抗Granulysin多克隆抗体的制备及鉴定

Preparation and identification of polyclonal antibody to granulysin

目的构建人颗粒溶素(granulysin,GNLY)质粒并在大肠杆菌中表达,纯化得到9kD目的蛋白片段,免疫新西兰大白兔制备兔抗颗粒溶素多克隆抗体。方法采用RT-PCR的方法获得编码人颗粒溶素的cDNA序列,将测序正确的片段克隆入pET-28a(+),构建pET28a(+)-GNLY表达载体。在大肠杆菌BL21(DE3)plysS中IPTG诱导下表达6×His-融合蛋白,纯化后免疫新西兰兔,制备兔抗人颗粒溶素血清,以Western Blotting和ELISA方法分析其特异性和效价。结果6×His融合蛋白免疫新西兰兔成功获得多克隆抗体,Western Blotting结果显示制备的抗体具有较高的特异性,ELISA显示效价为1∶6000。结论获得了纯化的颗粒溶素9kD蛋白和anti-GNLY多抗血清,为今后对疾病中CTL和NK细胞活性的研究打下了基础。

[Objective] The cDNA of human granulysin （GNLY） was cloned, then the 9 kD GNLY was expressed in E.coli, purified and immunized rabbit to prepare anti-granulysin polyclonal antibody. [Methods] The GNLY cDNA Was obtained by RT-PCR. Then the correctly identified sequence was cloned into the prokaryotic expression vector pET28 a （＋） -vector to construct pET28a （＋） -GNLY expression vector. 6xHis fusion protein was expressed in BI21 （DE3） plysS with IPTG induction. After purification, the fusion protein was used to immunize New Zealand rabbits to get the polyclonal anti-GNLY antibody. [Results] The ployclonal anti-GNLY antibody was successfully obtained by immunizing New Zealand rabbits with 6xHis fusion protein. The specificity of ployclonal anti-GNLY antibody was validated by Western Blotting. ELISA showed that the titers of anti-GNLY antibody are 1：6 000. [Conclusion] Purified 9 kD GNLY and anti-GNLY polyclonal antibody can be used for further study on CTL and NK cells activity in diseases.