[1]王鹏),任鹏飞),代丽萍),等.重组α2HS糖蛋白的原核表达、纯化及免疫学活性分析*[J].郑州大学学报(医学版),2012,(01):6.
 WANG Peng,REN Pengfei,DAI Liping,et al.Prokaryotic expression,purification and immunological analysis of recombinant alpha2HeremansSchmid glycoprotein[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2012,(01):6.
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重组α2HS糖蛋白的原核表达、纯化及免疫学活性分析*
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2012年01期
页码:
6
栏目:
论著
出版日期:
2012-01-20

文章信息/Info

Title:
Prokaryotic expression,purification and immunological analysis of recombinant alpha2HeremansSchmid glycoprotein
作者:
王鹏12)任鹏飞1)代丽萍12)王凯娟12)张建营12)#
1)郑州大学公共卫生学院流行病学教研室 郑州 450001 2)河南省肿瘤流行病学重点实验室 郑州 450052
Author(s):
WANG Peng12)REN Pengfei1)DAI Liping12)WANG Kaijuan12)ZHANG Jianying12)
1)Department of Epidemiology, College of Public Health, Zhengzhou University, Zhengzhou 450001 2)Henan Key Laboratory of Tumor Epidemiology,Zhengzhou 450052
关键词:
α2HS糖蛋白原核表达蛋白纯化抗原性
Keywords:
alpha2HeremansSchmid glycoproteinprokaryotic expressionprotein purificationantigenicity
分类号:
Q789
摘要:
目的:原核表达并纯化具有生物学活性的重组α2HS糖蛋白(AHSG)。方法:提取人肝癌细胞株HepG2细胞总RNA,逆转录得cDNA,PCR扩增出目的基因,与pMD18T载体连接后,转化至大肠杆菌DH5α中,大量扩增后提取克隆质粒,连接至原核表达载体pEP30a(+)中,转化BL21(DE3),经IPTG诱导蛋白表达,通过镍离子亲和层析的方法纯化重组蛋白,采用SDSPAGE电泳、免疫印迹杂交法对纯化产物进行分析鉴定。结果:成功构建了AHSG原核表达系统BL21(pET30aAHSG),最佳诱导条件为80 μmol/L IPTG诱导3 h;诱导表达的蛋白相对分子质量约54 600,主要以不溶性的包涵体形式存在,纯化后的重组蛋白质量浓度最高达2.3 g/L,经免疫印迹杂交鉴定有抗原性。结论:成功构建了AHSG原核表达系统,该系统可高效表达重组AHSG蛋白,该蛋白具有良好的抗原性。
Abstract:
Aim: To express and purify recombinant alpha2HeremansSchmid glycoprotein(AHSG) with biological activity.Methods:The total RNA from HepG2 cells was extracted and cDNA was synthesized.AHSG gene was amplified by RTPCR method,then cloned on pMD18T vector.The constructed clone plasmid was transformed into E.coli DH5α,then the fragments of AHSG acquired from that were ligated with pET30a(+) vector.The recombinant AHSG protein was expressed after the constructed expression plamid was tranformed into E.coli BL21(DE3) and purified by Nickel ion affinity chromatography.The purified products were identified and analyzed by SDSPAGE and Western blot, and the concentration of purified protein was measured.Results:The prokaryotic expression system BL21(pET30aAHSG) was constructed successfully.The most appropriate condition for the expression of target protein was 80 μmol/L IPTG for 3 h.A fusion protein approximately 54 600 was yielded, mainly existing in the form of insoluble inclusion bodies. The highest concentration of recombinant protein purified by Nickel ion affinity chromatography was up to 2.3 g/L.The recombinant protein had antigenicity identified by Western blot.Conclusion:The prokaryotic expression system of AHSG and protein purification system have been successfully constructed and the purified AHSG recombinant protein is highly antigenic and immunogenic.

参考文献/References:

[1]Mathews ST, Deutsch DD, Iyer G, et al. Plasma alpha2HS glycoprotein concentrations in patients with acute myocardial infarction quantified by a modified ELISA[J].Clin Chim Acta,2002,319(1):27 [2]陈学彬.人脑胶质瘤AHSG的相关性研究进展[D].苏州:苏州大学,2006. [3]Swallow CJ, Partridge EA, Macmillan JC, et al. α2 HSglycoprotein,an antagonist of transforming growth factor β in vivo, inhibits intestinal tumor progression[J].Cancer Res,2004,64(18):6402 [4]刘彦. 人肺鳞癌相关差异表达基因和基因表达谱的研究[D]. 北京: 中国协和医科大学,2007. [5]Yi JK, Chang JW, Han W, et al. Autoantibody to tumor antigen, alpha 2HS glycoprotein: a novel biomarker of breast cancer screening and diagnosis[J].Cancer Epidemiol Biomarkers Prev,2009,18(5):1357 [6]聂轶飞, 王凯娟, 代丽萍, 等. 肝癌相关抗原血清学自身抗体反应分析[J].中国公共卫生,2007,123(7):801 [7]Deng HW, Xu FH, Davies KM, et al. Differences in bone mineral density, bone mineral content, and bone areal size in fracturing and nonfracturing women, and their interrelationships at the spine and hip[J].J Bone Miner Metab,2002, 20(6):358 [8]Szweras M, Liu D, Partridge EA,et al.Alpha2HS glycoprotein/fetuin, a transforming growth factorbeta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling[J].J Biol Chem,2002,277(22):19991 [9]Kalaby L, Cseh K,Pajor A,et al.Correlation of maternal serum fetuim / alpha 2HSglycoprotein concentration with maternal insulin resistance and anthropometric parameters of neonates in normal pregnancy and gestational diabetes[J].Eur J Endocrinol,2002,147(2):243

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备注/Memo

备注/Memo:
*国家自然科学基金面上项目资助30872962 doi:10.3969/j.issn.16716825.2012.01.003 #通讯作者,男,1962年10月生,博士,教授,研究方向:肿瘤流行病学,Email:jianyingzhang@hotmail.com
更新日期/Last Update: 1900-01-01