抗CD40单链抗体的慢病毒载体的构建和表达
Construction and expression of a single chain antibody variable fragment of agonist anti-CD40 antibody lentiviral vector
文丽君 1赵李祥 2王征 1胡博 1赵昀 3刘海燕2
作者信息
- 1. 苏州大学医学部生物医学研究院,江苏苏州215123
- 2. 苏州大学医学部生物医学研究院,江苏苏州215123;苏州大学附属第一医院江苏省血液研究中心,江苏苏州215006
- 3. 苏州大学附属第一医院江苏省血液研究中心,江苏苏州215006
- 折叠
摘要
目的 克隆大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv),并构建可表达大鼠抗小鼠的抗CD40抗体的单链抗体(CD40-scFv)的重组慢病毒.方法 采用RT-PCR法从分泌大鼠抗小鼠抗CD40激活型抗体的杂交瘤细胞株(FGK-115)中克隆VH和VL基因,用重叠延伸PCR法将VH和VL拼接在一起,构建抗CD40抗体的scFv基因,将CD40-scFv基因连接至PEGM-T克隆载体,限制性内切酶酶切及测序鉴定；构建含有大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv)的慢病毒穿梭质粒,并与其它包装质粒一起通过磷酸钙法感染293T细胞；获得病毒颗粒并感染人慢性髓系白血病细胞MEG-01,检测其感染效率.结果 含大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv)的慢病毒穿梭质粒构建成功；通过磷酸钙感染293T细胞所得病毒上清可成功感染MEG-01细胞,流式细胞术检测阳性率可达96％.结论 成功克隆了大鼠抗小鼠的抗CD40激活型抗体的单链抗体(CD40-scFv)基因,并建立其重组慢病毒表达系统,为后续的实验及应用研究工作奠定了基础.
Abstract
Objective The purpose of this research was to construct a lentivira vector containing scFv of agonist anti-CD40 antibody gene, and detect the expression of scFv of agonist anti-CD40 antibody gene. Methods The VH and VL genes were cloned by RT-PCR from a rat anti-mouse hybridoma cell line FGK-115, which produced the agonist anti-CD40 monoclanal antibody. Splicing of overlapping extension PCR(SOE-PCR) was used to splice the VH and VL genes to construct CD40-scFv, then the CD40-scFv gene was connected to the PEGM-T vector, which was identified by the restriction enzyme digestion and sequencing; CEMO-scFv gene was subcloned into the transfer plamid of the lentivirus system, which was tansfected together with the packaging plasmids into 293T cells to obtain virus particles. The virus particles was used to infect MEC-01 cells, a kind of human chronic myeloid leukemia cells, then flow cyometric analysis was used to test the positive rate of infection. Results Lentivirus vector of CD40-scFv was constructed successfully. MEG-01 cells could be successfully infected by the recombinant lentivirus. The positive rate of infection was up to 96%. Conclusion The lentiviral vector of CD40-scFv was constructed successfully which would contribute to study the important role of CD40 in the follow-up experiments and immunotherapy research.
关键词
CD40/scFv/慢病毒载体Key words
CD40/scFv/lentivira vector引用本文复制引用
基金项目
教育部科学技术研究重点项目(208048)
出版年
2012
NETL
NSTL
维普
万方数据