9 ku颗粒溶素真核表达质粒的构建与分泌表达

目的:构建能分泌表达人9 ku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9 ku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9 ku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9 ku颗粒溶素.结论:成功构建能分泌表达9 ku颗粒溶素真核表达质粒pBudCE4.1-S9K.

Construction and secreting expression of eukaryotic expression plasmid carrying human 9 ku-granulysin gene

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 ku-granulysin gene fragment carrying leader peptide gene was cloned into pBudCE4.1 to construct recombinant plasmid pBudCE4.1-S9K.Identification of pBudCE4.1-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.