鸡颗粒溶素的原核表达及生物活性分析

Prokaryotic expression and bioactivity test of chicken granulysin

为了克隆表达纯化鸡颗粒溶素（chicken granulysin,ChGNLY）并进行活力鉴定。本试验以基因组数据库预测的ChGNLY基因为模板设计引物,对提取的鸡脾脏总RNA进行RT-PCR,扩增得到的ChGNLY基因插入pMD-19T载体中并测序。将测序正确的GNLY基因克隆至pGEX-6p-1,构建重组表达质粒pGEX-6p-1-GNLY,并将其转化至感受态BL21,IPTG诱导表达融合蛋白,SDS-PAGE、Western-blotting和蛋白质谱鉴定融合蛋白的正确性。CellTiter-Glo2.0Assay检测融合蛋白的生物学活性。另外,对GNLY的分子特性进行了生物信息分析。结果显示,测序结果显示克隆的ChGNLY片段长237bp,编码79个氨基酸,所表达的融合蛋白相对分子质量约为36 000。重组ChGNLY融合蛋白能特异性与人颗粒溶素抗体发生反应。纯化后的GNLY融合蛋白经CellTiter-Glo2.0Assay检测,发现其具有剂量依赖性的细胞毒活性。系统发生分析显示GNLY可明显分为4个群。结构分析发现,ChGNLY活性区域表面带有大量正电荷,且位点保守。结果表明,本试验利用原核表达系统成功表达了具有生物学活性的ChGNLY,为ChGNLY后续功能的研究奠定理论基础。

Express the chicken granulysin（ChGNLY）protein and detect its bioactivity after purification.The special primers for the ChGNLY fragment was designed based on the sequence in GenBank,and total RNA extracted from chicken spleen was used to RT-PCR.Then the recombinant expression vector pGEX-6 p-1-ChGNLY was constructed and transferred to E.coli（BL21）.Fusion protein was expressed under introduction of IPTG and identified by SDS-PAGE,Western-blotting and protein spectrum.The bioactivity of recombinant ChGNLY（rChGNLY）was measured by CellTiter-Glo~2.0 Assay.In addition,the molecular characteristics of GNLY were analyzed by bioinformatics method.The cloned fragment of ChGNLY was 237 bp and the corresponding 36 000 fusion protein was highly expressed.The rChGNLY was specifically bound to mouse anti-human GNLY antibody and a dose-dependent cytolytic effect of rChGNLY on cultured MDBK cells was detected through CellTiter-Glo~2.0 Assay.Phylogenetic analysis showed that GNLY can obviously divided into four groups in the various species.Structural analysis found active area of ChGNLY surface with much positive charge,and activated sites were conservative.In this study,the rChGNLY was successfully expressed using prokaryotic expression system,which will be beneficial for the further studies on its function.