Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous...Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.展开更多
A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signalpolypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vectorpEx31B of E.coli.The aut...A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signalpolypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vectorpEx31B of E.coli.The authors identified the recombinant plasmid,designated pEx31-IL11,by restriction endonu-cleases digestion and DNA sequencing.The resulting recombinant plasmids were then used to transform E.colistrain HB101,and expression in the PL promoter system,which is temperature-regulated,was achieved.The ex-pressed fusion protein amounts to 50% of total bacterial proteins.The hIL-11 protein expressed in E.coliwas fused to the N-terminal 99 amino acids of the MS2 polymerase to form the inclusion body.Theserecombinant proteins can be purified to about 80% by extracting inclusion body with urea.OneIL-6-dependent cell line 7 TD1 was used for bioassay.The recombinant hIL-11 protein was preliminarily puri-fied and renatured to a specific activity of 10~5U/mg,even in the presence of an excess of a neutralizing an-ti-IL-6 antibody.展开更多
基金research grant from the National NaturalScience Foundation of China (No. 39870380, 39670006) the ScienceFoundation of PLA (
文摘Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 (and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.
文摘A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signalpolypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vectorpEx31B of E.coli.The authors identified the recombinant plasmid,designated pEx31-IL11,by restriction endonu-cleases digestion and DNA sequencing.The resulting recombinant plasmids were then used to transform E.colistrain HB101,and expression in the PL promoter system,which is temperature-regulated,was achieved.The ex-pressed fusion protein amounts to 50% of total bacterial proteins.The hIL-11 protein expressed in E.coliwas fused to the N-terminal 99 amino acids of the MS2 polymerase to form the inclusion body.Theserecombinant proteins can be purified to about 80% by extracting inclusion body with urea.OneIL-6-dependent cell line 7 TD1 was used for bioassay.The recombinant hIL-11 protein was preliminarily puri-fied and renatured to a specific activity of 10~5U/mg,even in the presence of an excess of a neutralizing an-ti-IL-6 antibody.
