抗TfR-scFv-EGFP融合蛋白的构建，表达及特异性研究

发布时间：2018-09-18 13:13

【摘要】： 目的构建含抗转铁蛋白受体单链抗体(TfR-scFv)和EGFP融合蛋白基因的重组真核表达载体pTfRscfv-EGFP。并转染至COS-7细胞中进行表达,鉴定融合蛋白的生物学活性。为单链抗体-荧光融合蛋白在肿瘤示踪、定位、显像等方面的研究奠定基础。方法以Chi7579-pγ-Expr和Chi7579-pκ-Expr载体为模板,设计两对引物引入中间连接肽(Gly4Ser)3以及酶切位点HindⅢ和BamHⅠ,采用重叠延伸PCR的方法,分别扩增抗TfR单链抗体的轻、重链可变区(VL、VH)基因,二基因之间以连接肽(Gly4Ser)3的编码序列连接,从而构建具有前导肽的L-VH-Linker-VL单链抗体基因。将获得的单链抗体基因克隆至pGEM-T载体(scFv-T),转化大肠杆菌DH5α,进行蓝白筛选,挑取阳性克隆扩增。提取质粒以HindⅢ和BamHⅠ双酶切鉴定,并测序证实正确后,亚克隆至pEGFP-N1,获得pTfRscfv-EGFP,转染COS-7细胞,倒置相差荧光显微镜下观察绿色荧光蛋白表达,荧光分光光度计检测细胞分泌上清荧光强度,并通过流式细胞术(FCM)检测细胞分泌上清与乳腺癌细胞MCF-7及黑色素瘤细胞A375的结合活性。结果测序结果表明获得了正确的L-VH-Linker-VL单链抗体基因序列;亚克隆至pEGFP-N1后,HindⅢ和BamHⅠ双酶切可见约800bp的小片段和约4.5Kb的大片段,与理论计算值相符,表明pTfRscfv-EGFP真核表达载体构建成功;表达载体瞬时转染COS-7细胞,24小时后,可见荧光在细胞中呈指环状分布,细胞核无表达,为典型的分泌性表达模式,而pEGFP-N1空载体转染细胞中,荧光则呈弥漫性分布,以核中分布较多。收集转染后72h的细胞培养上清,用F4500荧光分光光度计检测其荧光强度,实验组细胞上清的荧光强度较对照组高出约两倍以上,证实抗TfR-scFv-EGFP融合蛋白在转染细胞中分泌性表达;FCM检测转染细胞培养上清与A375细胞及MCF-7细胞的结合,结果表明抗TfR-scFv-EGFP融合蛋白能够与上述天然高表达TfR的细胞特异性的结合,对照组细胞培养上清则未见与上述细胞有结合。结论成功构建抗TfR-scFv-EGFP融合蛋白的真核表达载体pTfRscfv-EGFP,融合蛋白可在COS-7细胞中分泌性表达,且具有与TfR阳性细胞结合的活性并发绿色荧光,本实验为进一步将该融合蛋白作为肿瘤示踪、定位、显像的特异性分子奠定了基础。
[Abstract]:Objective To construct a recombinant eukaryotic expression vector pTfRscfv-EGFP containing anti-transferrin receptor single-chain antibody (TfR-scFv) and EGFP fusion protein gene and transfect it into COS-7 cells to identify the biological activity of the fusion protein.
Methods Using Chi7579-pgamma-Expr and Chi7579-pkappa-Expr vectors as templates, two pairs of primers were designed to introduce intermediate ligand 3 (Gly4Ser) and the digested sites Hind III and BamH I. The light and heavy chain variable region (VL, VH) genes of anti-TfR single chain antibodies were amplified by overlapping extension PCR, and the two genes were linked by the coding sequence of the ligand peptide (Gly4Ser) 3. Then, the L-VH-Linker-VL single chain antibody gene with a propeptide was constructed. The obtained single chain antibody gene was cloned into pGEM-T vector (scFv-T), transformed into E.coli DH5a, screened with blue and white, and amplified with positive clones. Scfv-EGFP was transfected into COS-7 cells. The expression of green fluorescent protein was observed under inverted phase contrast fluorescence microscope. The fluorescence intensity of supernatant secreted by cells was measured by fluorescence spectrophotometer. The binding activity of supernatant secreted by cells to MCF-7 cells and A375 cells was detected by flow cytometry (FCM).
Results Sequencing results showed that the correct sequence of L-VH-Linker-VL single chain antibody gene was obtained. After subcloning to pEGFP-N1, a small fragment of about 800 BP and a large fragment of about 4.5 Kb could be seen in Hind III and BamH I digestion, which were in agreement with the theoretical value. The results showed that the eukaryotic expression vector of pTfRscfv-EGFP was successfully constructed. After transfection, the fluorescence showed a circular distribution in the cell nucleus and no expression in the cell nucleus, which was a typical secretory expression pattern. In pEGFP-N1 empty vector transfected cells, the fluorescence was diffusely distributed and distributed more in the nucleus. The fluorescence intensity of antiTfR-scFv-EGFP fusion protein was more than twice as high as that of the control group, which confirmed the secretory expression of antiTfR-scFv-EGFP fusion protein in the transfected cells. The binding of the supernatant of the transfected cells to A375 cells and MCF-7 cells was detected by FCM. The results showed that the antiTfR-scFv-EGFP fusion protein could specifically bind to the above natural TfR-expressing cells. Cell culture supernatant did not bind to the above cells.
Conclusion The eukaryotic expression vector pTfRscfv-EGFP against TfR-scFv-EGFP fusion protein has been successfully constructed. The fusion protein can be secreted in COS-7 cells, and has the activity of binding to TfR-positive cells with green fluorescence. This study laid a foundation for further using the fusion protein as a specific molecule for tumor tracing, localization and imaging.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R73-3

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