本文關(guān)鍵詞： 細(xì)胞間粘附分子-1 膜外Ⅰ～Ⅲ功能區(qū) 基因克隆 融合蛋白 蛋白純化　出處：《天津醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 細(xì)胞間粘附分子l(intracellular adhesion molecule-1 ICAM-1)是一種細(xì)胞表面單鏈糖蛋白,參與抗原識別、補體結(jié)合、細(xì)胞粘附功能。可溶性細(xì)胞間粘附分子l(soluble intracellular adhesion molecule-l sICAM-1)為細(xì)胞表面ICAM—1脫落所形成,其表達(dá)是導(dǎo)致Graves'病發(fā)生的重要因素之一,并且其血清水平對于判斷Graves'病的停藥和復(fù)發(fā)具有重要意義。ICAM-1的免疫功能主要集中在膜外Ⅰ～Ⅲ功能區(qū),本實驗室張志友等已完成了對該功能區(qū)基因的克隆及融合蛋白的表達(dá),并用所得到的融合蛋白初步免疫家兔制備了多抗。國外雖然有商品ICAM-1膜外區(qū)全長的抗原,但是性能不穩(wěn)定,不能滿足標(biāo)記純度的要求,且價格昂貴,因此如能進(jìn)一步得到ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白純品,用來做抗原標(biāo)準(zhǔn),即可建立檢測人血清sICAM-1含量的超微量分析技術(shù),進(jìn)而建立更經(jīng)濟(jì)實用的檢測方法,符合我國國情,將會有一定的經(jīng)濟(jì)意義和明顯的臨床應(yīng)用前景。 目的:1.原核表達(dá)GST-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)融合蛋白,純化后用凝血酶酶切融合蛋白去除GST標(biāo)簽,應(yīng)用免疫親和層析和高壓液相方法探索蛋白分離純化的最優(yōu)條件,以獲取ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白。 2.構(gòu)建重組質(zhì)粒ZPET28aICAM-1,原核表達(dá)只帶His標(biāo)簽的融合蛋白HisTag-ICAM-1,以獲取ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白。 方法:1.利用本室構(gòu)建并保存的重組ZpET42aICAM質(zhì)粒轉(zhuǎn)化宿主菌E.ColiBL21(DE3),IPTG誘導(dǎo)表達(dá),表達(dá)產(chǎn)物用His.Tag融合蛋白純化系統(tǒng)純化,得到GST-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)融合蛋白。應(yīng)用凝血酶對融合蛋白進(jìn)行酶切,SDS-PAGE電泳鑒定酶切結(jié)果。用飽和硫酸銨法從本室制備的含兔抗人ICAM-1的兔血清中粗提IgG,再用Protein A Sepharose CL-4B凝膠進(jìn)行IgG的進(jìn)一步提純,得到兔抗人ICAM-1的IgG純品,經(jīng)SDS-PAGE電泳鑒定后,用于CNBr-activited Sepharose CL-4B凝膠免疫親和層析,得到ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白;此外還探索應(yīng)用高壓液相方法對酶切后蛋白進(jìn)行分離純化。 2.構(gòu)建重組質(zhì)粒ZPET28aICAM-1,質(zhì)粒轉(zhuǎn)化宿主菌E.Coli BL21(DE3),IPTG誘導(dǎo)表達(dá),表達(dá)產(chǎn)物用His.Tag融合蛋白純化系統(tǒng)純化,得到His-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白。SDS-PAGE電泳鑒定蛋白純化結(jié)果并計算分子量,Western Blot檢測His-ICAM-1的免疫活性,考馬斯亮藍(lán)染色法定量,即可初步得到具有免疫學(xué)活性的ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白。 結(jié)果:1.GST-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)融合蛋白的免疫親和層析分離、純化 重組質(zhì)粒ZpET42aICAM轉(zhuǎn)化宿主菌后在最佳條件下表達(dá),表達(dá)產(chǎn)物用His.Tag融合蛋白純化系統(tǒng)純化。凝血酶在4℃酶切60小時,可以將GST-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)融合蛋白完全酶切開,SDS-PAGE電泳顯示酶切后得到兩條條帶,即:ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白,分子量為38.2kD和GST蛋白,分子量為28.0kD。開始免疫親和層析后從3ml兔抗血清中用飽和硫酸銨法和Protein A凝膠親和層析法提純兔抗人ICAM-1IgG純品,獲得約6mg,再用于CNBr-activitedSepharose CL-4B凝膠進(jìn)行的免疫親和層析,初步得到具有免疫學(xué)活性的ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白。 2.GST-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)融合蛋白的高壓液相法分離、純化 凝血酶酶切后經(jīng)SDS-PAGE電泳顯示兩條明顯的條帶,即GST和ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白,經(jīng)改變條件反復(fù)探索確定應(yīng)用HPLC法流動相為3%乙腈,0.02%TFA,0.02M乙酸銨,PH值為6.0,在此條件下進(jìn)樣收集樣品濃縮處理后,作SDS-PAGE和Western blot鑒定,發(fā)現(xiàn)雖有免疫學(xué)活性但是GST和ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白未完全分開,依然顯示兩條條帶。 3.His-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)融合蛋白的表達(dá)、純化及鑒定 構(gòu)建重組質(zhì)粒ZPET28aICAM-1,質(zhì)粒轉(zhuǎn)化宿主菌E.Coli BL21(DE3),以IPTG為誘導(dǎo)劑,濃度1mM,20℃誘導(dǎo)6小時。表達(dá)產(chǎn)物用His.Tag融合蛋白純化系統(tǒng)純化。純化后His-ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白,產(chǎn)率為4.0 mg/L培養(yǎng)基。 結(jié)論:1.成功地用抗體免疫親和層析方法分離ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白和GST,得到具有免疫學(xué)活性的ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白。 2.應(yīng)用HPLC方法分離ICAM-1膜外Ⅰ～Ⅲ功能區(qū)蛋白和GST,可以得到具有免疫學(xué)活性的蛋白,但是分離效果不甚理想,SDS-PAGE鑒定,依然顯示兩條條帶。 3.成功地構(gòu)建了重組質(zhì)粒ZPET28aICAM-1,Ni-NTA親和層析純化后可得到具有免疫學(xué)活性的ICAM-1膜外Ⅰ-Ⅲ功能區(qū)蛋白。因只帶His標(biāo)簽,可減少純化步驟和成本。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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