摘要
目的:构建鼠IFN-λ2重组腺病毒载体。方法:用人水疱口炎病毒诱导小鼠原代脾细胞表达IFN-λ2,通过RT-PCR获取IFN-λ2 cDNA,将其亚克隆至pShuttle-CMV载体,经PmeⅠ线性化后,与腺病毒的骨架载体pAdEasy在BJ5183菌中同源重组,转染293A细胞包装扩增,RT-PCR检测重组腺病毒的目的基因,TCID50法对构建的腺病毒进行滴度测定。用鼠IFN-λ2重组腺病毒感染小鼠肺腺癌细胞,蛋白质印迹法检测细胞中IFN-λ2蛋白的表达,倒置显微镜观察鼠IFN-λ2重组腺病毒对鼠肺腺癌细胞生长的抑制作用。结果:成功克隆鼠IFN-λ2的cDNA,序列与基因库公布序列完全一致;经酶切鉴定表明成功构建鼠IFN-λ2重组腺病毒载体;重组腺病毒载体在293A细胞中成功包装及扩增;包装成功的重组腺病毒中含有目的基因,病毒滴度为2×1010pfu/ml;蛋白质印迹显示感染细胞的上清中有鼠IFN-λ2蛋白的表达,倒置显微镜显示感染的鼠肺腺癌细胞生长受到抑制。结论:成功构建了鼠IFN-λ2重组腺病毒载体,并能介导鼠IFN-λ2蛋白的表达进而抑制鼠肺腺癌细胞的生长,为进一步开展抗肿瘤研究及基因治疗奠定基础。
Objective： To construct recombinant adenovirus vector containing mouse IFN-λ2 gene. Methods： Full-length of mIFN-λ2 cDNA was obtained by using RT-PCR from ceils of mouse spleen stimu- lated by vesicular stomatovirus and then subcloned to the vector of pShuttle- CMV. Generation of recombi- nants by cotransforming the Pme I -cut shuttle plasmids with pAdEasy-1 backbone vectors in BJ5183 ,virus was amplified in 293 cells. RT-PCR was used to identify mIFN-λ2 gene had transformed into recombinant adenovirus, and the method of TCID50 was used to measure the virus titer. The mouse lung adenocarcinoma cells were infected by the recombinant adenovirus, the expression of mIFN-λ2 was detected by Western bolt, and the inhibitory effect of mIFN-λ2 to mouse lung adenocarcinoma cells was observed by inverted micro- scope. Results： The sequence of mlFN-λ2 gene was identical with that reported in GenBank. The recombi- nant adenovirus vector was successfully constructed and verified by restriction endonucleases digestion. Re- combinant adenovirus was successfully produced and amplified in 293A cell line. The constructed recombi- nant adenovirus was verified containing mouse IFN-λ2 gene, and its virus titer was 2 × 10^10pfu/ml. Western bolt indicated mIFN-λ2 protein was expressed in the supematant of mouse lung adenocarcinoma cells infec- ted by the recombinant adenovirus. The inhibitory effect of mIFN-λ2 to mouse lung adenocarcinoma cells was identified by inverted microscope. Conclusion： The adenovirus vector of mIFN-λ2 was successfully con- structed. The mlFN-λ2 protein was expressed on mouse lung adenocarcinoma cells infected by the recombi- nant adenovirus and the growth of mouse lung adenocarcinoma ceils was inhibited. This result lays not only a solid foundation for a further study of the impact of the IFN-λ, on tumor but also for gene therapy.
出处
《江苏大学学报（医学版）》
CAS
2012年第2期120-124,共5页
Journal of Jiangsu University：Medicine Edition
基金
镇江市社会发展基金资助项目(SH2010036)
关键词
鼠
IFN-λ2
腺病毒载体
构建
鉴定
mouse
IFN-λ2
adenvirus vector
construction
indentification
