摘要
目的构建携带Math1基因的重组慢病毒载体,检测其滴度,检测其在293T细胞中的表达。方法 PCR扩增Math1基因,将其连入慢病毒载体pLenti-GFP中;在感受态细胞DH5α中培养扩增,并行Math1基因的测序鉴定;将重组的慢病毒四质粒共转染293T细胞,收获并浓缩病毒;感染293T细胞和提取细胞DNA后用实时定量PCR法检测病毒滴度。用逆转录PCR和Western blot法检测Math1基因在感染病毒的293T细胞中的表达。结果构建的慢病毒载体pLenti-Math1-GFP经测序分析证实基因序列正确。四质粒共转染293T细胞后,荧光显微镜下可见大量绿色荧光。包装后慢病毒测定滴度约为3×1011Tu/L。逆转录PCR和Western blot法均能检测Math1基因在感染病毒的293T细胞中的表达。结论成功构建携带Math1基因的重组慢病毒,并能在293T细胞中表达。
【Objective】 To construct a recombinant lentivirus vector with Math1,check lenriviral titer and detect its expression in 293T cells.【Methods】 Math1 amplified by PCR was inserted into pLenti-GFP vector;pLenti-Math1-GFP was cultured and amplified in the competence DH5α cells and Math1 gene sequence was identified.The 293T cells was transfected with recombinant Lentivirus 4 plasmid.The Lentivirus was harvested and condensed,then was used to infect 293T cells.The lentiviral titer was detected by realtime PCR menthod.The expression of Math1 was detected in 293T cells infected with lentivirus using reverse transcription PCR and Western blot.【Results】 The sequence of Math1 constructed into lentivirus vector was right.A lot of green fluorescence could be seen using fluorescence microscope after 293T cells was transfected with 4 lentivirus plasmid.The lentivirus titer was about 3×1011 Tu/L.The expression of Math1 could be detected in 293T cells infected with lentivirus using reverse transcription PCR and Western blot.【Conclusions】 Recombinant lentivirus vector with Math1 was constructed successfully and could be detected in 293T cells.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2011年第4期412-417,共6页
China Journal of Modern Medicine
基金
国家自然科学基金课题(No:81070782)
浙江省自然科学基金课题(No:Y2080334)
浙江省医药卫生科学研究基金课题(No:2008A056)
