Of Nt3 Prokaryotic Expression Vector Construction, Expression And Spermatogonial Stem Cells Was Prepared Transgenic Mice Experimental Study

Objective: To establish the high-expression recombinant plasmid pGEX-NT3 in order to obtain lots of human neurotropin 3 protein with biological activity.Methods: First we got the human genome from the marrow of a normal human. And the whole DNA sequence of neurotropin 3 was replicated by polymerase chain reaction and rejoined into the plasmid pGEX-1 λ T after digestion with restriction endonucleases. And then the pGEX-NT3 was transferred to E.coil JM109 by treated with Ca2+ and the bacteria taking up plasmid were selected on a plate with Amp. The recombinant plasmid was identified with restriction endonucleases and the sequence of the rejoined DNA was identified by auto-analysis. The E.coil JM109 with pGEX-NT3 was induced with IPTG and the fused protein with NT3 was analyzed by SDS-PAGE.Results: A 730bp DNA was obtained by PCR. And the gel analysis showed that the recombinant plasmid included the NT3 sequence that wasconsistent with the reported literature except for a mutation in 198th base of NT3. The bacteria with pGEX-NT3 expressed a special 54kD fused NT3 protein and mostly formed, inclusion body.Conclusions: We successfully established the recombinarit plasmid pGEX-NT3.The results indicated the fused protein could be efficiently expressed in the eukaryotic cell.