中文摘要：

目的：构建能分泌表达人9ku颗粒溶素（granulysin）的真核质粒，为下一步利用颗粒溶素基因治疗肿瘤奠定基础．方法：以含有颗粒溶素cDNA序列的质粒为模板，通过聚合酶链反应（PCR）扩增获得含分泌肽编码基因的9ku的颗粒溶素基因片段后，定向插入真核表达载体pBudCE4．1中，获得重组表达质粒pBudCE4．1-S9K，通过PCR，双酶切及插入片段序列测定对重组质粒进行鉴定；采用RT—PCR，免疫细胞化学与Dot—ELISA法检测pBudCE4．1-S9K在RAW264．7细胞的瞬时表达与分泌．结果：成功扩增出含分泌肽编码基因的9ku的颗粒溶素基因片段；经酶切，PCR及测序鉴定证明得到读码框正确的重组质粒；转染细胞证实可分泌表达9ku颗粒溶素．结论：成功构建能分泌表达9ku颗粒溶素真核表达质粒pBudCE4．1-S9K．

英文摘要：

AIM： To construct secreting eukaryotic expression plasmid carrying human 9 ku-granulysin gene and to lay a foundation for the study of granulysin as anti-tumor gene therapy agent. METHODS： After amplified by PCR from the plasmid including granulysin complementary deoxyribonucleic acid （cDNA） , 9 kugranulysin gene fragment carrying leader peptide gene was cloned into pBudCFA. 1 to construct recombinant plasmid pBudCFA. 1- S9K. Identification of pBudCFA. 1-S9K was conducted by PCR , restriction enzyme digestion and sequencing. The recombinant plasmid was transfected into RAW264. 7 cells, and its transient expression was detected by RT -PCR, immunocytochemistry and Dot-ELISA. RESULTS： 9 ku-granulysin gene fragment including leader peptide gene was obtained ; it was confirmed that gene was inserted into eukaryotic expression plasmid correctly; 9 ku-granulusin can be expressed and secreted in transfected cells. CONCLUSION： Eukaryotic expression plasmid pBudCE 4.1-S9K carrying human 9 ku-granulysin gene was constructed successfully.