中文摘要：

目的：构建人颗粒溶素（Granulysin,GNLY）基因的表达载体并在大肠杆菌中表达。方法：抽提体外培养的单个核细胞总RNA,通过RT-PCR的方法获得含有222bp的颗粒溶素cDNA,克隆到pMD18-T载体,测序正确后再亚克隆到质粒pET28a（＋）中,构建重组表达质粒pET28a（＋）-GNLY,在大肠杆菌中诱导表达并经Ni亲和层析纯化。用SDS-PAGE和Westernblot对表达产物进行鉴定。采用MTT法检测GNLY融合蛋白的生物学活性。结果：酶切结果证实,成功地构建了GN-LY原核表达载体,并在大肠杆菌中获得稳定的表达,表达产物的相对分子质量（Mr）同预期值相一致。结论：成功构建了重组表达质粒pET28a（＋）-GNLY,并在大肠杆菌BL21（DE3）plysS中表达,获得了具有良好的抗原性和特异性的GNLY融合蛋白,为下一步研究人GNLY的功能奠定了基础。

英文摘要：

Objective： To construct a recombinant expression vector of human granulysin gene and to express it in E coli. Methods：Total RNA from cultured cells and obtained a 222bp cDNA segment with granulysin-specific primers by RT-PCR were extracted. Then the cDNA segment of granulysin was inserted into pET28a （ ＋ ） plasmid. Expression of the recombinant protein was induced, which was purified by Ni-NTA affinity chromatography and approved by SDS-PAGE and Western blot. The bioactivity of granulysin fusion protein was measured by MTT assay. Results： Restriction enzyme digestion showed that the recombinant prokaryotic expression plasmid pET28a（ ＋ ）-GNLY was constructed and expressed in E coil. The relative molecular mass （Mr ）of the expression product was identical with the predicted value. Conclusion：The recombinant expression plasmid pET28a（ ＋ ）-GNLY has been constructed success- fully in E coil BL21 （DE3）plysS and the target protein are expressed with a fine antigenicity, which is helpful for the further study of the biological function of granulysin.