Preparation And Activity Of LHRH-TAT-p53 Fusion Protein

Tumors are caused by the abnormal differentiation of cells due to the normal tissues in vivo under the induction of external factors,and the malignant tumor is called cancer in medicine and clinical medicine.According to the results of WHO's prediction of global cancer in 2014,it shows that the number of cancer patients worldwide will increase rapidly.In the coming decades,the total number of various types of cancer is likely to break above 22 million annually,and because of cancer,the number of deaths will increase to about 13 million.With the increase of people suffering from cancer worldwide,anti-tumor treatment methods are innovated constantly.p53 is widely recognized as a special gene that inhibits cancer.The reason is that scientists found that abnormal p53 gene in the body of more than 50% tumors patients.p53 gene is relative to 50% tumors of humans,what include liver cancer,mammary cancer,bladder cancer,gastric cancer and so on.p53 plays a critical role during cells cycle regulation,apoptosis and tumor formation,deterioration.Therefore,it is significantly important that learning all basic structure and function of p53 gene for treating tumors.The main purpose of this study is to prepare cancer-inhibitory factor p53 that possess function of tumor targeting and transmembrane shuttle through recombining gene with the protein core peptide of p53 can inhibit tumor cells and LHRH have tumor cell targeting and TAT with shuttle cell membrane function.This study lays the foundation for anti-tumor therapy drugs.In this study,the pET28a-TAT-GFP plasmid was used as a PCR template to design PCR primers containing the LHRH gene sequence,and the LHRH-TAT-GFP gene fragment was amplified by PCR.Then the LHRH-TAT-GFP gene fragment was cloned into prokaryotic expression vector pET28(a)with Nco?and HindIII double digestion to construct recombinant expression plasmid pET28a-LHRH-TAT-GFP.Subsequently,transform into competent E.coli BL21(DE3)cells.Transforming to the expression bacteria E.coliBL21(DE3)when the sequencing result is correct.Several common temperatures were used to optimize the induction,the optimum temperature was determined to be 30°C,and the optimal induction time was determined based on the optimal temperature is 4 hour.Western Blot analysis indicated the target protein is mainly expressed in soluble form.Through constant exploration to chromatographic purification of recombinant protein,successfully obtaining high-purity and high-concentration protein.The purification process is: Supernatant of bacterial liquid with sonication through low temperature centrifugation added ammonium sulfate with a final concentration of 1.25 mol/L.After standing at 4°C,the supernatant was centrifuged to perform Phenyl-HP hydrophobic chromatography.The target protein was eluted with 30 mm/LTris solution.Through Q anion exchange chromatography,the final target protein was dialyzed against 1×PBS in 30mm/LTris,0.25mol/L NaCl solution.After concentration,LHRH-TAT-GFP recombinant protein with a relative molecular weight of approximately 30 kD and a concentration of 1.62mg/mL was successfully obtained.Under the fluorescence microscope,through the detection of the ability of the fusion protein to enter into the cell,the LHRH-mediated ability and TAT protein transduction ability was visualized by the fluorescence specificity of the green fluorescent protein GFP.The distribution of the recombinant protein in the cells was observed by fluorescence microscopy,demonstrating that the recombinant protein can be transported into the cells by the mediating action of LHRH and the transmembrane action of TAT.The recombinant p53 gene containing restriction enzyme sites was digested with the previously constructed recombinant plasmid pET28a-LHRH-TAT-GFP and then ligated to obtain the recombinant plasmid pET28a-LHRH-TAT-p53.After double digestion with PCR and pET20 b The connection was transformed into the competent E.coli DH5?.After the sequencing result was correct,it was transformed into E.coli BL21(BL21)cells and the expressed bacteria were successfully obtained.The inducing expression conditions were optimized.The optimal induction temperature was 32?.The optimal inducer concentration was 0.8mmol/L.The induction time was 5 hour.After induced expression,12% SDS-PAGE analysis showed that there was a target protein at 54 kD.Explore the purification process,first use the characteristics of the histidine-containing protein tag,choose to use metal chelate chromatography column for crude purification,and then use anion column for purification,the final target protein in 6mol/LUrea,0.5mol/L NaCl solution The target protein was renatured and the recombinant protein pET20b-LHRH-TAT-p53 was finally obtained for activity detection.A certain concentration of protein was added to 96 wells of the well-laid cells to observe cell growth changes.After 12 hours of protein addition,it was found that hela cells and BGC cells were obviously dead,but there was no significant change in the growth state of normal cells MDCK cells,and it was confirmed that p53 had Inhibition effect on cancer cells.CCK-8 method was used to detect the inhibitory effect of the protein on the cells,and it was found that the inhibition rate of the protein on the two kinds of cancer cells increased as the concentration increased.