9 ku granulysin Construction of eukaryotic expression plasmid expression and secretion of.docVIP

 PAGE \* MERGEFORMAT 15 9 ku granulysin Construction of eukaryotic expression plasmid expression and secretion of Authors: Yong-lin, ZHU Dao Yin, Yi Zheng-Jun, Yang Chun, Xu Lei [Keywords:] granulysin; eukaryotic expression plasmid; secretory expression Construction and secreting expression of eukaryotic expression plasmid carrying human 9 kugranulysin gene [Abstract] AIM: To construct secreting eukaryotic expression plasmid carrying human 9 kugranulysin gene and to lay a foundation for the study of granulysin as antitumor 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 pBudCE4.1 to construct recombinant plasmid pBudCE4.1S9K. Identification of pBudCE4.1S9K 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 DotELISA. RESULTS: 9 kugranulysin gene fragment including leader peptide gene was obtained; it was confirmed that gene was inserted into eukaryotic expression plasmid correctly; 9 kugranulusin can be expressed and secreted in transfected cells. CONCLUSION: Eukaryotic expression plasmid pBudCE4.1S9K carrying human 9 kugranulysin gene was constructed successfully. [Keywords] granulysin; eukaryotic expression plasmid; secreting expression [Abstract] Objective: To construct 9 ku secreting expression of human granulysin (granulysin) the eukaryotic plasmids for further use granulysin lay the foundation for cancer gene therapy. Methods: The cDNA sequence containing granulysin plasmid as a template, by polymerase chain reaction (PCR) amplified with the gene encoding secreted peptides of 9 ku granulysin gene fragments, the orientation into the eukaryotic expression vector pBudCE4.1, the availability of recombinant expression plasmid pBudCE4.1S9K, by PC