| 题名 | 大蹼铃蟾孔道形成毒素类似蛋白Bm -ALP3的克隆及原核表达 |
| 作者 | 周凯峰 |
| 学位类别 | 硕士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 大蹼铃蟾 孔道形成毒素样蛋白 RACE 原核表达 |
| 其他题名 | The Molecular Cloning and Prokaryotic Expression of Pore-forming Toxin-like Protein Bm-ALP3 from Bombina Maxima |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 病原微生物感染宿主依赖其毒力因子，其中孔道形成毒素家族是最大的一类毒力因子。令人惊讶的是，大量的孔道形成毒素样蛋白在脊椎动物中广泛存在，然而它们的生物学功能未知。本实验室前期工作在大蹼铃蟾分泌物中发现了一种孔道形成毒素类似蛋白与三叶因子组成的复合物βγ-CAT，并对其功能进行了一定的探讨。前期工作还新发现了一种孔道形成毒素类似蛋白Bm-ALP3(Bombina maxima Aerolysin-like Protein 3)，其作为βγ-CAT α亚基的同源分子，也存在于大蹼铃蟾皮肤分泌物中，但其完整cDNA序列及生物学功能未知。 实验室前期工作得到Bm-ALP3的部分序列，为了对其进行后续的结构鉴定及功能分析，本实验主要进行了以下两个方面的研究：（1）利用RACE的方法克隆得到Bm-ALP3 cDNA分子全长，将Bm-ALP3序列与同源序列进行比对，并模拟了Bm-ALP3的三维结构，结果证实Bm-ALP3是一种孔道形成毒素类似蛋白。（2）原核表达Bm-ALP3融合蛋白，制备抗Bm-ALP3的抗体。首先将接近全长的Bm-ALP3部分序列与表达载体pET32a连接得到重组载体，重组载体转化到原核表达菌BL21大量表达目标融合蛋白，利用金属螯合层析分离纯化得到目标融合蛋白。然后利用得到的融合蛋白免疫兔，按照制备多克隆抗体的方法制备兔抗血清，Western-blot验证了兔抗血清中具有抗融合蛋白和天然Bm-ALP3的多克隆抗体。 本实验成功得到了Bm-ALP3 cDNA全长，并制备了抗Bm-ALP3的多克隆抗体，为后续深入研究其生物学功能及可能的作用机制奠定基础。 |
| 英文摘要 | Pathogens infect hosts by virulence factors, and the bacterial pore-forming toxin (PFT) superfamily is the largest family of bacterial toxins. Surprisingly, numerous PFT-like proteins have been found in vertebrates, but we know little about their biological functions. In earlier stage work of our laboratory, we have identified a complex called βγ-CAT which consists of a PFT-like protein subunit and two trefoil factor subunits in the Bombina maxima skin secretions, and have done a preliminary study on it. We have also identified a new PFT-like protein Bm-ALP3(Bombina maxima Aerolysin-like Protein 3), which also exists in the Bombina maxima skin secretions as a homolog of βγ-CAT α subunit，but we have not identified its complete cDNA sequence and have not studied its biological functions. In earlier stage work of our laboratory, we have got the incomplete sequence of Bm-ALP3. For doing the follow-up study on its structure identification and biological functions analysis, we have done two aspects of the research: (1) we obtained the complete cDNA sequence by using the method of RACE. We have done sequence alignment with homologs and predictied the 3D structure of Bm-ALP3. Our result shows that Bm-ALP3 is a PFT-like protein. (2) We expressed the Bm-ALP3 fusion protein in prokaryotic expression system and prepared anti-Bm-ALP3 antibody. We linked nearly full-length cDNA sequence of Bm-ALP3 with the pET-32a vector, and transformed the recombinant vector into the engineered bacteria BL21, followed by target fusion protein expression and metal-chelated affinity purification. Then we used the target protein to stimulate rabbit and prepared rabbit antiserum. We have detected the existence of anti-fusion-protein polyclona antibody and anti-Bm-ALP3 polyclona antibody in the rabbit antiserum by Western-blot. In summary, we obtained the full-length cDNA sequence of Bm-ALP3 successfully and the preparation of anti-Bm-ALP3 polyclona antibody provide a basis for further study on the function and mechanism of Bm-ALP3. |
| 语种 | 中文 |
| 公开日期 | 2014-07-01 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7922]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 周凯峰. 大蹼铃蟾孔道形成毒素类似蛋白Bm -ALP3的克隆及原核表达[D]. 北京. 中国科学院研究生院. 2014. |
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