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.
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.