本文選題：Fap1　切入點：粘附素　出處：《河南科技大學》2010年碩士論文

【摘要】：目的:成熟的糖蛋白Fap1是一個副血鏈球菌細胞表面粘附素,對人類最復雜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調控。已有研究表明位于fap1基因座下游的Gtf1和Gtf2參與Fap1第一步糖基化,可將N-乙酰葡萄糖胺(GlcNAc)轉移至Fap1肽段上。但對于Fap1糖基化的后續(xù)步驟是如何進行這一問題至今還是未知。本研究針對位于fap1基因座上游的nss基因在Fap1糖基化中的功能進行了探求。 方法:(1)構建nss基因突變株及回復突變株:采用基因置換技術以野生型Streptococcus parasanguinis FW213菌株為出發(fā)菌株構建nss基因突變株;并采用基因互補技術(將nss基因克隆入大腸桿菌和副血鏈球菌穿梭表達載體中,并轉化nss基因阻斷突變株)對突變株進行基因互補;運用Western Blotting和BactELISA檢測nss基因突變株、回復突變株與副血鏈球菌野生型菌株的表型,即Fap1蛋白的糖基化情況。 (2)在異源宿主大腸桿菌系統(tǒng)中鑒定與Fap1糖基化相關基因nss的功能:構建一系列含有不同擬糖基轉移酶基因的重組質粒,將這些重組質粒分別與含有Fap1△RII(缺失RII重復區(qū)的Fap1蛋白)的重組質粒以及含有Gtf1和Gtf2的重組質粒共轉化到大腸桿菌中,運用Western Blotting檢測在不同的基因存在的情況下Fap1△RII蛋白的糖基化情況。 (3)利用氣相色譜質譜分析法確定nss基因的特性:從大腸桿菌中純化帶有nss的重組蛋白rFap1 (+Nss)和不含nss的重組蛋白rFap1 (-Nss);然后利用氣相色譜質譜分析法檢測Fap1△RII蛋白的單糖組分。 結果:(1)nss基因突變株不能與抗Fapl多糖表位的特異性抗體F51和D10反應,但nss基因的回復突變株可以恢復野生型副血鏈球菌FW213的表達,均可于特異性抗體F51、E42、D10反應。 (2)只有在加入Nss后,經Gtf1和Gtf2修飾的Fap1在凝膠中的遷移速度變慢,而其他擬糖基轉移酶的加入并沒有使Fap1在凝膠中的遷移速度發(fā)生變化。 (3)被Gtf1、Gtf2和Nss共同修飾的重組Fap1蛋白出現了兩個峰值,分別為N-乙酰葡萄糖胺(GlcNAc)和葡萄糖(Glucose),而在缺失Nss,只有Gtf1、Gtf2修飾的重組Fap1蛋白中只出現一個峰值,即N-乙酰葡萄糖(GlcNAc)。 結論:Nss參與并調控Fap1糖基化的第二步;Nss是一個葡萄糖基轉移酶。
[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，

本文編號：1723208