细胞间粘附分子1 Ⅰ～Ⅲ功能区蛋白的表达、分离与纯化

发布时间：2018-03-01 23:17

本文关键词： 细胞间粘附分子-1 膜外Ⅰ～Ⅲ功能区基因克隆融合蛋白蛋白纯化　出处：《天津医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 细胞间粘附分子l(intracellular adhesion molecule-1 ICAM-1)是一种细胞表面单链糖蛋白,参与抗原识别、补体结合、细胞粘附功能。可溶性细胞间粘附分子l(soluble intracellular adhesion molecule-l sICAM-1)为细胞表面ICAM—1脱落所形成,其表达是导致Graves'病发生的重要因素之一,并且其血清水平对于判断Graves'病的停药和复发具有重要意义。ICAM-1的免疫功能主要集中在膜外Ⅰ～Ⅲ功能区,本实验室张志友等已完成了对该功能区基因的克隆及融合蛋白的表达,并用所得到的融合蛋白初步免疫家兔制备了多抗。国外虽然有商品ICAM-1膜外区全长的抗原,但是性能不稳定,不能满足标记纯度的要求,且价格昂贵,因此如能进一步得到ICAM-1膜外Ⅰ～Ⅲ功能区蛋白纯品,用来做抗原标准,即可建立检测人血清sICAM-1含量的超微量分析技术,进而建立更经济实用的检测方法,符合我国国情,将会有一定的经济意义和明显的临床应用前景。目的:1.原核表达GST-ICAM-1膜外Ⅰ～Ⅲ功能区融合蛋白,纯化后用凝血酶酶切融合蛋白去除GST标签,应用免疫亲和层析和高压液相方法探索蛋白分离纯化的最优条件,以获取ICAM-1膜外Ⅰ～Ⅲ功能区蛋白。 2.构建重组质粒ZPET28aICAM-1,原核表达只带His标签的融合蛋白HisTag-ICAM-1,以获取ICAM-1膜外Ⅰ～Ⅲ功能区蛋白。方法:1.利用本室构建并保存的重组ZpET42aICAM质粒转化宿主菌E.ColiBL21(DE3),IPTG诱导表达,表达产物用His.Tag融合蛋白纯化系统纯化,得到GST-ICAM-1膜外Ⅰ～Ⅲ功能区融合蛋白。应用凝血酶对融合蛋白进行酶切,SDS-PAGE电泳鉴定酶切结果。用饱和硫酸铵法从本室制备的含兔抗人ICAM-1的兔血清中粗提IgG,再用Protein A Sepharose CL-4B凝胶进行IgG的进一步提纯,得到兔抗人ICAM-1的IgG纯品,经SDS-PAGE电泳鉴定后,用于CNBr-activited Sepharose CL-4B凝胶免疫亲和层析,得到ICAM-1膜外Ⅰ～Ⅲ功能区蛋白;此外还探索应用高压液相方法对酶切后蛋白进行分离纯化。 2.构建重组质粒ZPET28aICAM-1,质粒转化宿主菌E.Coli BL21(DE3),IPTG诱导表达,表达产物用His.Tag融合蛋白纯化系统纯化,得到His-ICAM-1膜外Ⅰ～Ⅲ功能区蛋白。SDS-PAGE电泳鉴定蛋白纯化结果并计算分子量,Western Blot检测His-ICAM-1的免疫活性,考马斯亮蓝染色法定量,即可初步得到具有免疫学活性的ICAM-1膜外Ⅰ～Ⅲ功能区蛋白。结果:1.GST-ICAM-1膜外Ⅰ～Ⅲ功能区融合蛋白的免疫亲和层析分离、纯化重组质粒ZpET42aICAM转化宿主菌后在最佳条件下表达,表达产物用His.Tag融合蛋白纯化系统纯化。凝血酶在4℃酶切60小时,可以将GST-ICAM-1膜外Ⅰ～Ⅲ功能区融合蛋白完全酶切开,SDS-PAGE电泳显示酶切后得到两条条带,即:ICAM-1膜外Ⅰ～Ⅲ功能区蛋白,分子量为38.2kD和GST蛋白,分子量为28.0kD。开始免疫亲和层析后从3ml兔抗血清中用饱和硫酸铵法和Protein A凝胶亲和层析法提纯兔抗人ICAM-1IgG纯品,获得约6mg,再用于CNBr-activitedSepharose CL-4B凝胶进行的免疫亲和层析,初步得到具有免疫学活性的ICAM-1膜外Ⅰ～Ⅲ功能区蛋白。 2.GST-ICAM-1膜外Ⅰ～Ⅲ功能区融合蛋白的高压液相法分离、纯化凝血酶酶切后经SDS-PAGE电泳显示两条明显的条带,即GST和ICAM-1膜外Ⅰ～Ⅲ功能区蛋白,经改变条件反复探索确定应用HPLC法流动相为3%乙腈,0.02%TFA,0.02M乙酸铵,PH值为6.0,在此条件下进样收集样品浓缩处理后,作SDS-PAGE和Western blot鉴定,发现虽有免疫学活性但是GST和ICAM-1膜外Ⅰ～Ⅲ功能区蛋白未完全分开,依然显示两条条带。 3.His-ICAM-1膜外Ⅰ～Ⅲ功能区融合蛋白的表达、纯化及鉴定构建重组质粒ZPET28aICAM-1,质粒转化宿主菌E.Coli BL21(DE3),以IPTG为诱导剂,浓度1mM,20℃诱导6小时。表达产物用His.Tag融合蛋白纯化系统纯化。纯化后His-ICAM-1膜外Ⅰ～Ⅲ功能区蛋白,产率为4.0 mg/L培养基。结论:1.成功地用抗体免疫亲和层析方法分离ICAM-1膜外Ⅰ～Ⅲ功能区蛋白和GST,得到具有免疫学活性的ICAM-1膜外Ⅰ～Ⅲ功能区蛋白。 2.应用HPLC方法分离ICAM-1膜外Ⅰ～Ⅲ功能区蛋白和GST,可以得到具有免疫学活性的蛋白,但是分离效果不甚理想,SDS-PAGE鉴定,依然显示两条条带。 3.成功地构建了重组质粒ZPET28aICAM-1,Ni-NTA亲和层析纯化后可得到具有免疫学活性的ICAM-1膜外Ⅰ-Ⅲ功能区蛋白。因只带His标签,可减少纯化步骤和成本。
