Construction Of The Engineering Pichia Pastoris For HSA-G₄SKEEEKG₄S-ONC And Biological Activity Analysis Of The Expressed Fusion Protein

With the development of biotechnology,genetic engineering pharmacy based on genetic recombination technology has become an important development trend of the medical industry.Among them,onconase?ONC?is a ribonuclease that has a strong effect on tumor cells.However,the ONC used in clinical trials comes from natural extracts,the source is limited,and the yield is extremely low,which limits its wide application.It is imperative to solve this problem by genetic engineering.On the other hand,human serum albumin?HSA?is the most abundant protein in the blood and has a wide range of biological effects.If genetic engineering methods can be used to prepare high expression recombinant HSA,it will have important medical and economic significance.If we can further combine the anti-tumor effect of ONC with the extensive biological effects of HSA,a fusion protein with high activity and high expression characteristics will produce huge economic,social and medical value.To this end,the research team has constructed the Pichia pastoris engineering strain of the fusion protein HSA-ONC,and obtained the highly expressed fusion protein HSA-ONC.To further increase the expression level of HSA-ONC,anti-tumor activity and selectivity to tumor cells,it is intended to achieve the above objectives by adding a specific linker peptide between HSA and ONC.Based on the existing fusion protein-binding peptide?GGGGS?_n,KEEEK was added to form a G₄SKEEEKG₄S linker peptide with high positive and negative ionization and high hydrophilicity.It is expected that the linker peptide does not affect the activity of the two proteins to which it is linked,but also contributes to maintaining or even increasing the expression levels of both proteins and the selectivity to tumor cells.In the construction of the fusion protein HSA-G₄SKEEEKG₄S-ONC,the pPIC9K?HSA and the recombinant plasmids pPIC9K?ONC constructed in our laboratory were used to add the linker G₄SKEEEKG₄S between HSA and ONC.The specific method is as follows:the primers are design,and the HSA-G₄SKEEEKG₄S-ONC gene sequence is synthesized by overlapping PCR technology,and then construction of recombinant?amplification?plasmid pMD20-T?HSA-G₄SKEEEKG₄S-ONC,which is transfer into E.coli DH5?,then screening and identificing for positive clone.Then,the correct positive bacteria were cultured,and the plasmid was extracted,and the recombinant plasmid pPIC9K?HSA-G₄SKEE EKG₄S-ONC was constructed and transferred into E.coli DH5?,and the positive clones was identified and screened.Then,taking the identified strain to expand the culture,extracting the recombinant?expression?plasmid and transferring it into P.pastoris GS115 for constructing GS115?pPIC9K?HSA-G₄SKEEEKG₄S-ONC P.pastoris,and screened and identified for positive clone.The positive P.pastoris engineering GS115?pPIC9K?HSA-G₄SKEEEKG₄S-ONC was used for subsequent experiments.To express the fusion protein HSA-G₄SKEEEKG₄S-ONC,GS115?pPIC9K?HSA-G₄SKEEEKG₄S-ONC P.pastoris was expanded and cultured,and induced by 1%methanol.After induction for 5 days,the fermentation broth was centrifuged,and the supernatant was taken for detection of the expression of the fusion protein HSA-G₄SKEEEKG₄S-ONC by SDS-PAGE.To isolated and purify HSA-G₄SKEEEKG₄S-ONC,the supernatant of the fermentation broth containing the fusion protein HSA-G₄SKEEEKG₄S-ONC was subjected to non-denaturing polyacrylamide gel electrophoresis?PAGE?,wherein the separation gel concentration was 10%,and the voltage was 90V,at an ambient temperature of 4°C.After electrophoresis,the target protein is cut according to the target protein in the gel plate,homogenized,the target protein is washed out,the supernatant is collected by centrifugation,and freeze-dried in vacuo to obtain the fusion protein HSA-G₄SKEEEKG₄S-ONC.The powder was obtained and saved at-80°C.In the deter mination of the biological activity of the fusion protein HSA-G₄SKEEEKG₄S-ONC, the fusion protein HSA-G₄SKEEEKG₄S-ONC at a concentration of 0?M?0.1?M?0.2?M?0.5?M?0.8?M?1?M?1.5?M?2?M were deal with the rat hepatoma cell CBRH-7919 which were cultured in vitro for 24 h.Then detected the cell viability by MTT chromogenic assay.The results showed that the cell activity gradually decreased with the increase of drug concentration,and it was concentration dependent.Flow cytometry to detect cell cycle phase distribution and apoptotic rate.The results showed that the rat hepatoma cell CBRH-7919 treated with the fusion protein HSA-G₄SKEEEKG₄S-ONC had a higher proportion of cells in the G0/G1 phase than in the control group,but in the S phase and G2/M phase the proportion of cells begins to decrease.The apoptosis rate of the fusion protein HSA-G₄SKEEEKG₄S-ONC treatment group was significantly higher than that of the control group.Western Blot was used to detect the expression changes of c-Jun and BAX proteins in cells.The results showed that the expression of cell proliferation-related protein c-Jun was down-regulated,and the expression of apoptosis-related protein BAX was up-regulated.It is indicates that the fusion protein inhibits the activity of liver cancer cells by regulating cell proliferation/apoptosis-related protein expression.In summary,the G₄SKEEEKG₄S linker peptide was designed and the recombinant plasmid pMD20-T?HSA-G₄SKEEEKG₄S-ONC,recombinant plasmid pPIC9K?HSA-G₄SKEEEKG₄S-ONC and the P.pastoris engineered GS115?pPIC9K?HSA-G₄SKEEEKG₄S-ONC were constructed.Then the fusion protein HSA-G₄SKEEEKG₄S-ONC was expressed.Isolation and purification of the protein and detection of its biological effects,found that the fusion protein inhibits the proliferation of CBRH-7919 and promotes its apoptosis,and inhibited the activity of hepatoma cells by regulating cell proliferation/apoptosis-related protein expression.