Light Chain LC And TAT-EGFP-HCS Of Botulinum Toxin Expression And Biological Function

Objective: Neuroblastoma is the most common solid tumor in children and is highly malignant in patients with distant metastasis and/or MYCN oncogene amplification older than 12 months.Neuron tumors have a high rate of recurrence after surgery due to their specific location,thus the mortality rate is extremely high,along with the morbidity rate.At present,the drugs for treating neuroneoplastic tumors have poor targeting,making it difficult to obtain the desired therapeutic effects,and the drugs themselves also have a high incidence of adverse side effects.In addition,many drugs with good prospect cannot play their role efficiently because they cannot pass through the blood-brain barrier.Therefore,it is an urgent task to develop a low-toxicity,high-efficiency,and environmentally-friendly targeted drug for the treatment of neuronal tumors.Botulinum neurotoxin(Bo NT)is an exotoxin produced by anaerobic clostridium botulinum with seven serotypes of A-G(5).The structural and functional domains of the Botox neurotoxin(Bo NT)is an exotoxin produced by Clostridium anaerobicum and has seven serotypes of A-G(3).The structural and functional domains of Bo NT are very conserved,and their precursors are single polypeptide chains with a molecular weight of approximately 150 k D.The precursor is decomposed by an endogenous or exogenous proteolytic enzyme into an active double-stranded structure linked by two sulfur bonds:the light chain(LC,50 KD)is a catalytic domain with endopeptidase activity;the heavy chain(HC,100 KD)It is a cell-binding and translocation domain that mediates binding between specific receptors and neurotoxins on the cell membrane.According to reports in the literature,botulinum toxin inhibits the release of the neurotransmitter acetylcholine at the human neuromuscular junction,resulting in a series of flaccid paralysis symptoms.In addition,botulinum toxin can block the "information transmission" between nerves and muscles,relax the excessively contracted muscles,disappear wrinkles,and be widely used in beauty.Although botulinum toxin has many applications in the treatment of central diseases,as biological macromolecules,it is difficult to pass the blood-brain barrier,which greatly limits their application.Many neurological diseases are located in the central nervous system,and in order to get the drugs they carry into the nervous system,the system must solve the problem of how to pass the blood-brain barrier.Although botulinum toxin has been reported as a targeting vector for neurological diseases,there are many toxic side effects.Recent studies have shown that TAT peptide fusion proteins can enter the brain through the blood-brain barrier through peripheral blood.The TAT peptide consists of 11 amino acid residues of the transduction domain of the HIV Tat protein.Compared to traditional intracerebral injections,TAT-mediated protein transduction technology delivers biomacromolecules to the brain without causing infection and trauma to brain tissue.A number of TAT fusion proteins have been reported to date,such as TAT-14-3-3,TAT-Bcl-x L,TAT-globulin and TATPARK7.They are able to effectively cross the BBB and show significant neuroprotection in animal models of brain disease.In this study,p ET-28a-TAT-EGFP-HCS and p ET-28a-TAT-EGFP-LC plasmids were constructed and purified,and the biological activity was verified.Protein EGFP,TAT-EGFP,TAT-EGFP-LC,TAT-EGFP-HCS was co-incubated with PC12 cells,BV2 cells,C6 cells and He La cells to verify whether TAT-EGFP-LC can penetrate the cell membrane and transmembrane ability of PC12 BV2 cells,C6 cells and He La cells;The ability of TAT-EGFP-HCS to penetrate the heart,brain,liver and kidney tissue in mice.Methods:1.Plasmid construction,expression,detection and activity assay of TAT-EGFP-HCS,SNAP-25,LC and TAT-EGFP-LC The plasmid was digested,and the digested p ET-28 a TAT-EGFP and HCS were ligated,and then transformed into DH5 a competent cells for monoclonal amplification,amplification and double digestion.Electrophoretic identification.The ligated plasmid p ET-28 a TAT-EGFP-HCS was transformed,and the single clone was used for amplification culture,and the expansion culture was continued,followed