兰州大学机构库 >生命科学学院
补体C3及其裂解片段C3b的电镜结构研究和重组Pra1表达载体构建
Alternative TitleStructural analysis of C3 and C3b by NS electron microscopy and the vector construction of recombinant Pra1
张学斌
Thesis Advisor朱莉
2018-05-11
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name学士
Keyword补体C3 补体C3b 蛋白纯化 负染电镜 单颗粒分析 Pra1
Abstract

补体系统是天然免疫至关重要的组成部分,补体C3是补体系统的中枢,三条补体活化途径均涉及补体C3的裂解,C3b是补体C3主要的裂解片段之一,参与下游广泛的免疫反应。本文利用离子交换层析、SDS-PAGE电泳、Western Blot等技术纯化鉴定补体C3,探索出一套完整成熟的从血浆中分离纯化补体C3的实验方案。我们利用负染电镜、电镜单颗粒分析等技术解析补体C3及C3b的空间结构,利用Eman2、Relion、Scipion等软件分析电镜数据,计算出3类补体C3三维结构模型,但仅有一种正确;分析补体C3b的二维图像,与晶体结构二维投影比较发现溶液中有较多的二聚体。本文的研究结果对补体C3、C3b及其相关蛋白复合体的结构机制研究具有重要意义。

近年来发现白色念珠菌蛋白Pra1可以结合补体C3及其裂解片段C3b,是白色念珠菌感染破坏人体免疫系统的重要途径。本文构建了pPIC9k-pra1重组表达载体,利用毕赤酵母表达系统在体外表达Pra1蛋白,以pPIC9k载体上的α分泌因子作为信号肽,插入6×His标签方便下游的筛选。本文的工作为今后研究补体C3-Pra1、C3b-Pra1复合体结构以及Pra1对补体C3、C3b的作用机制奠定实验基础,为治疗白色念珠菌感染提供新的思路。

Other Abstract

Complement C3 is the pivotal component of the complement system which is important for innate and adaptive immunity. Complement is activated via three pathways, forming C3 convertases that cleave C3 and generate the activation fragment C3b, which participates in a wide range of downstream immune responses. In this study, complement C3 was purified and identified using ion exchange chromatography, SDS-PAGE, Western Blot techniques. A complete and mature experimental protocol for the isolation and purification of complement C3 from plasma was explored. The spatial structure of complement C3 and C3b was revealed under negative staining electron microscopy and single particle analysis. We used Eman2, Relion, Scipion, and other software to analyze electron microscopy data and calculated three types of complement C3 3D structural models, but only one was correct. We found that there are more dimers in the solution comparing the 2D images of C3b with the 2D projection of the crystal structure. This will lay the foundation for the structural and functional mechanism of some protein complex consist of C3, C3b and related proteins.

In recent years, Candida albicans protein Pra1 has been found to bind complement C3 and its cleavage fragment C3b, which is an important way for Candida albicans infection to destroy the human immune system. In this paper, the pPIC9k-pra1 recombinant expression vector was constructed and Pra1 protein was expressed in vitro using Pichia pastoris expression system. The α-secretory factor on the pPIC9k vector was used as the signal peptide. The 6×His tag was inserted to facilitate the downstream screening. It laid an experimental foundation for further study on the structure of C3-Pra1, C3b-Pra1 complex and the mechanism of their interaction, provide new target for the treatment of Candida albicans infection.

URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221726
Collection生命科学学院
Recommended Citation
GB/T 7714
张学斌. 补体C3及其裂解片段C3b的电镜结构研究和重组Pra1表达载体构建[D]. 兰州. 兰州大学,2018.
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