α-N-乙酰半乳糖胺酶的表达及活性检测

Overexpression and characterization of a bacterial α-N-acetylgalactosaminidase

为了建立新型α-N-乙酰半乳糖胺酶的筛选、检测方法,实验中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Bl21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,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.

The coding sequence of alpha-N-acetylgalactosaminidase （A4） was amplified from ge- nomic DNA of Chryseobacterium meningosepticum and subcloned into pET24a, which was then transformed into BL21 （DE3） for overexpression of His-A4. The overexpressed His-A4 enzyme was purified by using affinity chromatography and its activity was comparable to that previously reported by using a conventional method with an artificial substrate. To better measure the activity of α-N-acctylgalactosaminidase in real application, we established a novel method in which we directly used the surface antigen of red blood cell as substrate and applied ELISA to the detection of 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 manner.