Construction of a Retplase, Its Mutants and Expression in E. Coli and CHO Cells
|Keywords||t-PA rPA PCR|
In this trial, according to the structure and the function of tissue typeplasminogen activator, wild type of tissue type plasminogen activator cDNA wasused as template. rPA cDNA, a mutant of tPA, was amplified by polymerase chainreaction and cloned. Three mutants, based on the rPA, were also obtained bychanging KHRR296 to 299AAAA, 473A to 473S, and KHRR296, 473A to299AAAA, 473S. After they were cloned into prokaryotic and eukaryoticexpression vectors, plasmids pErA, pErA (K), pErA (KA), pErA (A) and pCSRA,pCSRK were obtained. All these plasmids were separately transformed into cellfor identification of expression. And the plasmids pCSRA, pCSRK weretransformed into CHO-dhfr-cells for stable expression. It was shown that ①theproduct from E Coli was reactive with the antibodies against tPA, after refolding,the product was shown to be fibrinolytic. ②eukaryotic expression vectors weretranscribed in cos-7 cells as showed in FAPA assay. This indicated that the changeof the amino acids did not affect the fibrinolytic activity. ③ expressionCHO-dhfr-cell clones of rPA and its mutant rPA (K) were established, the westernblotting analysis showed that the expressed product had specific reactivity withthe antibody and was fibrinolytic. Both the level and the duration of expressionare stable. It has laid a foundation for establishment of stable expressioneukaryotic cell line, further purification, in vivo activity assay, and commercialpreparation of tPA and for its potential clinical application.