百日咳杆菌粘着素的分子克隆、表达、纯化及生物学特性研究

大肠杆菌中克隆了编码百日咳杆菌粘着素（Ｐｅｒｔａｃｔｉｎ，Ｐｒｎ）１．９ｋｂ的结构基因，获得了４株表达Ｐｒｎ的重组菌株，以融合蛋白的形式，对Ｐｒｎ进行了表达。Ｐｒｎ融合蛋白的表达量占细胞总蛋白量的４６．４％。融合蛋白纯度达８９．２％。免疫印迹证明，抗天然Ｐｒｎ的单克隆抗体ＢＰＥ３能识别重组菌中及纯化的Ｐｒｎ融合蛋白。Ｐｒｎ融合蛋白能与抗百日咳杆菌Ⅰ相血清发生特异凝集反应。Ｐｒｎ融合蛋白及重组菌免疫小鼠后，对百日咳杆菌毒力株１８３２３的脑内攻击，保护率达６０～６２．５％。

Molecular Cloning,Expressing,Purification and Characterization of Recombinant Pertactin of Bordetella Pertussis

The 1.9 kb structural gene encoding pertactin (Prn) was amplified by Polymerase Chain Reaction (PCR),also cloned,and expressed as a fusion protein in Escherichia coli DH5α.The expression level of recombinant Prn (rPrn) is 46.4% of total cell protein.The purity of rPrn purified by Amylose Resin Affinity Chromatography is 89.2% on SDS-PAGE.The rPrn was probed in recombinant E.coli DH5α by Western Blotting with monoclonal antibody BPE3 against the natural Prn,and agglutinated with antiserum against the B.pertussis phase Ⅰ.Mice immunized with rPrn and E coli expressing rPrn showed a significant levels (60-62.5%) of protection against an intracerebral challenge with virulent B.pertussis 18323. The rPrn should be considered as it is a new effective immunogen component for engineering pertussis vaccine.A cleavage site for the signal peptidase was found at the protein residue 32 or 34 from the N-terminus (or at 102 base from ATG in DNA seguence)of Prn. 