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目的:构建人4-磷酸-磷脂酰肌醇-5-激酶(phos-phatidylinositol-4-phosphate5-kinases-like1,PIP5KL1)质粒并在大肠杆菌中表达,纯化得到相对分子质量(Mr)为42000的融合蛋白,免疫新西兰大白兔制备兔抗PIP5KL1多克隆抗体。方法:采用PCR的方法获得编码人PIP5KL1的cDNA序列,将其克隆入pET-32a,构建pET32a-PIP5KL1表达载体。在大肠杆菌BL21(DE3)中IPTG诱导下表达6×His-融合蛋白,纯化后免疫新西兰兔,制备兔抗人PIP5KL1血清,以Westernblot和ELISA方法分析其特异性和效价。结果:PIP5KL1融合蛋白免疫新西兰兔成功获得多克隆抗体,West-ernblot结果显示制备的抗体具有较高的特异性,ELISA显示效价为1∶100000。结论:获得了纯化的PIP5KL1融合蛋白和anti-PIP5KL1多抗血清,为今后对新基因PIP5KL1功能研究打下了基础。
OBJECTIVE: To construct the plasmid pPHOS-4-phosphate5-kinases-like1 (PIP5KL1) and express it in Escherichia coli (E.coli). The purified product has a molecular weight of 42,000 Fusion protein was used to immunize New Zealand white rabbits to prepare rabbit anti-PIP5KL1 polyclonal antibody. Methods: The cDNA encoding human PIP5KL1 was obtained by PCR and cloned into pET-32a to construct pET32a-PIP5KL1 expression vector. The 6 × His-fusion protein was expressed under the induction of IPTG in E.coli BL21 (DE3), and purified to immunize New Zealand rabbits to prepare rabbit anti-human PIP5KL1 serum. The specificity and titer of the recombinant protein were analyzed by Western blot and ELISA. RESULTS: Polyclonal antibody was successfully obtained from New Zealand rabbits immunized with PIP5KL1 fusion protein. The results of West-ernblot assay showed that the prepared antibody has high specificity. The ELISA titer was 1: 100,000. Conclusion: The purified PIP5KL1 fusion protein and anti-PIP5KL1 multi-antiserum were obtained, which laid the foundation for the future study on the function of PIP5KL1.