The roles of the single-chain variable fragment specific for human acidic fibroblast growth factor

摘要

人酸性成纤维细胞生长因子（FGF-1或aFGF）是一类具有肝素结合区域的生长因子家族的一员。这些生长因子具有广泛的生物学作用，在肿瘤发生和迁移中起着重要的作用。提示aFGF有可能作为肿瘤治疗的一个靶标。为了探索aFGF与肿瘤的关系，我们构建了特异性抗aFGF的单链抗体（scFv）。用这个抗体有可能阻断aFGF的作用而达到抑制肿瘤生长的目的。 为了研究scFv的功能，将scFv基因插入哺乳动物细胞表达载体pEGFP—C1，并转染到细胞中，通过免疫荧光证明scFv可以结合细胞内源性的aFGF，并且scFv的过表达可以使细胞变小变圆。进一步通过流式细胞术证明scFv的过表达可以使293T和MCF7细胞阻断在G1期。这些结果表明scFv具有抑制细胞生长的作用。进一步我们通过ECM830体内电转仪将scFv基因导入肿瘤组织，结果表明scFv在肿瘤组织的表达可以抑制实体瘤的生长。 然而，aFGF非肿瘤特异性抗原，抗aFGF—scFv作为药物应用时可能会“敌我不分”，另一方面，scFv本身主动进细胞的能力较弱。基于肿瘤细胞表面高表达hEGF受体的特点和绿脓杆菌外毒素A的Ⅱ区（PEⅡ）主动跨细胞膜的特点，对scFv基因进行了修饰，通过Overlap PCR技术在单链抗体基因的N端融合了hEGFfr和PEⅡ基因序列，将重组后的hEGFfr—PEⅡ—scFv克隆到原核表达载体pET-28a（+），并将重组质粒pET28a—hEGFfr—PEⅡ—scFv转化入大肠杆菌BL21（DE3），在1 mM IPTG条件下成功诱导出LHRH—PEⅡ—scFv蛋白，分子量约为44 kDa。通过Ni—柱亲和层析得到了纯化的hEGFfr—PEⅡ—scFv蛋白，但是其活性较低。为了得到具有较高生物学活性的蛋白，我们将hEGFfr—PEⅡ—scFv克隆到酵母表达载体pPIC9K并转化到酵母菌GS115，通过G418筛选得到稳定整合hEGFfr—PEⅡ—scFv基因的菌株，下一步的表达工作正在进行当中。这些工作为开发抗aFGF—scFv作为一种抗肿瘤药物奠定了一定的基础。

目录

文摘

英文文摘

论文说明：Abbreviations

声明

1 Introduction

1.1 Monoclonal antibodies for therapy

1.2 The structure of the single-chain variable fragment

1.3 scFv involved in tumor therapy research progress

1.4 Intrabodies and intracellular delivery of antibodies

1.5 Gene therapy and its applications

1.6 The roles of human acidic fibroblast growth factor(aFGF)

1.7 Pseudomonas exotoxin A domain Ⅱ(PEII)

1.8 Human epidermal growth factor receptor binding fragment(hEGFr)

1.9 Purpose and significance of this study

2 Materials and Methods

2.1 Cell lines，bacterial strains，yeast strain and vectors

2.2 Reagents

2.3 Flow cytometry

2.4 Animal model

2.4.1 Eleetroporation

2.4.2 Histological analysis

2.5 Expression of fusion protein into E.coli

2.5.1 PCR amplification of hEGFfr-PEll-scFv fusion gene

2.5.2 Double enzyme digestion

2.5.3 Ligation reaction

2.5.4 Transformation

2.5.5 Expression and analysis of recombinant hEGFfr-PEII-scFv protein

2.5.6 Purification of His-tagged fusion protein by Ni-NTA affinity chromatography

2.5.7 Refolding and dialysis

2.5.8 SDS-PlAGE and Western blot analysis

2.5.9 Antigen binding activity of the hEGFfr-PEII-scFV protein detected by ELISA assay

2.6 Expression of fusion protein into Pichia Pastoris

2.6.1 PCR amplification of His·Tag-hEGFfr-PEII-scFv fusion gene

2.6.2 Double enzyme digestion

2.6.3 Ligation reaction

2.6.4 Transformation

2.6.5 Preparation of transforming DNA

2.6.6 Electroporation of P.pastoris

2.6.7 In Vivo screening of multiple inserts

2.6.8 PCR analysis of P.pastoris integrants

2.6.9 Expression of recombinant Pichia strains

2.6.10 Analysis by SDS-Polyacrylamide Gel Electrophoresis and Western Blot

3 Results and Analysis

3.1 scFv expression led to cell cycle G1 phase arrest

3.2 Electroporation of scFv into solid tumor

3.3 Construction of hEGFfr-PEII-scFv for E.coli expression

3.4 Expression of hEGFfr-PEII-scFv in E.coli BL21(DE3)

3.5 Antigen binding activity of the hEGFfr-PEII-scFv

3.6 Construction of hEGFfr-PEAII-scFv for yeast expression

3.7 Screen for Geneticin resistant transfotruants

3.8 Recombination and integration in P.pastoris

4 Discussion

5 Conclusions

References

Acknowledgments

Publication