by SDS-PAGE electrophoresis and Western-Blot;electrophoresis and Western-Blot verification,and the protein purification experiment was performed.After a large amount of expression,the bacteria are collected,purified,and purified by a HIS-tagged protein purification column.After purification,the protein is salted out using a dialysis bag,and then stored;The plasmids p ET-28a-TAT-EGFP-LC,p ET-28a-LC and p ET-28a-SNAP-25 were constructed in the same manner,and then expressed and purified to obtain a protein,followed by LC,TAT-EGFP-LC digestion and SNAP-25 viability assay,using the purchased botulinum toxin SNAP-25 as a control,followed by SDS-PAGE electrophoresis,Western-Blot identification,specific detection using SNAP-25 antibody,verification of protein LC,TAT-EGFP-LC enzyme Cut vitality.The PC12 cells were cultured,and the protein LC and TAT-EGFP-LC were added to the cells to incubate with the cells.The protein liquid was extracted and identified by Western-Blot.It was examined whether the protein LC could cross the cell membrane,and whether the enzyme could act to cleave the SNAP-25 in the PC12 cells.2.Transmembrane ability of protein EGFP,TAT-EGFP-HCS,TAT-EGGP-LC,TAT-EGFP to PC12,BV2,C6 and Hela cells.PC12 cells,BV2 cells,C6 cells and He La cells were cultured separately.After the cells were grown,they were passaged in a six-well plate,and then protein EGFP,TAT-EGFP,TAT-EGFP-HCS and TAT-EGFP-LC were co-incubated with the cells.Experiment,adding fresh medium,fluorescence microscopy,observation of EGFP,TAT-EGFP,TAT-EGFP-HCS and TAT-EGFP-LC transmembrane,and analyzing the results of transmembrane by flow cytometry.3.Effects of protein EGFP,TAT-EGFP-HCS,TAT-EGGP-LC and TAT-EGFP on apoptosis of PC12 cells The proteins TAT-EGFP-HCS,TAT-EGFP,EGFP,TAT-EGFP-LC were incubated with PC12 cells,samples were collected,and flow cytometry was performed to verify these protein pairs.4.Distribution of protein TAT-EGFP-HCS in mouse tissues The protein TAT-EGFP-HCS was injected into the tail vein of C57BL/6 mice,and the heart,liver,liver and kidney of the mice were taken,and frozen sections were prepared.The protein was permeated through the microscope.Results:1.The plasmids ET-28a-TAT-EGFP-HCS,p ET-28a-SNAP-25,p ET-28a-TAT-EGFP-LC and p ET-28a-LC were successfully constructed and identified by SDS-PAGE electrophoresis after expression.The theoretical values ? ? are consistent,indicating successful expression,and it is proved that the proteins SNAP-25,TAT-EGFP-HC,LC and TAT-EGFP-LC have good biological activity in vitro;2.Co-incubation with cells by protein EGFP,TAT-EGFP,TAT-EGFP-HCS,TAT-EGFP-LC,fluorescence microscopy showed that TAT-EGFP-HCS on nerve cells PC12 cells,BV2 cells,C6 cells and He La cells The transmembrane effect is relatively strong,while TAT-EGFP-LC has a weak penetrating ability and EGFP protein has no cell transmembrane ability,which confirms that TAT-EGFP-HCS has specific cell transmembrane effect.The results of FACS were similar to fluorescence microscopy results.3.Protein TAT-EGFP-HCS,TAT-EGFP,EGFP,TAT-EGFP-LC were co-incubated with PC12 cells,and the protein TAT-EGFP-LC was detected by flow cytometry to induce early apoptosis of PC12 cells.Protein EGFP,TAT-EGFP,and TAT-EGFP-HCS had no effect on cell apoptosis.4.The distribution of TAT-EGFP-HCS protein in the brain,liver,heart and kidney of mice was compared.It was found that the penetration ability of mouse brain tissue was stronger,and the penetration ability to other tissues was significantly weaker than that of brain tissue.Conclusion:1.The biological activities of protein EGFP,TAT-EGFP and TAT-EGFP-HCS expressed in vitro showed no obvious cytotoxic side effects.After incubation with cells,the cells still grew well,while the protein TAT-EGFP-LC could cause apoptosis;2.In vitro experiments showed that TAT-EGFP-HCS has a specific ability to penetrate nerve cells and has better penetrating ability than TAT-EGFP,while TAT-EGFP-LC has poor penetrating ability;in non-neuronal cells He La The ability of transmembrane in cells is weaker than that of TAT-EGFP;3.The expressed botulinum toxin heavy chain TAT-EGFP-HCS has a distinct neuro-directed protein.In mice,the protein TAT-EGFP-HCS specifically penetrates the blood-brain barrier and enters the mouse brain.