The Study On Recombinant Human Chorionic Gonadotropin (HCG) Expression In E.coli

In the study,for exploring the expression of human chorionic gonadotropin in the DH5? of E.coli,we mainly used PGEX-4T-1 plasmid to construct the recombinant plasmid.Then the recombinant plasmid was imported into E.coli BL 21,and we explored its expression ability.First of all,we used a small amount of preparation methods of DNA extraction to extract expression plasmid PGEX-4T-1 and the plasmid containing the HCG gene respectively.We found a sequence of genes that we need mostly,which is as a template of PCR technology to amplificate these genes,and designed the upstream and downstream primers with restricition endonucleases Sal ? and Bgl ? enzyme tanent points.The PCR products were purified and then used Sal ? and Bgl ? for double enzyme;the enzyme products have the same sticky end and carrier.Then PGEX – 4 T-1 was used different enzyme digestion,besides these enzyme products carrier and HCG with the same sticky end of enzyme products were connected.We successf?lly constructed the recombinant plasmid.Then recombinant plasmid was injected into E.coli BL 21(DE 3),which in LB solid medium containing ampicillin screened positive bacteria colony,amplification and then put forward the double enzyme identification of plasmid,and PCR identification.Finally the recombinant plasmid was send to sequencing detection,after a series analysising,we confirmed that the gene sequences were correct,recombinant plasmid construction was successf?l.PGEX-4T-1-HCG,recombinant plasmid was successf?lly construced.These recombinant plasmids were injection into E.coli BL21(DE3),and then used IPTG(isopropyl-B-D-galactose and glycosides)induced its expression.The products were deal with SDS-PAGE electrophoresis analysis.SDS-PAGE and western blot res?lts showed that products of expression by the E.coli BL21(DE 3)were correct with the aim protein,the molecure weight was the protein which we want,and the 37 KD is match aim protein.though the single factor experiment,optimization of E.coli expression condition of culture medium,culture temperature and culture time,IPTG concentration and the concentration of bacteria,and other conditions,to improve the output of target protein.