抗活化血小板表面受体全套单链抗体噬菌体展示文库的构建和筛选-内科血液学专业毕业论文.docxVIP

抗活化JllLd,板表面受体全套单链抗体噬菌体展示文库的构建和筛选 抗活化JllLd,板表面受体全套单链抗体噬菌体展示文库的构建和筛选 提要 ．r 566始争 内容摘要 本研究从活化血小板免疫小鼠的脾淋巴细胞中分别克隆出小鼠 免疫球蛋白的重、轻链可变区(V。和vL)基因，将V。和V。在体外拼接 成单链抗体(ScFv)基因后转入噬菌体载体，使单链抗体分子展示在 噬菌体表面，从而成功构建了抗血小板表面受体全套单链抗体噬菌体 展示文库。以血小板膜表面数种受体糖蛋白(GP)11 b、IIIa、11 b／ Ilia复合物及重组凝血酶受体PAR一1(rPARl)为靶抗原从该文库中筛 选各自的特异性单链抗体。靶抗原GP II b、GPIIIa、GP II b／Ilia以亲合 层析方法从血小板裂解液中提取，rPARl以重组蛋白的形式在大肠杆 菌中表达，表达产物具有与天然PAR-1分子相似的活性和功能。 从文库中筛选出的单链抗体克隆经测序证实符合抗体可变区结 构特点，对其中抗rPARl的单链抗体克隆APAR31进行了表达，得到 了有抗原结合能力的单链抗体分子，同时也证实了所建噬菌体文库的 有效性。上述工作为噬菌体展示技术在血栓与止血领域的应用奠定了 基础。 关键词： 噬菌体展示 单链抗体 血小板 作者：江淼 导师：阮长耿教授 抗活化IfIL 抗活化IfIL J]、扳表面受体全套单链抗体噬菌体展示文库的构建和筛选 提要 Construction anti-actived human platelet phage display ScFv library and screening specific ScFv antibody against platelet membrane receptors Abstract The variable region of heavy—chain and light—chain(VH and VL、of immunoglobulin gene were amplified from the spleen cells of Bal b／c mouse immunized with actived human platelets， VH and VL are joined to construct single-chain Fv fragment(ScFv)gene in vitro．The recombinant ScFv gene was ligated into phagemid vector,then the SeFv were displayed in Filamentous phage． Thus，the anti-actived human platelet phage display ScFv library was constructed． From the library,the specific ScFv antibody against several platelet membrane receptors such as GP II b．GPIIIa,GP II b／IIIa and recombinant thrombin receptor PAR．I(rPARl)have been screened．Target antigens，GPIIb，OPIIIa and GPIIb／IIIa were purified from solubilized platelet solution using affinity chromatography．The rPARl was expressed in E．eoli．The function of recombinant peptide is similar to native PAl01． The gene of the selected clones were sequenced，their sequence were conformed to mouse Ig gene．The gene of high affinity anti—rPARl specific phage ScFv clone APAR3 1 was expressed and the expressed ScFv fragment retain binding activity to target antigen．These results also demonstrated preliminarily the availability of the library． It is useful for application of phage display tec