同种异型抗原激活并套式PCR法克隆人颗粒溶素Mr 15000与Mr 9000活性片段  

The cDNA cloning of human granulysin Mr 15000 and Mr 9000 active segments from CTL activated by allogenic antigen

作　　者：伊正君[1] 朱道银[1] 杨春[1] 何永林[1] 刘晔华[1] 

机构地区：[1]重庆医科大学基础医学院微生物学与免疫学教研室,重庆400016

出　　处：《重庆医科大学学报》2005年第6期777-780,共4页Journal of Chongqing Medical University

基　　金：国家自然基金项目资助(30400375)。

摘　　要：目的:采用套式PCR克隆经同种异型抗原激活后的人细胞毒性T淋巴细胞(CTL)表达的颗粒溶素(Granulysin, GLS)Mr 15000与Mr 9000活性片段的编码序列并进行序列测定与分析,为进一步表达纯化该活性片段和研究GLS的免疫杀伤机制奠定基础。办法:抽提经过培养并激活的健康人CTL淋巴细胞总RNA,经逆转录后以套式PCR方法扩增出GLS的Mr 15000 与Mr 9000活性片段的编码序列,插入pET32a(+)载体,转化大肠杆菌TOP10,阳性克隆经PCR及酶切鉴定后进行测序分析。结果:成功扩增出了包含GLSMr 15000与Mr9000活性片段完整编码序列的cDNA,与预期大小相同,PCR、酶切及测序鉴定证明得到了读码框正确的pET32a-Mr 15000与pET32a-Mr 9000重组子。序列分析表明GLS基因编码产物在第119 位存在氨基酸多态性。结论:人天然颗粒溶素Mr 15000与Mr 9000活性片段编码序列能够通过以上方法获得和克隆并用于后续研究。Objective： To clone, sequence and analyze the coding sequences of the Mr 15000 and Mr 9000 active segments of natural granulysin derived from human CTLs activated by allogenic antigen ,in order to establish the basis for further purifying and investigating the immune - impairing mechanism of granulysin. Methods： The coding sequences of the Mr 15000 and Mr 9000 granulysin gene were amplified from the total RNA of activated CTLs of healthy person after reverse transcription by Nested - PCR, inserted into pET32a （ ＋ ） vectors and then transformed into E. Coli TOP10, respectively. The recons were identified by PCR, endonuclease digestion and sequencing. Results： The whole coding sequences of the Mr 15000 and Mr 9000 active segments were successfully cloned as expected. The accurate pET32a- Mr 15000 and pET32a- Mr 9000 recons were obtained through Colony -PCR, endonuclease digestion and sequencing. There lied polymorphism on the 119 th amino acid of the product encoded by GLS gene. Conclusion： The coding sequences of the Mr 15000 and Mr 9000 active segments of human granulysin can be obtained and cloned by the methods mentioned above and can be used for subsequent research.

关 键 词：同种异型抗原 颗粒溶素 细胞毒性T淋巴细胞 套式PCR 

分 类 号：R378.91[医药卫生—病原生物学]