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目的:构建新型噬菌体展示载体,评价其展示效果,为构建scFv库寻找新的展示体系。方法:PCR分离、扩增M13KO7中pⅨ蛋白编码基因,克隆入噬菌体展示载体PHB-gⅢ,替换原载体中的gⅢ-CT段,构建载体PHB-pⅨ,并将相对分子质量约50×103的碱性磷酸酶的编码基因分别克隆入两展示载体,观察其展示碱性磷酸酶能力的差别。结果与结论:成功构建了展示载体PHB-pⅨ,该载体具有与PHB-gⅢ相当的展示碱性磷酸酶的能力,且相同滴度的PHB-gⅢ噬菌体DNA含量略高于PHB-pⅨ噬菌体DNA含量,提示PHB-gⅢ系统展示外源蛋白时存在感染能力下降。PHB-pⅨ系统具有用于展示噬菌体抗体库的良好应用前景。
OBJECTIVE: To construct a new phage display vector, evaluate its display effect and find a new display system for constructing scFv library. Methods: The gene encoding pⅨ protein in M13KO7 was amplified by PCR, cloned into phage display vector PHB-gⅢ, replaced with gⅢ-CT fragment in original vector, and constructed vector PHB-pⅨ. The relative molecular mass of about 50 × 103 base The genes coding for the phosphatase were cloned into two display vectors, respectively, to observe their differences in the ability to display alkaline phosphatase. RESULTS AND CONCLUSION: The display vector PHB-pIX was constructed successfully. The vector has the same capability of displaying alkaline phosphatase as PHB-gIII, and the PHB-gⅢ phage DNA content of the same titer is slightly higher than that of the PHB-pIX phage DNA , Suggesting that the PHB-gIII system has the ability to infect the foreign protein when it is displayed. The PHB-pIX system has good prospects for the display of phage antibody libraries.