[Abstract]:Intercellular adhesion molecule L (intracellular adhesion molecule-1 ICAM-1) is a cell surface glycoprotein scFv, involved in antigen recognition, complement binding, cell adhesion function. Soluble intercellular adhesion molecule L (soluble intracellular adhesion molecule-l sICAM-1) is a cell surface ICAM - 1 off form, its expression is one of the important factors in Graves'disease, and its serum level to determine the immune function of Graves' disease withdrawal and relapse of.ICAM-1 has an important significance mainly concentrated in the outer membrane I-III function area, the friends of the Zhang Zhi lab has completed the functional areas for gene cloning and expression of fusion protein, and the fusion protein primary immunization of rabbits preparation of the polyclonal antibody. Although foreign commodity ICAM-1 ectodominant full-length antigen, but the performance is not stable, can not meet the mark purity requirements, and the price is expensive, So as to further ICAM-1 outer membrane protein I to function area of pure products, used as a standard antigen, can establish trace analysis techniques to detect the content of sICAM-1 in serum, and then establish the detection method is more economical and practical, in line with China's national conditions, there will be some economic significance and obvious clinical application prospect.
Objective: 1. the prokaryotic expression of GST-ICAM-1 extracellular I-III function fusion protein purified by fusion protein digested with thrombin to remove GST tag by immunoaffinity chromatography and high pressure liquid optimal conditions for separation and purification of protein exploration phase method, to obtain the ICAM-1 outer membrane protein I-III functional areas.
2. the recombinant plasmid ZPET28aICAM-1 was constructed, and the prokaryotic expression only with His labeled fusion protein HisTag-ICAM-1 was used to obtain the protein I to III functional region outside the ICAM-1 membrane.
