High level expression of recombinant human serum albumin in CHO cells and optimization of its culture technology / 中国生物制品学杂志

@#ObjectiveTo achieve efficient expression of human serum albumin(HSA)in Chinese hamster ovary(CHO)cells and optimize its culture technology,so as to lay a foundation of the large-scale production of HSA.MethodsThe eukaryotic expression vector of HSA was constructed by gene recombination technology,and then electrotransfected into fully suspended CHO cells. The monoclonal cell lines with stable and high expression of HSA were screened by G418 and limited dilution method. By adding glucose,sodium butyrate and supplementalmedium to the basal medium,the cell culture process was optimized to improve the expression of HSA. Finally,the scale-up culture verification was carried out in a 5 L bioreactor.ResultsThe recombinant expression vector pcDNA3.1-HSA was successfully constructed and expressed in fully suspended CHO cells. After two monoclonal screening,the secondary monoclonal cell lines CHO-rHSA-7H2A9 and CHOrHSA-7H2D12 were obtained with high HSA expression of 29. 37 mg/L and 25. 26 mg/L respectively. The HSA expression level reached about 100. 00 mg/L by optimizing the culture process and wasfinally increased to 166. 16 mg/L in the 5 L bioreactor,which was about 30 times higher than that in the supernatant of the first monoclonal cells.Conclusion The high level expression of HSA in CHO cells was achieved,which laid a foundation of the further large-scale production of HSA in the field of biological products and solving the market supply problems.

High-level expression of Actin of Paeonia lactiflora in Escherichia coli / 中草药

Objective: To construct the prokaryotic expression vector of Actin gene and to establish the high-level expression of Actin of Paeonia lactiflora in Escherichia coli. Methods: Based on cDNA sequence of Actin of P. lactiflora and the polyclonal sites on the prokaryotic expression vector pET-32a (+), a pair of PCR primers whose 5' end was inserted with BamH I and Hind III, respectively were designed. Subcloning of Actin was carried out using RT-PCR technique. The recombinant plasmid pMD18-T-PlActin and the prokaryotic expression vector pET-32a (+) was digested by BamH I and Hind III and the objective gene and the empty vector were purified. After ligation and transformation, the recombinant pET-32a (+)-PlActin, which was characterized by colony PCR, plasmid PCR, and double enzyme digestion, was transformed into BL21 (DE3), and the engineering expression strain was obtained. The expression of recombinant Actin protein in E. coli was induced at different concentration of inducer IPTG, different bacteria density, and different expression time. After SDS-PAGE and Coomassie brilliant blue R250 staining, the dry gel was prepared and the expression level of recombinant Actin protein was analyzed. Results: Subcloning sequencing result showed that the sequence of PlActin was exactly same with the objective sequence and the 5' and 3' ends were successfully inserted with BamH I and Hind III sites. The prokaryotic expression vector of Actin gene pET-32a (+)-PlActin was constructed successfully. The best concentration of IPTG was 0.1 mmol/L and the bacteria density A600 was 0.4 to 0.6. The optimal expression time was 4 h. Conclusion: The prokaryotic expression vector of Actin gene pET-32a (+)-PlActin is constructed and the high-level expression of Actin of P. lactiflora in E. coli is established.

High level expression of human truncated insulin like growth factor 1 in E.coli / 中国免疫学杂志

Objective: To establish an efficient expression system for human truncated insulin like growth factor 1 〔Des(1 3)IGF1〕as fusion protein in Escherichia coli(E.coli). Methods: The cDNA of Des(1 3)IGF1 was cloned into an fusion protein expression plasmid, pMTY4, using gene recombinant technique. The protein was purified by ion exchange chromatography and identified by SDS polyacrylamide gel electrophoresis, radioimmunoassay(RIA), N terminal amino acid sequence and biological activity. Results: A prokaryotic expression vector was constructed and the fusion protein containing MS2 polymerase fragment, thrombin recognition site and human Des(1 3)IGF1 was expressed in E.coli at high level. It was showed that the purified recombinant Des(1 3)IGF1 released from the fusion protein after digestion with thrombin was identical to the native Des(1 3)IGF1.Conclusion:This is an effective method for obtaining human recombinant Des(1 3)IGF1 and it is very important for further study of Des(1 3)IGF1.

HIGH LEVEL EXPRESSION AND ACTIVITY DETECTION OF SINGLE CHAIN IMMUNOTOXIN 183B_2ScFvPE38 AGAINST OVARIAN CARCINOMA / 解剖学报

Objective To prepare the immunotoxin protein (183B 2ScFvPE38) which might be useful in immuno guided therapy for ovarian carcinoma and study the activity of the protein. Methods The methods of ELISA and cytotoxicity were used to study the immunotoxin after induced with IPTG and the activity of the immunotoxin. Results The expressed fusion proteins were detected mostly as inclusion bodies at high level, and soluble immunotoxins were also observed. The results showed liable activity of antibody part and toxic part. Conclusion The recombinant fusion protein 183B 2ScFvPE38 keeps the activity of both components and might be of great use in the future to deal with ovarian carcinoma. [