人血型B抗原模拟多肽P1/Fas-Mip3β双表达重组质粒的构建及鉴定

Construction and identification of blood type B antigen mimetic polypeptide-Mip3β double expression recombinant plasmid

目的构建并鉴定人血型B抗原模拟多肽、巨噬细胞炎症蛋白3β(Mip3β)双表达重组质粒。方法抗血型B抗体筛选噬菌体随机12肽库,筛选阳性噬菌体克隆P1,设计特异性引物扩增P1噬菌体DNA,同样设计特异性引物扩增PBluscript-Fas基因的跨膜区及胞内段,与P1酶切连接,最后与质粒PORF5-mMip3βv21扩增巨噬细胞炎症蛋白3β基因酶切连接,获得血型B抗原模拟多肽、Mip3β双表达重组质粒并进行鉴定,并在人黑色素瘤细胞株B16中转染与表达。结果和结论成功制备了血型B抗原模拟多肽、Mip3β双表达重组质粒,并在黑色素瘤细胞株B16中成功表达。

Objective To construct and identify blood type B antigen mimetic polypeptide-macrophage infliammatory protein 3β （Mip3β） double expression recombinant plasmid. Methods The positive phage clone P1 was obtained using phage random 12-mer peptide library. Specific primers were designed to amplify the phage DNA of P1 and transmembrane domain and inner segment of PBluscript-Fas gene. The products of the amplification were linked into Mip3βv21 to construct blood type B antigen mimetic polypeptide-Mip3β double expression recombinant plasmid. The recombinant plasmid was transfected into human melanoma cell line B16 to identify its expression. Results and conclusion Blood type B antigen mimetic polypeptide-Mip3β double expression recombinant plasmid is successfully obtained and expressed in human melanoma cell line B16.