by the recombinant adenovirus Ad-FⅦ was high. Conclusion The high expression of FⅦ mediated by recombinant adenovirus will contribute to the production of FⅦ with high biological activity in mammalian cells.
出处
《军事医学》
CAS
CSCD
北大核心
2015年第3期203-205,共3页
Military Medical Sciences
基金
国家863计划资助项目(2012AA021902)
病原微生物生物安全国家重点实验室资助项目(SKLPBS1103)
广东省教育部产学研结合资助项目(2011A090200071)
关键词
人凝血因子Ⅶ
重组腺病毒载体
哺乳动物细胞
蛋白表达
human coagulation factor Ⅶ
adenovirus vector
mammalian cells line
protein expression
