通用辅助T细胞表位转化生长因子融合蛋白的原核表达

通用辅助T细胞表位（PADRE）- 转化生长因子β1（TGFβ1）融合蛋白的原核表达及Western-blotting(9000字)

摘  要：TGFβ1为一种强烈的免疫抑制因子，同时还可以诱导调节性T细胞的生成而介导免疫耐受，其在家畜的持续性及隐性感染中发挥重要的作用，本实验拟应用基因工程技术制备PADRE-TGFβ1融合蛋白，作为TGFβ1自体疫苗，来诱导机体产生抗TGFβ1抗体，探讨抑制TGFβ1是否有利于机体免疫系统清除持续性感染的病原体。方法：采用实验室构建的pET28-PADRE- TGFβ1表达质粒转染E.coli BL21，测序证实序列无误后，进行原核表达，表达产物以SDS-PAGE、western-blotting等方法鉴定。结果：SDS-PAGE证实原核表达产物相对分子质量为20kD，western-blotting检测证实TGFβ1的抗原表位可正确暴露。结论:本研究成功构建并表达了PADRE-TGFβ1融合蛋白质，并初步证实了表达产物具有TGFβ1的抗原性，为下一步进行TGFβ1自体疫苗的制备打下了基础。
关键词：转化生长因子β1；通用辅助T细胞表位；融合蛋白；原核表达

Prokaryotic expression and Werstern-Blotting of PADRE - TGFβ1 Fusion Protein

Abstract：As a strong immune inhibitory factor, TGFβ1 can induce the generation of regulatory T cells which mediate immune tolerance. It play an important role in continuity and latent infection of livestock. In this study, PADRE-TGFβ1 fusion protein was prepared, as TGFβ1 autologous vaccine to induce antibodies against TGFβ1, TGFβ1 inhibition of the immune system is beneficial to clear the persistent infection of pathogens. Methods: The use of laboratory constructed pET28-PADRE-TGFβ1 expression plasmid transfected into E.coli BL21, correct sequence was confirmed after the prokaryotic expression product by SDS-PAGE, western-blotting. Results: SDS-PAGE confirmed that the prokaryotic expression products relative molecular mass of 20 kD, western-blotting detection confirmed TGFβ1 epitopes can be correctly exposed. Conclusion: The successfully constructed and expressed PADRE-TGFβ1 fusion protein, Preliminary expression products confirmed that the fusion protein have the antigenicity of TGFβ1. It lay the foundation for TGFβ1 for the next step on the preparation of autologous vaccine foundation.
Key words : transforming growth factor β1; Pan DR epitope; fusion protein; prokaryotic expression
 

目    录    
□□摘要……………………………………………………………………………1
□□关键词……………………………………………………………………………1
□□1前言……………………………………………………………………………2
1.1     TGFβ1的分子结构…………………………………………………………2
1.2     TGFβ1的功能研究进展……………………………………………3
1.3     PADRE的研究进展…………………………………………………4
1.4 目的意义…………………………………………………………………5
□□2材料与方法……………………………………………………………………6
2.1  试剂和仪器……………………………………………………………6
2.2  试剂的配制……………………………………………………………6
    2.1.1  LB培养基制……………………………………………………………7
    2.2.2 卡那霉素(硫酸盐) 配制………………………………………………7
    2.2.3  1×Tris甘氨酸电泳缓冲液……………………………………………7
2.3 方法…………………………………………………………………………8
2.2.1 重组质粒的诱导表达………………………………………………8
2.2.2 SDS-PAGE电泳检测方法………………………………………………9
2.2.3 凝胶的染色……………………………………………………………10
2.2.4 电转移步骤………………………………………………………10
2.2.5  Western Blotting 操作………………………………………………10
3结论 …………………………………………………………………………11
3.1 大肠杆菌的talin1．GST蛋白表达及纯化结果…………………………11
3.2 Werstern-Blotting 检测结果…………………………………………12
4讨论………………………………………………………………………12
参考文献  ……………………………………………………………………13