副血链球菌粘附素Fap1糖基化相关基因-nss的功能研究

发布时间：2018-04-08 20:27

  本文选题：Fap1　切入点：粘附素　出处：《河南科技大学》2010年硕士论文

【摘要】：目的:成熟的糖蛋白Fap1是一个副血链球菌细胞表面粘附素,对人类最复杂的生物膜牙菌斑形成非常重要。Fap1糖基化由fap1基因座附近的基因簇所调控。已有研究表明位于fap1基因座下游的Gtf1和Gtf2参与Fap1第一步糖基化,可将N-乙酰葡萄糖胺(GlcNAc)转移至Fap1肽段上。但对于Fap1糖基化的后续步骤是如何进行这一问题至今还是未知。本研究针对位于fap1基因座上游的nss基因在Fap1糖基化中的功能进行了探求。 方法:(1)构建nss基因突变株及回复突变株:采用基因置换技术以野生型Streptococcus parasanguinis FW213菌株为出发菌株构建nss基因突变株;并采用基因互补技术(将nss基因克隆入大肠杆菌和副血链球菌穿梭表达载体中,并转化nss基因阻断突变株)对突变株进行基因互补;运用Western Blotting和BactELISA检测nss基因突变株、回复突变株与副血链球菌野生型菌株的表型,即Fap1蛋白的糖基化情况。 (2)在异源宿主大肠杆菌系统中鉴定与Fap1糖基化相关基因nss的功能:构建一系列含有不同拟糖基转移酶基因的重组质粒,将这些重组质粒分别与含有Fap1△RII(缺失RII重复区的Fap1蛋白)的重组质粒以及含有Gtf1和Gtf2的重组质粒共转化到大肠杆菌中,运用Western Blotting检测在不同的基因存在的情况下Fap1△RII蛋白的糖基化情况。 (3)利用气相色谱质谱分析法确定nss基因的特性:从大肠杆菌中纯化带有nss的重组蛋白rFap1 (+Nss)和不含nss的重组蛋白rFap1 (-Nss);然后利用气相色谱质谱分析法检测Fap1△RII蛋白的单糖组分。 结果:(1)nss基因突变株不能与抗Fapl多糖表位的特异性抗体F51和D10反应,但nss基因的回复突变株可以恢复野生型副血链球菌FW213的表达,均可于特异性抗体F51、E42、D10反应。 (2)只有在加入Nss后,经Gtf1和Gtf2修饰的Fap1在凝胶中的迁移速度变慢,而其他拟糖基转移酶的加入并没有使Fap1在凝胶中的迁移速度发生变化。 (3)被Gtf1、Gtf2和Nss共同修饰的重组Fap1蛋白出现了两个峰值,分别为N-乙酰葡萄糖胺(GlcNAc)和葡萄糖(Glucose),而在缺失Nss,只有Gtf1、Gtf2修饰的重组Fap1蛋白中只出现一个峰值,即N-乙酰葡萄糖(GlcNAc)。 结论:Nss参与并调控Fap1糖基化的第二步;Nss是一个葡萄糖基转移酶。
[Abstract]:Objective : The mature glycoprotein Fap1 is one of the surface adhesin of Streptococcus sanguis , which is very important to the formation of the most complex biofilm plaque in human . The Fap1 glycosylation is regulated by the gene cluster near the fap1 gene locus . The research shows that Gtf1 and Gtf2 located downstream of the fap1 locus are involved in the first step glycosylation of Fap1 , and N - acetylglucosamine can be transferred to the Fap1 peptide segment . However , the following steps to the glycosylation of Fap1 are still unknown . The present study explored the function of the nss gene located upstream of the fap1 gene locus in the glycosylation of Fap1 .

Methods : ( 1 ) The nss gene mutation strain and the revertant mutant were constructed . The nss gene mutation was constructed by using the wild type Streptococcus sanguinis FW213 strain as the starting strain , and the nss gene was cloned into the shuttle expression vector of Escherichia coli and Streptococcus sanguis , and the nss gene was transformed to block the mutant strain . The nss gene was used to detect the phenotype of the nss gene mutation strain , the revertant mutant and the wild type strain of Streptococcus sanguis , that is , the glycosylation of the Fap1 protein .

( 2 ) The function of Fap1 glycosylation related gene nss was identified in heterologous host E . coli system : a series of recombinant plasmids containing different glycosyltransferase genes were constructed . These recombinant plasmids were co - transformed with recombinant plasmids containing Fap1 鈻，

本文编号：1723208