| onconase(ONC),Amphibious enzyme,or P-30 is a protein initially purified from extracts of Rana pipiens oocytes,it was homologous to pancreatic RNase A,onconase causes tumor cell death.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)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 significance,If we can further combine the anti-tumor effect of ONC with the extensive biological effects of HSA,Combines the anti-tumor properties of ONC with the biological effects of HSA will produce huge medical significance。To this end,the research Based on the high expression of HSA,the HSA-ONC fusion protein was constructed by conceiving and designing the linker,which not only satisfies the biological effects of the onconase,but also meets the requirements for expression.In order to enable the successful expression of the fusion protein,KNNNK was added to form a G4SKNNNKG4 S linker with negative ionization and high hydrophilicity on the basis of the flexible linker(GGGGS)n,thereby improving the water solubility of the fusion protein.It is easier to carry out the separation and purification of the protein.It is hoped that the linker peptide will not affect the biological activity of the two proteins to which it is linked,but also help to maintain or even increase the expression levels of both proteins and the selectivity to tumor cells.In this experiment,the recombinant plasmid pPIC9K∕HSA and pPIC9K∕ONC were used in the laboratory,and the previously designed linker G4SKNNNKG4 S was added between the fusion protein HSA-G4SKNNNKG4S-ONC.The specific method is as follows: the primers are design,and the HSA-G4 SKNNNK G4S-ONC gene sequence is synthesized by overlapping PCR technology,and then ligated with the amplification vector pMD20-T to form recombinant(amplification)plasmid pMD20-T∕HSA-G4 SKNNNK G4S-ONC,which is transfer into E.coli DH5α,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-G4SKNNNKG4S-ONC was constructed and transferred into E.coli DH5α,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-G4SKNNNKG4S-ONC P.pastoris,and screened and identified for positive clone.The positive P.pastoris engineering GS115∕pPIC9K∕HSA-G4SKNNNKG4S-ONC was used for subsequent experiments.The P.pastoris GS115∕pPIC9K∕ HSA-G4 S KNNNKG4S-ONC was expanded and cultured,then cultured by fermentation,induced by 1% methanol,induced by 5 days,centrifuged after fermentation,and the supernatant was taken for detection of the expression of the fusion protein HSA-G4SKNNNKG4S-ONC by SDS-PAGETo isolated and purify HSA-G4SKNNNKG4S-ONC the supernatant of the fermentation broth containing the fusion protein HSA-G4SKNNNKG4S-ONC was subjected to non-denaturing polyacrylamide gel electrophoresis(PAGE)wherein the separation gel concentration was 10%,and the voltage was 90 V,at 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-G4SKNNNKG4S-ONC.The powder was obtained and saved at-80 °C.Then detecting whether the fusion protein HSA-G4SKNNNKG4S-ONC has biological activity,the specific method isthe fusion protein HSA-G4SKNNNKG4S-ONC at a concentration of 0μM、0.1μM、0.2μM、0.5μM、0.8μM、1μM、1.5μM、2μM were deal with the rat normal liver cell BRL-3A and the rat hepatoma cell CBRH-7919 which were cultured in vitro for 24 h.After 48 hours of cultivation,Then detected the cell viability by MTT chromogenic assay.The results showed that the semi-lethal dose(IC50)of fusion protein to the rat hepatoma cell CBRH-7919 was 0.64 ± 0.060μM,The activity of cells showed a gradual decrease with the increase of drug concentration,and showed concentration dependence,Then,the distribution of cell cycle phase and apoptosis rate were detected by flow cytometry.The results showed that the proportion of cells in the G0/G1 phase of the rat hepatoma cell line CBRH-7919 treated with the fusion protein HSA-G4SKNNNKG4S-ONC was higher than that of the control group.The proportion of cells in the S phase and G2/M phase began to decrease,and the apoptosis rate of the fusion protein HSA-G4SKNNNKG4S-ONC was significantly increased compared with the control group.Western Blot was used to detect the expression changes of Caspas8 and BAX proteins in cells.The expression of Caspas8 and BAX protein in the cells was detected by Western Blot.The results showed that the expression of apoptosis-related proteins Caspas8 and BAX were up-regulated,indicating that the fusion protein inhibits the activity of hepatoma cells by regulating the expression of apoptosis-related proteins.In summary,the G4SKNNNKG4 S linker peptide was designed and the recombinant plasmid pMD20-T∕HSA-G4SKNNNKG4S-ONC,recombinant plasmid pPIC9K∕HSA-G4SKNNNKG4S-ONC and the P.pastoris engineered GS115∕pPIC9K∕HSA-G4SKNNNKG4S-ONC were constructed.Then the fusion protein HSA-G4SKNNNKG4S-ONC was expressed,The protein was isolated and purified and its biological effects were detected.It was found that the fusion protein inhibits the activity of hepatoma cells by regulating the expression of apoptosis-related proteins in rat hepatoma cells. |
