Construction Of The Engineering Pichia Pastoris For HSA-G₄SRKDRRG₄S-ONC And Biological Activity Analysis Of The Expressed Fusion Protein

During the last two decades,modern biotechnology has developed rapidly and made significant progress and breakthroughs.Especially in the medical industry,the development of genetic engineering pharmaceuticals has made a significant contribution to human health.Among them,Leopard frog anti-tumor enzyme?ONC?,an enzyme of natural tumor cells belongs to ribonuclease,has shown strong lethality against tumour cells.It has been well advanced in both in vivo and in vitro tumor research,however the ONC used in clinical trials was extracted from frog eggs and early embryos which has limited sources,and is difficult to obtain,and have relatively low yields,making it hard to fulfill its clinical needs.On the other hand,human serum albumin?HSA?was known to have a high expression property.Combining the effects of ONC and HSA to produce a fusion protein with high activity and high expression will produce huge social benefits and promote the medical industry to further.In previous work,our research group had obtained a highly expressed fusion protein HSA-ONC from the fusion protein HSA-ONC.To further increase the expression level of HSA-ONC,anti-tumor activity,and selectivity to tumor cells,we intended to achieve the above object by adding a specific linker peptide between HSA and ONC.In this study,the RKDRR sequence fragment was inserted to form a G₄SRKDRRG₄S linker peptide with high positive and negative ionization and high hydrophilicity based on the existing fusion protein linker peptide?GGGGS?n..This study was accomplish by four successive steps:firstly,constructing the engineered strain;then,expressing the fusion protein HSA-G4SRKDRRG4S-ONC;and isolating and purifying the fusion protein;at last,the biological activity of the fusion protein HSA-G₄SRKDRRG₄S-ONC was determined..It is expected that the linker peptide does not affect the activity of the two proteins,but also contributes to maintaining or even increasing the expression levels of both proteins and the selectivity against tumor cells.In the construction of the fusion protein HSA-G4SRKDRRG4S-ONC,the recombinant plasmid pPIC9K?HSA and pPIC9K?ONC were constructed and the recombinant plasmid pPIC9K?HSA-was constructed by overlapping PCR and positive clones.G₄SRKDRRG₄S-ONC and Pichia pastoris GS115?pPIC9K?HSA-G₄SRKDRRG₄S-ONC,followed by expression of fusion protein HSA-G₄SRKDRRG₄S-ONC,first GS115?pPIC9K?HSA-G4SRKDRRG4S-ONC Pichia pastoris Enlarged culture was carried out to induce expression.After 5 days of induction,the fermentation broth was centrifuged,and the supernatant was taken for detection of the expression of the fusion protein HSA-G₄SRKDRRG₄S-ONC by SDS-PAGE.Then,when the fusion protein HSA-G₄SRKDRRG₄S-ONC is isolated and purified,the supernatant of the fermentation broth containing the fusion protein HSA-G4SRKDRRG4S-ONC is subjected to non-denaturing polyacrylamide gel electrophoresis?PAGE?,and after electrophoresis,according to the target protein in the gel The target protein was cut out in the plate,homogenized,the target protein was washed out,the supernatant was collected by centrifugation,and lyophilized in vacuo to obtain a fusion protein HSA-G₄SRKDRRG₄S-ONC powder,which was stored at-80°C for use.Finally,when measuring the biological activity of the fusion protein HSA-G4SRKDRRG4S-ONC,the fusion protein HSA-G4SRKDRRG4S-ONC was treated with0?M,0.1?M,0.2?M,0.5?M,0.8?M,1?M,1.5?M,2?M concentration for 24 h.The hepatocellular carcinoma cell line CBRH-7919 was assayed for cell viability after 48 h treatment by MTT assay.The results showed that the semi-lethal dose(IC₅₀)of rat hepatoma cell line CBRH-7919 was 0.39±0.14?M.The distribution of cell cycle phase and apoptosis rate were detected by flow cytometry.The results showed that the proportion of cells in G0/G1 phase of CBRH-7919,which was treated with fusion protein HSA-G₄SRKDRRG₄S-ONC,was higher than that of the control group.The proportion of cells in S phase and G2/M phase began to decrease.The apoptosis rate of fusion protein HSA-G4SRKDRRG4S-ONC treatment group was significantly higher than that of the control group.The expression of Caspas8 and BAX protein in cells was detected by Western Blot.The expression of apoptosis-related proteins Caspas8 and BAX was up-regulated,indicating that the fusion protein inhibits the activity of hepatoma cells by regulating the expression of apoptosis-related proteins.In summary,in this study we designed the G4SRKDRRG4S linker peptide,isolated and purified the fusion protein HSA-G4SRKDRRG4S-ONC and tested its biological effects,found that the fusion protein is more toxic to rat hepatoma cells than normal cells,and through regulating cells apoptosis-related protein expression and inhibits liver cancer cell activity.