Methods: 1. the recombinant plasmid ZpET42aICAM was constructed in our lab and the preservation of the transformed E.coli E.ColiBL21 (DE3), IPTG induced expression product with His.Tag fusion protein purification system, GST-ICAM-1 membrane I-III function fusion protein. Application of thrombin on fusion protein by enzyme digestion, SDS-PAGE enzyme digestion electrophoresis results. By using saturated ammonium sulfate from the preparation containing the Rabbit anti human ICAM-1 rabbit serum in the crude extract of IgG Protein and A Sepharose, further purified by IgG CL-4B gel, IgG purified Rabbit anti human ICAM-1, was identified by SDS-PAGE electrophoresis, CNBr-activited Sepharose used CL-4B gel immuno affinity chromatography, ICAM-1 film the outer I-III function area protein; in addition to explore the application of high pressure liquid chromatography method of digested protein was purified.
2. the recombinant plasmid ZPET28aICAM-1 was transformed into host bacteria E.Coli BL21 (DE3), IPTG induced expression product with His.Tag fusion protein purification system, His-ICAM-1 membrane I-III function protein.SDS-PAGE electrophoresis identification of protein purification results and calculation of molecular weight, Western Blot detection of His-ICAM-1 immunoreactivity, Kaumas Coomassie blue staining quantitative method you can get ICAM-1, preliminary film has immunological activity of I ~ III functional region protein.
Results: the isolation and purification of the fusion protein of the functional area of 1.GST-ICAM-1 and III outside the membrane by immunoaffinity chromatography
The recombinant plasmid ZpET42aICAM was transformed into host bacteria expression under the optimum conditions, the expression product with His.Tag fusion protein purification system. Purified thrombin digestion for 60 hours at 4 DEG C can be GST-ICAM-1 extracellular enzyme, I-III function region fusion protein enzymes completely open, SDS-PAGE electrophoresis showed that the digested two bands, namely: ICAM-1 membrane protein outside I - III functional areas, the molecular weight of 38.2kD and GST protein, the molecular weight of 28.0kD. began immunoaffinity chromatography from 3ml rabbit sera with saturated ammonium sulfate and Protein A gel affinity chromatography purification of Rabbit anti human pure ICAM-1IgG, about 6mg, and then used to immunize CNBr-activitedSepharose CL-4B gel affinity chromatography, ICAM-1 the outer membrane has immunological activity of I ~ III preliminary functional region protein.
Separation and purification of the fusion protein of I ~ III functional region outside the membrane of 2.GST-ICAM-1 by high pressure liquid chromatography
Thrombin digestion after SDS-PAGE electrophoresis showed two bands, namely GST and ICAM-1 outer membrane protein I-III function area, the change condition of repeated exploration HPLC in determining the mobile phase of 3% 0.02M ammonium acetate, acetonitrile, 0.02%TFA, pH 6, under this condition the sample collected sample concentration after treatment SDS-PAGE and Western blot found that although the immunological identification, but GST and ICAM-1 outer membrane protein function I ~ not completely separate, still showed two bands.
Expression, purification and identification of the fusion protein of I ~ III functional region outside the membrane of 3.His-ICAM-1
The recombinant plasmid ZPET28aICAM-1 was constructed, and the plasmid was transformed into E.Coli BL21 (DE3), and IPTG was used as the inducer. The concentration of 1mM was induced at 20 C for 6 hours. The expression product was purified by His.Tag fusion protein purification system. After purification, the yield of the first to third functional region of His-ICAM-1 membrane was 4 mg/L medium.
Conclusion: 1.. We successfully separated the outer membrane I and III functional region proteins and GST from ICAM-1 by immunoaffinity chromatography, and obtained immunoreactive ICAM-1 extracellular domain I to III functional region proteins.
2., using the HPLC method to separate the ICAM-1 domain functional protein and GST from outer membrane, we can get the protein with immunological activity, but the separation effect is not ideal. SDS-PAGE identification still shows two bands.
3., a recombinant plasmid ZPET28aICAM-1 was successfully constructed. After purification with Ni-NTA affinity chromatography, ICAM-1 immunoreactive extracellular domain I - III protein was obtained. Because only His tag can reduce the purification step and cost.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【相似文献】