重组人乙酰胆碱受体原核表达条件的优化及初步应用  

Optimization and Primary Application of Prokaryotic Expression of Human AChR α-subunit 1-210 in E.coli

作　　者：陈利春[1] 汪凌云[2] 孙玉秀[2] 陈兵[2] 徐从贞[2] 鲁云霞[2] 

机构地区：[1]安徽省合肥市妇幼保健院,合肥230001 [2]安徽医科大学生物化学与分子生物学教研室,合肥230032

出　　处：《生物技术通报》2009年第8期109-113,共5页Biotechnology Bulletin

摘　　要：在已建立的重组人乙酰胆碱受体表达质粒的基础上进一步优化试验条件,初步应用于ELISA分析实验性重症肌无力模型(EAMG)中抗体的滴度。取构建好的pET28a(+)-hAChRα1-210经小规模诱导,确定IPTG诱导的最佳浓度为0.006mmol/L,最佳时间为5h,最佳温度为37℃;大规模诱导培养后离心,超声得到菌体沉淀,1mol/L尿素洗涤后8mol/L尿素溶解,过Ni2+亲和层析柱,紫外吸收及Folin酚法确定表达量较多而蛋白最纯的4℃透析过夜,PEG8000浓缩后免疫雌性近交系Lewis鼠构建EAMG,ELISA检测分析模型大鼠血清中抗体的效价。结果表明,制备了大量较纯的rhAChR,并成功应用于ELISA法分析EAMG模型中抗体的滴度。因此,获得的纯化rhAChR可用于构建EAMG模型及鉴定。It was to explore the conditions for high expression of human AChR et-subunit 1-210 in E. coli（BL21 ） , the induction conditions such as concentration of IPTG, induction temperature and time were improved. Results indicated that the highly expressed human AChR et-subunit 1-210 were obtained at 0. 006 mmol/L IPTG for 5 h under 37℃. After large scale of induction culture, puri- fied recombinant proteins were obtained through centrifugation and sonication of bacteria, then solved with 8 M urea, and went through Ni2＋ affinity chromatography. Immunized female Lewis rats were involved in to induce EAMG, to analyze antibody titer of serum in model group. The results exhibited that recombinant proteins were pure and can be used to construct EAMG model to determine anti- body titer successfully.

关 键 词：pET28a(+)-hAChRα1-210 IPTG Ni2+亲和层析 PEG8000 EMAG 

分 类 号：R746.1[医药卫生—神经病学与精神病学]