Study On Pichia Pastor Of Expression For HSA-IFNα-2b Of Different Linker And Bioactivities

Objective Human serum albumin is a protein with higher levels of plasma, which can combine with many substances in the body. Albumin is also a good drug carrier. In the body can effectively extend the drug half-life, but will not to produce immunogenic. This experiment want to make different linker of pPIC9-HSA-IFNa-2b, and then the pPIC9K-HSA-IFNa-2b was cut with restrictive endonuclease sal I, At the same time, the lined plasmid was transformed into Pichia. Pastor SMD1168by electroporation, positive transformants express the target protein by induce of methanol under the right conditions, the supernatant analyzed by SDS-PAGE and Western-Blot, and then the expression level of the fusion albumin interferon was identified by the enzyme-linked immunosorbent assay, and finally using the cytopathic effect inhibition assay (Wish cells-VSV virus) to measure its antiviral activity. Methods Different linker of the human serum albumin interferon by overlapping PCR between albumin and interferon a-2b, the number of amino acids joints are15,10,5under the same conditions, the first joint of each human albumin interferon induced to express, filter out the highest expression level, and then we take three the highest cloning to express by the methanol induction under the same conditions, It was measured by the amount of expression, According to the difference of expression, with a500mL shake flask culture, the preliminary separation and purification of proteins, Finally, we utilized the cytopathic effect inhibition assay for the determination of the size of its activity, respectively, to calculate the specific activity. Results The constructing of three recombinants of the adapter protein expressed in Pichia.pastoris by overlapping PCR, the expression of the15amino acids linker, the10amino acids linker and the5amino acids linker of proteins concentration is194mg/L,135mg/L, and177mg/L, Three kinds of linkers of human albumin interferon specific activity are much lower than specific activity of interferon a-2b, the smaller differences of three kinds of linkers of human albumin interferon.It may affect the active site of interferon a-2b is the presence of albumin.Conclusion we found that the expression level of the human albumin interferon of the15amino acids linker of protein is highest. Followed by five amino acids linkers between albumin and interferon connection (GGGGS)n with a flexible peptide, the connection peptide of15amino acids linker is (GGGGS)3, the connection peptide of10amino acids linker is (GGGGS)2, These studies provides some theoretical guidance for the industrial production.