MR-1S融合蛋白的表达、纯化与保存及其抗血清的制备

检验医学 ›› 2012, Vol. 27 ›› Issue (10): 835-839.

MR-1S融合蛋白的表达、纯化与保存及其抗血清的制备

卢仁泉,张菁,高翔,郭林

复旦大学上海医学院肿瘤学系、复旦大学附属肿瘤医院检验科，上海 200032

收稿日期:2012-07-03 修回日期:2012-08-02 出版日期:2012-10-30 发布日期:2012-10-12
通讯作者: 高翔，联系电话：021-64175590-2204。
作者简介:卢仁泉，男，1975年生，硕士，主管技师，主要从事临床免疫学和分子生物学工作。

Expression，purification and preservation of myofibrillogenesis regulator 1S recombinant protein and its antiserum preparation

Department of Oncology，Shanghai Medical College，Fudan University，Department of Clinical Laboratory，Shanghai Cancer Center，Fudan University，Shanghai 200032，China

Received:2012-07-03 Revised:2012-08-02 Online:2012-10-30 Published:2012-10-12

摘要/Abstract

摘要： 目的　通过人肌纤生成调节因子1（hMR-1S）基因重组表达获得MR-1S-His融合蛋白，纯化后制备抗MR-1S抗血清；同时确定该蛋白保存的稳定体系，为研究MR-1S蛋白理化性质及其功能提供关键的原料。方法　利用原核表达载体pET21a（+），构建pET21a-MR-1S重组质粒，转化大肠杆菌BL21（DE3），异丙基硫代半乳糖苷(IPTG)诱导MR-1S-His融合蛋白表达，亲和纯化后Western blot鉴定；进一步用该融合蛋白免疫新西兰兔，制备抗MR-1S的多克隆抗体。同时通过在不同温度条件下，对4种不同的稳定体系中保存后蛋白活性测定进行比较，确定最合适的稳定保存体系及条件。结果　构建了重组质粒pET21a-MR-1S，并在BL21中进行可溶性表达，经Western blot鉴定确认；免疫新西兰兔获得了抗MR-1S抗血清；确定了合适的稳定体系及保存条件。结论　在非变性条件下实现了可溶性MR-1S融合蛋白的表达和稳定保存，进一步制备的抗MR-1S抗血清为研究MR-1S的性质及功能奠定了基础。

关键词: 肌纤维生成调节因子1, 分子克隆, 抗体

Abstract: Objective　To obtain the myofibrillogenesis regulator 1S (MR-1S)-His recombinant protein through the expression of MR-1S gene and prepare anti-MR-1S antiserum after purification. To identify the stable system to preserve the recombinant protein，and to provid the reference for the investigation of MR-1S protein characteristics and biological function. Methods　The recombinant plasmid pET21a-MR-1S was constructed with pronucleus expression vector pET 21a(+) and transferred into Escherichia coli BL21 (DE3)，and the expression of MR-1S recombinant protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG). The MR-1S recombinant protein was purified by affinity chromatography and identified by Western blot. The anti-MR-1S polyclonal antibodies were prepared by the rabbit-immunized technique. With comparison of the protein activities of 4 stable systems under different temperatures，the most suitable stable preservation system and condition were picked out. Results　Recombinant plasmid pET21a-MR-1S was established，and the MR-1S-His recombinant protein was expressed solubly in Escherichia coli BL21(DE3) by the identification of Western blot. The anti-MR-1S antisera were obtained by immunization rabbit，and the satabilization system and preservation condition were definited. Conclusions　Under undenatured conditions，the soluble MR-1S recombinant protein is expressed and can be preserved stably，and it lays the foundation for MR-1S characteristices and biological function studies.

Key words: Myofibrillogenesis regulator 1, Molecular cloning, Antibody

卢仁泉，张菁，高翔，郭林. MR-1S融合蛋白的表达、纯化与保存及其抗血清的制备[J]. 检验医学, 2012, 27(10): 835-839.

LU Renquan，ZHANG Jing，GAO Xiang，GUO Lin. Expression，purification and preservation of myofibrillogenesis regulator 1S recombinant protein and its antiserum preparation[J]. , 2012, 27(10): 835-839.

参考文献

[1]LI TB，Liu XH，Feng S，et al. Characterization of MR-1，a novel myofibrillogenesis regulator in human muscle[J].Acta Biochim Biophys Sin(Shanghai)，2004，36(6)：412-418.
[2]Lee HY，Xu Y，Huang Y，et al.The gene for paroxy-smal non-kinesigenic dyskinesia encodes an enzyme in a stress response pathway[J]. Hum Mol Genet，2004，13(24)：3161-3170.
[3]徐菲菲，刘秀华，王彦珍，等. 肌原纤维调节因子-1在心肌肥大中的作用研究[J]. 中国病理生理杂志，2006，22(3)：443-447.
[4]Liu X，Li T，Sun S，et al. Role of myofibrillogene-sis regulator-1 in myocardial hypertrophy[J]. Am J Physiol Heart Circ Physiol，2006，290(1)：H279-H285.
[5]Ren K，Jin H，Bian C，et al. MR-1 modulates proliferation and migration of human hepatoma HepG2 cells through myosin light chains-2(MLC2)/focal adhesion kinase(FAK)/Akt signaling pathway[J]. J Biol Chem，2008，283(51)：35598-35605.
[6]Chen DH，Matsushita M，Rainier S，et al. Presence of alanine-to-valine substitutions in myofibrillogenesis regulator1 in paroxysmal nonkinesigenic dyskinesia： confirmation in 2 kindreds[J].Arch Neurol，2005，62(4)： 597-600.
[7]Hempelmann A，Kumar S，Muralitharan S，et al. Myofibrillogenesis regulator 1 gene (MR-1) mutation in an Omani family with paroxysmal nonkinesigenic dyskinesia[J]. Neurosci Lett，2006，402(1-2)：118-120.
[8]王金华，边春景，赵春华. MR1 siRNA抑制人肝癌细胞BEL-7402的增殖[J]. 基础医学与临床，2009，29(1)：1-5.
[9]卢仁泉，胡娟，吴兴中，等. HE4融合蛋白基因的构建、表达及鉴定[J]. 中国实验诊断学，2010，14(1)：17-19.
[10]李天伯，胡洋，冯爽，等. 人肌纤生成调节因子1融合蛋白在大肠杆菌的表达和抗体制备[J].中国医学科学院学报，2005，27(1)：42-47.
[11]李天伯，胡洋，王以光，等. mMR-1基因的克隆和在毕赤酵母中分泌表达[J].生物工程学报，2005，21(1)：25-29.
[12]Kleber-Janke T，Becker WM. Use of modified BL21 (DE3) Escherichia coli cells for high-level expression of recombinant peanut allergens affected by poor codon usage[J]. Protein Expr Purif，2000，19(3)： 419-424.
[13]樊绮诗，吕建新. 分子生物学检验技术[M]. 第2版. 北京：人民卫生出版社，2003：119-122.