融合蛋白CBD-SPA“Z”基因的构建、表达与活性鉴定
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摘要
针对金黄色葡萄球菌蛋白A(SPA)层析柱的应用问题,将SPA的Z结构域基因与纤维素结合结构域(CBD)重组,并在Z结构域C端引入1个半胱氨酸(Cys),构建并表达出一种新型免疫亲和材料。利用基因工程技术将质粒pEZZ18中的Z基因插入到含有CBD的质粒pET35b(+)质粒中,构建出原核表达载体pET35b(+)-ZCys,经IPTG诱导表达,获得CBD-Protein Z融合表达蛋白。pET35b(+)-Z-Cys在E.coli BL21中正确表达,所表达的融合蛋白具有与哺乳动物IgG抗体结合的生物学活性和与纤维素结合的活性。结果证明CBD-Protein Z蛋白具有Z结构域与CBD结构域的活性,预计能在免疫亲和层析中用于IgG抗体的纯化。
To solve the problems of staphylococcal protein A( SPA) application,recombining the fusion protein of cellulose binding domain( CBD) with Z peptide of SPA,introducing a cysteine at the C-terminal of Z peptide,and construction and expression of immune affinity material were investigated. Prokaryotic expression vector pET35b( +)-Z-Cys was constructed by using the genetic engineering to clone the Z gene of pEZZ18 plasmid and inserted into the pET35b( +) plasmid with CBD gene,thus obtaining the fusion protein CBD-protein Z by IPTG. The plasmid pET35b( +)-Z-Cys was correctly expressed in E. coli BL21 and the fusion protein retained the bio-functional effects of Z peptide and CBD. CBD could bind to cellulose and Z peptide bind to immunoglobulin G( IgG) of many mammalians,via Fc region of IgG.
To solve the problems of staphylococcal protein A( SPA) application,recombining the fusion protein of cellulose binding domain( CBD) with Z peptide of SPA,introducing a cysteine at the C-terminal of Z peptide,and construction and expression of immune affinity material were investigated. Prokaryotic expression vector pET35b( +)-Z-Cys was constructed by using the genetic engineering to clone the Z gene of pEZZ18 plasmid and inserted into the pET35b( +) plasmid with CBD gene,thus obtaining the fusion protein CBD-protein Z by IPTG. The plasmid pET35b( +)-Z-Cys was correctly expressed in E. coli BL21 and the fusion protein retained the bio-functional effects of Z peptide and CBD. CBD could bind to cellulose and Z peptide bind to immunoglobulin G( IgG) of many mammalians,via Fc region of IgG.
引文
[1]乐易林,邵蔚蓝.嗜热真菌纤维素酶的CBD与海栖热袍菌的纤维素酶融合表达[J].微生物学通报,2009,36(6):837-841.
[2]孙丹,曹宇,白钢,等.纤维素磁性微球的制备及其生物活化研究[J].离子交换与吸附,2009,25(1):17-24.
[3]徐建生,成大荣,孙怀昌,等.细菌表面表达载体的构建与鉴定[J].河南科技大学学报:农学版,2004,24(4):14-19.
[4]Tomas M,Abrahmsén L,Nilsson B,et al.Staphylococcal protein A consists of five IgG-binding domains[J].Eur J Biochem,1986,156(3):637-643.
[5]Khalili M,Liwo A,Scheraga H A.Kinetic studies of folding of the B-domain of staphylococcal protein A with molecular dynamics and a united-residue(UNRES)model of polypeptide chains[J].J Mol Biol,2006,355(3):536-547.
[6]李志会,岳盈盈,李鹏,等.金黄色葡萄球菌蛋白A的原核表达及生物亲和特性研究[J].山东大学学报:医学版,2009,47(10):28-31.
[7]Nelson D E,Grishin V N.Folding domain B of protein A on a dynamically partitioned free energy landscape[J].PNAS,2008,105(5):1489-1493.
[8]逄少堃,梁浩,宋淑亮,等.葡萄球菌蛋白A活性机制与现代应用[J].生命的化学,2008,28(6):748-751.
[9]Himmel M E,Ding S Y,Johnson D K,et al.Biomass recalcitrance:engineering plants and enzymes for biofuels production[J].Science,2007,315:804-807.
