Clone And Expression The CMP-Sialic Acid Transporter Gene From CHO Cell In E.coli

Objective:The gene of CMP-sialic acid transporter(CMP-sat) obtained from the Chinese Hamster Ovary(CHO) cell was cloned and expressed in the E.coli.It was the good foundation to study this gene and protein translated by the gene deeply.Methods:1.Extract the total RNA from CHO cell.2.Obtain the gene of CMP-sialic acid transporter by RT-PCR.3.Construct the clone vector(CMP/pMD 18-T) and sequence the target gene.4.Construct the expression vector(CMP/pET42a) containing the CMP-sat gene.5.CMP/pET42a was expressed by inducing of IPTG.The expressed product of CMP-sialic acid transporter gene was analyzed by SDS-PAGE.Results:1.The RNA extracted from CHO cell was detected by electrophoresis.From the 1%agarose electrophoresis,18S and 28S bands were found,and depth of 28S was two times of 18S.2.From the 1%agarose electrophoresis of RT-PCR and PCR,1000bp band was found,and the size was same with the result of CMP-sat in the Genbank.The gene of CMP-sat was gotten successfully by RT-PCR.3.The target gene constructed in the clone vector(CMP/pMD18-T) was sequenced,and the base pair were same with the sequence of CMP-sat gene from CHO in the Genbank.4.The target gene in the vector CMP/pET42a was induced by IPTG and expressed.Expressed product was analyzed by SDS-PAGE and the 36.4KD band was found.This result was digit and analyzed by computer,and target protein is accounted for 38.5 percent of total bacterial protein. Conclusions:Make use of molecular biological technology,author clone successfully the gene of CMP-sialic acid transporter from the CHO cell,and target gene was expressed in the E.coli system.