抗肺癌单链抗体偶联绿色荧光蛋白基因的构建及表达研究

【作者】 徐飞虎；

【导师】 龚兴国；

【作者基本信息】 浙江大学 ， 生物物理学， 2003， 硕士

【摘要】 绿色荧光蛋白作为一种新型的标记分子，可应用于活体细胞的实时检测。目前已有相当多的文献报道关于将GFP基因与特定抗体或细胞因子基因结合进行融合表达的研究，证明融合蛋白具有GFP的荧光性质和配体蛋白质的生物功能，可用于对肿瘤的检测、药物筛选、细胞因子受体的分布以及功能等方面的研究，应用前景广阔。 目前国内关于将绿色荧光蛋白与其他细胞因子融合表达的报道较少。相关的报道有抗肝癌单链抗体融合GFP在真核细胞中的表达，以及重组IL-18-GFP融合蛋白活性研究等。本论文拟将抗肺癌单链抗体连接绿色荧光蛋白进行融合表达，预期目标蛋白应同时具有抗体活性及光激发绿色荧光双重功能。 本实验经PCR方法从mRNA逆转录产物中克隆到抗肺癌抗体的重链可变区和轻链可变区，并利用重叠延伸PCR方法将其构建为单链抗体scfv形式。利用酶切连接将scfv基因序列插入到绿色荧光蛋白基因3’-端构成gfp-scfv重组基因，并转化大肠杆菌表达。经IPTG诱导表达后并SDS-PAGE蛋白电泳，与阴性菌对照，在预期分子量范围内出现一明显条带。利用Ni-NTA树脂纯化蛋白，出现两个明显的峰。SDS-PAGE电泳分析表明，其中一个峰的蛋白条带符合预期蛋白分子量。396nm激发显示绿色荧光，证实目的基因在宿主中得到表达。不过，表达出的蛋白是否具有抗体活性还需要进一步的分析研究。更多还原

【Abstract】 Use of the green fluorescent protein(GFP) of Aequorea victoria as a reporter has provided sensitive new approaches for real time monitoring in living cells. More and more reports have demonstrated that when GFP fused to target antibody, the host cell can express bifunctional fusion protein, which has the fluorescent character of GFP and the antibody activity. As an alternative to immunofluorescence microscopy, the expression of GFP gene fusions has been used for tumor monitoring, high-throughput screening drugs or researching the function of the acceptor of certain cell factor.In this paper, a expression vector that contains GFP gene and anti-lung cancer ScFv gene is construct. First, the variable region of heavy chain and light chain is cloned from cDNAs through PCR. Then, VL gene is amplified and modified with linker primer, Equal mole of VH and modified VL are then subjected to SOE ligation and ScFv gene is amplified with Vhbak and Vlfor. Sequencing results indicate that no point mutation and frame-shifting mutation are brought about during PCR amplification. The GFP gene is cloned into pPROEX HTb plasmid between the Nco I/Xba I sites. After modified by PCR using sfvl and vlfor primers so that the amplified fragment contains Bgl II /Xho I sites, the PCR product and the recombinant vector are digested with Bgl II +Xho I , the modified scfv gene is inserted into the C-terminal of GFP gene between Bgl II /Xho I sites, then transforming the recombinant vector into E.coli.Compared to the negative bacterial, the constructed plasmid gave a high level of expression similar to the anticipated protein after IPTG induction. It appears two sharp peaks through the purification of Ni-NTA resin, the analysis of SDS-PAGE shows that one of the molecule weight of the fusion protein is similar to the target protein. The host cells fluoresce bright green under 396nm wavelenth, it means the gene has expressed in the cells, but whether the target protein has the biological function needs further research.更多还原