The cDNA cloning of human granulysin Mr 15000 and Mr 9000 active segments from CTL activated by allogenic antigen
- VernacularTitle: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
- Author:
Zhengjun YI
;
Daoyin ZHU
;
Chun YANG
;
Yonglin HE
;
Yehua LIU
;
- Publication Type:Journal Article
- Keywords:
Allogenic antigen;
Granulysin;
CTL;
Nested - PCR
- From:
Journal of Chongqing Medical University
2003;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
