带有小鼠CD40L基因的汉滩病毒糖蛋白表达载体的构建
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摘要
目的:构建汉滩病毒包膜糖蛋白基因的真核表达载体,并加入可增强免疫应答效应的细胞因子CD40L基因,检测其可否在真核细胞中表达。方法与结果:参照GenBank中汉滩病毒M基因和小鼠CD40L的全基因序列设计引物,通过聚合酶链反应(PCR)获得M和CD40L基因片段,将其与pCI-neo载体相连,测序证实该载体构建成功后,将此真核表达载体以脂质体转染法转染至哺乳动物细胞CHO-K1中,利用间接免疫荧光法(IFA)检测发现M基因和CD40L基因可以同时表达于CHO-K1细胞中。结论:构建了带有CD40L基因的汉滩病毒包膜糖蛋白重组质粒并获得表达,为深入研究汉滩病毒感染后包膜糖蛋白引起的特异性免疫应答规律奠定了实验基础。
Objective: The eukaryotic expression vector of Hantaan virus glycoprotein was constructed and added with mouse CD40L gene that could strengthen mouse immune response, in order to detect whether or not it could express in the eukaryotic cell. Methods & Results: Primers were designed referring to M gene sequence of Hantaan virus and mouse CD40L gene in the Gen Bank.Then M gene and mouse CD40L gene were obtained through PCR, and cloned into eukaryotic expressing vector pCI-neo vector. After sequenced, the eukaryotic expressing vector was transfected into eukaryotic cells CHO-K1 with liposome method.It was found that specific fluorescence appeared in the CHO-K1 after transfection by indirect immunofluorescence assay(IFA). Conclusion: The results showed that the eukaryotic expressing vector of Hantaan virus glycoprotein conjugated with mouse CD40L was successfully constructed and expressed, to deeply investigate the immune response of Hantaan virus glycoprotein.
Objective: The eukaryotic expression vector of Hantaan virus glycoprotein was constructed and added with mouse CD40L gene that could strengthen mouse immune response, in order to detect whether or not it could express in the eukaryotic cell. Methods & Results: Primers were designed referring to M gene sequence of Hantaan virus and mouse CD40L gene in the Gen Bank.Then M gene and mouse CD40L gene were obtained through PCR, and cloned into eukaryotic expressing vector pCI-neo vector. After sequenced, the eukaryotic expressing vector was transfected into eukaryotic cells CHO-K1 with liposome method.It was found that specific fluorescence appeared in the CHO-K1 after transfection by indirect immunofluorescence assay(IFA). Conclusion: The results showed that the eukaryotic expressing vector of Hantaan virus glycoprotein conjugated with mouse CD40L was successfully constructed and expressed, to deeply investigate the immune response of Hantaan virus glycoprotein.
引文
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[3]Barr T A,Carlring J,Heath A W.Co-stimulatory agonists as immunological adjuvants[J].Vaccine,2006,24(17):3399-3407.
[4]Jiang H,Du H,Wang L M,et al.Hemorrhagic fever with renal syndrome:pathogenesis and clinical picture[J].Front Cell Infect Microbiol,2016,6:1.
[5]Gupta S,Termini J M,Kanagavelu S,et al.Design of vaccine adjuvants incorporating TNF superfamily ligands and TNF superfamily molecular mimics[J].Immunol Res,2013,57(1-3):303-310.
[6]Kornbluth R S,Stone G W.Immunostimulatory combinations:designing the next generation of vaccine adjuvants[J].J Leukoc Biol,2006,80(5):1084-1102.
[2]Yoshimatsu K,Yoo Y C,Yoshida R,et al.Protective immunity of Hantaan virus nucleocapsid and envelope protein studied using baculovirus-expressed protein[J].Arch Virol,1993,130(3-4):365-376.
[3]Barr T A,Carlring J,Heath A W.Co-stimulatory agonists as immunological adjuvants[J].Vaccine,2006,24(17):3399-3407.
[4]Jiang H,Du H,Wang L M,et al.Hemorrhagic fever with renal syndrome:pathogenesis and clinical picture[J].Front Cell Infect Microbiol,2016,6:1.
[5]Gupta S,Termini J M,Kanagavelu S,et al.Design of vaccine adjuvants incorporating TNF superfamily ligands and TNF superfamily molecular mimics[J].Immunol Res,2013,57(1-3):303-310.
[6]Kornbluth R S,Stone G W.Immunostimulatory combinations:designing the next generation of vaccine adjuvants[J].J Leukoc Biol,2006,80(5):1084-1102.