【作者】 林毅刚；

【导师】 夏钢；

【作者基本信息】 华东师范大学 ， 基因组学， 2011， 硕士

【摘要】 α-N-乙酰半乳糖胺酶是血型转换的工具酶,可以降解A型红细胞表面抗原、消除其抗原抗体反应,在输血治疗中,可以避免血型配型带来的不便。本实验室从报道的α-N-乙酰半乳糖胺酶中选取活性最好的酶,构建α-N-乙酰半乳糖胺酶的原核表达载体,表达并纯化α-N-乙酰半乳糖胺酶,检测酶活性。利用定点突变技术,在关键位点进行突变,用高通量筛选技术筛选突变文库,期望得到较高活性的α-N-乙酰半乳糖胺酶。本课题的研究工作主要包括以下两部分：1.α-N-乙酰半乳糖胺酶的构建为了建立新型α-N-乙酰半乳糖胺酶的筛选、检测方法,我们提取脑膜金黄杆菌的基因组DNA,以此为模板PCR扩增出α-N-乙酰半乳糖胺酶(A4)。将A4克隆至pET-24a载体,转化B121表达菌株进行蛋白表达。使用亲和层析方法纯化His-A4酶,选择显色底物验证酶活性。同时,我们改进了传统的ELISA方法,直接将红细胞膜包被于ELISA检测平板中,以红细胞膜表面抗原作为直接底物,用ELISA方法检测酶活性。此研究建立了新型ELISA实验方法,以此方法验证了A4酶的活性,证明了此酶能够有效降低红细胞表面抗原抗体反应,且具有浓度和时间依赖性。2.α-N-乙酰半乳糖胺酶突变文库的筛选自然界中的α-N-乙酰半乳糖胺酶活性较低,不足以大规模生产酶用于通用型血的制备。在本实验中,使用定点突变技术结合高通量筛选方法,期望得到活性较高的糖苷酶。我们突变了酶的6个氨基酸位点分别是96、98、179、181、225、228,筛选了2400个左右的重组子,从实验中可知,179、181、225、228位置突变后酶的活性大幅度下降,因此这四个位点对酶的活性至关重要。96、98这两个位点突变后,有4个突变酶活性相当,它们突变后活性改变的机理需要进一步研究。更多还原

【Abstract】 Alpha-a-N-acetylgalactosaminidase belongs to an important family of enzymes that can be used to degrade surface antigen on the surface of A-type red blood cells to eliminate the antigen-antibody reaction. It can help to relieves difficulties associate with typing and crossmatching in blood transfusion. The best activity of a-N-acetylgalactosaminidase was selected and the recombinant plasmid was constructed. With the expression and purification of a-N- acetylgalactosaminidase, its enzyme kinetics were measured. The key site of the enzyme was mutated by PCR directed mutagenesis. We hoped to get the higher efficiency of a-N-acetylgalactosaminidase through high-throughput screening. The main objectives are as follows:1. The construction ofa-N-acetylgalactosaminidaseThe coding region of an a-N-acetylgalactosaminidase (A4) was PCR amplified from genomic DNA of Chryseobacterium meningosepticum and subcloned into pET24a-A4 for overexpression His-A4. The overexpressed His-A4 enzyme was purified using affinity chromatography and has activity that is comparable with literature reported using a conventional method with an artificial substrate. To better measure the activity of a-N-acetylgalactosaminidase in real application, we established a novel method that directly use the surface antigen of red blood cell as substrate and use ELISA to detect the un-cleaved antigen. The activity of His-A4 was evaluated in the new ELISA method and was demonstrated to be able to decrease the blood cell surface antigen-antibody reaction in concentration- and time-dependent fashion.2. The screening of a-N-acetylgalactosaminidase mutation librariesThe major obstacle to use glycosidase in the preparation of universal blood is their low enzymatic activity. We hoped to get the higher efficiency enzyme by directed mutagenesis and high-throughput screening. The site of amino acid were 96,98,179,181,225 and 228 separately. We screened about 2,400 recombinant bacteria. The results showed that 179,181,225 and 228 these four sites are the key sites for the enzyme because the activity of a-N-acetylgalactosaminidase has been lost after the mutation. We got the four recombinant bacterias. Their activities were comparable to the wild bacteria. The mechanism of the changing enzyme activity should be further studied.更多还原