人血管紧张素Ⅱ2型受体基因慢病毒表达载体的构建

Construction of a lentiviral vector expressing human angiotensin Ⅱ type 2 receptor gene

目的构建携带人血管紧张素Ⅱ2型受体(angiotensinⅡtype 2 receptor,AGTR2)基因的慢病毒表达载体。方法运用基因重组技术,将AGTR2基因克隆至慢病毒载体pLVX-IRES-ZsGreen1中,获得慢病毒表达载体pLVX-AGTR2-IRES-ZsGreen1,XhoI、BamHI双酶切反应及测序分析加以鉴定。在Lipofectamine 2000的介导下将慢病毒表达载体质粒和包装质粒(pHelper1.0、pHelper2.0)共转染人胚肾细胞系(293T),包装慢病毒载体并测定滴度。反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)法检测AGTR2基因在感染病毒的293T细胞中的表达水平。结果所获AGTR2基因测序证明与GenBank中序列一致;成功构建慢病毒表达载体pLVX-AGTR2-IRES-ZsGreen1并获得相应的慢病毒。收集、浓缩病毒后测定其滴度为1.2×108 TU/mL。构建的携带AGTR2基因的慢病毒表达载体可有效转染293T细胞,RT-PCR结果表明AGTR2基因呈高表达。结论成功构建AGTR2慢病毒表达载体,为进一步研究其功能建立了实验基础。

Objectives To construct a lentiviral vector expressing human angiotensin Ⅱ type 2 receptor （A GTR2） gene.Methods Recombinant technology was employed to clone AGTR2 gene to lentiviral vector pLVX-IRES-ZsGreen1 to construct lentiviral expression vector pLVX-A GTR2-IRES-ZsGreen1.XhoI and BamHI enzyme digestion and sequencing analysis were used for identification.Lentiviral expression vector plasmid and lentiviral packaging materials （pHelper1.0,pHelper2.0） were cotransfected into human embryonic kidney 293T cells by Lipofectamine 2000 to produce lentiviral particles.Virus titer was measured.Expression of AGTR2 was detected in 293T cells infected with lentivirus using reverse transcription polymerase chain reaction （RT-PCR）.Results Sequencing analysis confirmed that the cloned A GTR2 gene was coincident with the sequence registered in GenBank.The lentiviral vector pLVX-AGTR2-IRES-ZsGreen1 expressing A GTR2 gene was successfully established.The titer of the lentiviral vector particles was 1.2×10s TU/mL.RT-PCR demonstrated that the expression level of A GTR2 in the lentivirus-infected 293T cells increased significantly.Conclusions A GTR2 expression vector has been successfully constructed,which provides a foundation to explore the functions of A GTR2.