[10]张益谋,邱海霞,稽慧珍.葡萄球菌A蛋白基因的克隆及其IgG受体区新型表达载体的构建[J].生物技术通报,2010(12):201-205.
[11]唐雪珍.微米级亲和凝胶载体的制备及其亲和吸附蛋白质研究[D].上海:华东理工大学,2011.
[12]伍学勤,吴岑,凌晓红,等.一种安全制备活化琼脂糖树脂的方法[J].生物化学与生物物理进展,2002,29(2):328-331.
[13]Cao Y,Zhang Q,Wang C,et al.Preparation of novel immunomagnetic cellulose microspheres via cellulose binding domain protein A linkage and its use for the isolation of interferonα-2b[J].J Chromatogr A,2007,1149:228-235.
[14]白芳,张奇,王龙,等.纤维素磁性微球在病原微生物检测中的应用研究[J].离子交换与吸附,2010,26(5):431-438.
[15]周亚丽,劳兴珍,袁玲,等.免疫球蛋白结合结构域的研究进展[J].药物生物技术,2012,19(3):274-277.
[16]Jendberg L,Tashiro M,Tejero R,et al.The mechanism of binding staphylococcal protein A to immunoglobulin G does not involve helix unwinding[J].Biochemistry,1996,35:22-31.
[17]夏海锋,吴璞强,金雄华,等.一种高效结合IgG的重组蛋白A及其工程菌的构建方法:中国,102373225A[P].2012-03-14.
[2]孙丹,曹宇,白钢,等.纤维素磁性微球的制备及其生物活化研究[J].离子交换与吸附,2009,25(1):17-24.
[3]徐建生,成大荣,孙怀昌,等.细菌表面表达载体的构建与鉴定[J].河南科技大学学报:农学版,2004,24(4):14-19.
[4]Tomas M,Abrahmsén L,Nilsson B,et al.Staphylococcal protein A consists of five IgG-binding domains[J].Eur J Biochem,1986,156(3):637-643.
[5]Khalili M,Liwo A,Scheraga H A.Kinetic studies of folding of the B-domain of staphylococcal protein A with molecular dynamics and a united-residue(UNRES)model of polypeptide chains[J].J Mol Biol,2006,355(3):536-547.
[6]李志会,岳盈盈,李鹏,等.金黄色葡萄球菌蛋白A的原核表达及生物亲和特性研究[J].山东大学学报:医学版,2009,47(10):28-31.
[7]Nelson D E,Grishin V N.Folding domain B of protein A on a dynamically partitioned free energy landscape[J].PNAS,2008,105(5):1489-1493.
[8]逄少堃,梁浩,宋淑亮,等.葡萄球菌蛋白A活性机制与现代应用[J].生命的化学,2008,28(6):748-751.
[9]Himmel M E,Ding S Y,Johnson D K,et al.Biomass recalcitrance:engineering plants and enzymes for biofuels production[J].Science,2007,315:804-807.
[10]张益谋,邱海霞,稽慧珍.葡萄球菌A蛋白基因的克隆及其IgG受体区新型表达载体的构建[J].生物技术通报,2010(12):201-205.
[11]唐雪珍.微米级亲和凝胶载体的制备及其亲和吸附蛋白质研究[D].上海:华东理工大学,2011.
[12]伍学勤,吴岑,凌晓红,等.一种安全制备活化琼脂糖树脂的方法[J].生物化学与生物物理进展,2002,29(2):328-331.
[13]Cao Y,Zhang Q,Wang C,et al.Preparation of novel immunomagnetic cellulose microspheres via cellulose binding domain protein A linkage and its use for the isolation of interferonα-2b[J].J Chromatogr A,2007,1149:228-235.
[14]白芳,张奇,王龙,等.纤维素磁性微球在病原微生物检测中的应用研究[J].离子交换与吸附,2010,26(5):431-438.
[15]周亚丽,劳兴珍,袁玲,等.免疫球蛋白结合结构域的研究进展[J].药物生物技术,2012,19(3):274-277.
[16]Jendberg L,Tashiro M,Tejero R,et al.The mechanism of binding staphylococcal protein A to immunoglobulin G does not involve helix unwinding[J].Biochemistry,1996,35:22-31.
[17]夏海锋,吴璞强,金雄华,等.一种高效结合IgG的重组蛋白A及其工程菌的构建方法:中国,102373225A[P].2012-03-14.