大肠杆菌L-门冬酰胺酶Ⅱ作为多肽呈现载体的可行性研究  

Study on the Possibility of Foreign Peptide Display on the Surface of Escherichia coli L-AsparaginaseⅡ

作　　者：林洁[1] 黄永城[1] 王学军[1] 吴洁[1] 刘景晶[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京210009

出　　处：《药物生物技术》2006年第3期159-165,共7页Pharmaceutical Biotechnology

基　　金：国家自然科学基金项目(No.30270298)

摘　　要：为了研究大肠杆菌L-门冬酰胺酶Ⅱ(L-asparaginaseⅡ,AnsB)能否作为多肽呈现载体。选取乙肝病毒表面抗原(HBSAg)preS2结构域的核心抗原表位区作为模型多肽,构建了两种表达载体pET28a-AnsB-preS2L和pET28a-AnsB-preS2C,分别转化至表达宿主菌E.coliBL-21,LB培养基(Kanr)培养,乳糖诱导高效表达,通过渗透休克,DEAE 52-cellulose柱层析纯化获得了在AnsB C端融合有线状和环状模型多肽的嵌合酶AnsB-preS2L和AnsB-preS2C,两种嵌合酶分别保留了AnsB 81%、25%的催化活力,两者的pH稳定性均与AnsB相符。ELISA检测结果显示AnsB-preS2L与抗preS2抗体的结合能力较AnsB-preS2C强。证明AnsB确实能够作为一种表面呈现载体将线性外源多肽以融合表达的方式呈现在酶分子的表面,最终形成一种在每个亚基表面呈现有多肽的嵌合酶。最后,用富含带电序列的8肽(KRKRKKSR)替换核心抗原表位区作为模型多肽,进一步验证了AnsB作为多肽呈现载体的可行性和通用性。L-asparaginaseU（AnsB） of Escherichia coli, a tetramer of identical subunits, was tested as a vector to display foreign peptides on the surface of each enzyme subunit. The main epitope of the preS2 domain on the HBSAg was chosen as a model of foreign peptide. Two expression plasmids, named pET28a AnsB preS2L and pET28a AnsB preS2C were constructed. They were transformed into IT,. coli/3I;21（DE3） and the two kinds of chimeric enzyme were expression and targeted to the periplasm of E. coli after inducing with lactose. These periplasm fusion proteins were extracted by osmotic shock and furthermore purified with DEAE52-cellulose. The purified chimeric enzyme AnsB preS21, fusing linear model peptides on the C-terminal of enzyme exhibited approximately 81% activity of the native enzyme. On the contrast, AnsB preS2C fusing cyclic model peptides only exhibited about 25% activity of the native one. They have the same pH stability as the native AnsB. ELISA asay demonstrated that the chimeric enzyme AnsB preS21 could bind with anti-preS2 antibody much more tightly than AnsB preS2C. After being reduced with DTT, AnsB preS2C binds with the antibody much more easily and tightly than before. Finally, through choosing the basic octopeptides （KRKRKKSR） as the new model peptide instead of the main epitope of the preS2, and the purified chimeric enzyme AnsB-KRKRKKSR could bind with the Heparin tightly. All of these results demonstrated that L-asparaginaseⅡ could be used as a vector to display linear peptides on the surface of enzyme with the help of linker peptides, and the displayed peptide has the native bioactivity and immunoactivity. These could be utilized as a rapid pepsean technique for antigen epitope mapping.

关 键 词：L-门冬酰胺酶Ⅱ 嵌合酶 表面呈现 外源多肽 抗原表位 表位扫描 

分 类 号：Q78[生物学—分子生物学]