Study On The Expression And Purification Of IL-18 And IL-18BP's Fusion Protein In Different Vector

IL-18 is a special member of the IL-1 family,which closely related to IL-1 in structure and require easpase-1 as an activing agent.Interleukin(IL)-18 was identified as an cell factor that induces the produce of interferon-gamma,enhances NK cell cytotoxicity,mprove the activity of natural killer cells as well as Thl immunoreation type and and fulls of antieancer effects and so on.Interleukin-18 binding protein(IL-18BP)is a novel glycoprotein and belongs to the immunoglobulin superfamily.So far,six IL-18BP isoforms have been found in various eDNA libraries.IL-18BP is regarded as a natural antagonist for IL-18 which efficiently inhibits the biological functions of IL—18 in vitro an d in vivo.Formerly,the research and analysis of IL-18 and the IL-18BP were only restricted in some vector,has not carry specially on almming at the nucleus system,and at gainning the fusion protein,also has not carry specially on unified analysis to these two natural antagonist compounds-IL-18 and IL-18BP in the induction expresses and purification,has not summarize mixed theirs cushion environment,inducting condition of the fusion protein,washes,the elution environment for a common pattern.IL-18 and IL-18BP exist in animal's vivo,therefore it appears very meaningful to research under the identical environment.The experiment uses analysis vertically and horizontally,longitudinal is form PCR to the purification,crosswise is form IL-18 to IL-18BP,although they has the certain similitude in structure,but their difference is very far,the scrabble between them that can use the common pattern is possibly to provide the help for their action mechanism,the research which keeps in balance in the animal in vivo's interaction. IL-18BP is the natural antagonist compound of IL-18,because they have the immune bidirectional adjust action,therefore in disease's process through beth's balanced,achieve the treatment and prevent disease's function,then this experiment's research is possibly to have the certain extent significance in the common environment - balance of animal in vivo.Simultaneously,it also will provide the new scientific research basis for the foundation veterinary science and the veterinarian immunology,will have the important influence.The experimental study used PCR to amplify the eDNA of human interleukin 18 and Interleukin-18 binding protein and then that were cloned into the expression vector PET28a and Pgex-6p-1,transformed into the E.coli BL21.expressed and purified the reeomabinant human interleukin 18 and Intefleukin-18 binding protein in prokaryofic system(BL21).This article discuss experimental technique and matters needing attention in experiment process about PCR,the enzyme outed,Connection,transformation,inducted expression and purification.Thus,it demonstrates clearer mentality form PCR to purification about prokaryofic system, Simultaneously it has also carried on certain analysis to the protein sequence,Obtains data and so on extinction coefficient through the analysis,Again data union which provides with the chromatographic analysis system,calculated the protein density,finally carried on the protein the concentration,Preservated in - 80℃.It has also withdrawn the Clone from the fungus, preserves,with the aim of facilitating in the future uses.The experiment has designed the primers through the software-gene gunner,According to the gene characteristic and the nature,chose two enzymes to cut the position spot -BamH I and EeoRI.Because they have the common cushion environment,then we had determined that PET28a and Pgex-6p-1 are the vectors.According to mycelium's nature,we optimized the Clone's extraction efficiency.The experiment to enhance the connection's efficiency,uses the over night connection,guarantees the connection's succession.The transforming process avoided the vibration,the over time heat's strucking and so on a series of disturbance factor,enabled the experiment to be able to carry on smoothly.When inducting expression,we controlled the time strictly,the vaccine OD value and so on,avoided some tings in the expression process that it is losing the clone,the bacteriophage disturbance,other fungus categories developed and so on situation occurrences.Regarding the best induction density's fumble,we used vertically and horizontally the overlapping gradient to try to find out as far as possible the condition.that induces the fusion protein.In order to avoid the influence of the absorbing peak to RNA and the protein's degeneration when we used the chromatographically analyzes system,we had joined the RNA enzyme and the reducing agent beforehand in the buffer solution,according to information which obtains form the chromatographic analysis system,we can deputes the protein further.The research of interleukin-18 and Interleukin-18 binding protein has very big help in the treatment of tumor disease,own immunity vigorous sickness,infectious disease,transplant anti-host sickness,metabolism syndrome,allergic disease and their pathogenesis,the purified protein regardless of makeing the immunity active determination,specific monoclonal antibodies preparation,useing in the cell experiment,using in the animal experimentation,as well as,useing in the illness treatment and the illness research that gets sick has the very broad significance. Treating disease Specially the tumor treatment,it provide the new way.