Recombinant Adenovirus Infected HaCat Cells Express Human Kerotinocyte Growth Factor (hKGF) / 中国生物工程杂志

Human kerotinocyte growth factor (hKGF) gene amplified by PCR was inserted into the shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hKGF, which was linearized with Pme I and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 to obtain the recombinant adenoviral plasmid pAdEasy-hKGF. The recombinant adenoviral plasmid was then transfected into HEK-293 cell lines via Lipofectamine 2000 to package and amplify the recombinant adenovirus containing hKGF gene detected by PCR. The recombinant adenovirus produced could effectively infect HaCat cells. The result of Western blotting showed that HaCat cells infected with the recombinant adenovirus expressed and secreted hKGF protein.

Expression of Recombinant Snake Venom Cystatin in Yeast Pichia pastoris and Its Effects on B16F1 Melanoma Invasion in vitro / 中国生物工程杂志

To investigate the biological role of snake venom cystatin(sv-cystatin) in tumor progression, the cDNA of sv-cystatin amplified by PCR from pUC18-cystatin plasmid was cloned into methanol-inducible expression vector pPICZ?A. The linearized recombinant plasmid pPICZ?A-cystatin was transfered into Pichia pastoris, strain GS115 by electrophoration. Transfermants with phenotype Mut+ selected were identified by PCR analysis and induced in 1.0% methanol. The reombinant sv-cystatin protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of expression product was about 14 kDa and approximately 16 mg/L of recombinant sv-cystatin was produced from one of GS115-cystatin transformants. The chromatography purified protein could reduce the activity of papain. The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly (P

Study on recombinant chicken ? interferon production in yeast Pichia pastoris and detection for its antiviral activity / 中国免疫学杂志

Objective:To obtain eukaryotic expressing protein of chicken interferon ? (ChIFN-?) and research its anti-virus activity.Methods: Chicken interferon ? mature protein gene was cloned and amplified by reverse transcripition-polymerase chain reaction(RT-PCR) from the total mRNA in the lymphocyte of chicken blood stimulated with ConA for 4～10 hours.The gene was inserted into the expression vector pPICZa-A,which had been cleaved by EcoR I and Xba I.The recombinant vector was linearized by Sac I and transferred into yeast Pichia pastoris,strain X33,anti-virus activity of the recombinant cytokine was detected by the classical experiment cell pathological effect inhibition assay.Results:The result showed that the preparation of recombinant interferon had higher anti-virus activity(10?48 U/ml).Conclusion: The recombinant chicken interferon ? with anti-virus bioactivity has been obtained.