Expression Of Mycobacterium Tuberculosis ESAT6 In Pichia Pastoris And Optimization Of Fermentation Condition

Tuberculosis is a kind of people and animals infectious disease together that caused by Mycobactermm tuherculosis.Three major infectious diseases becoming our times to threaten human health with AIDS and malaria.In recent years because the population flowed the increase,the bacillus tuberculosis multiple drug resistance appearance and the BCG vaccine long-term multiplication,hands down from generation to generation to cause the protection antigen unceasingly to lose,the variation,finally made the tuberculosis the epidemic situation to aggravate once again,proposed the new challenge for tuberculosis preventing and controlling.So it is imperative to develop new,safe,high-efficient tuberculosis vaccine.The ESAT6 protein(early secretion protein antigen target 6) is one kind of secretion protein which filtrates from the mycobacterium tuberculosis short-term raise purifies,has special immunity original,can stimulate the organism to have the protection immune response.ESAT6 molecular weight is 6KD,its genetic total length of code is 288bp,can encode 95 amino acids.It only exists in Mycobacterium tuberculosis and a few kinds of pathogenic branch bacillus,and more than 90%of the non-pathogenic branch bacillus bacterial strain lacks this protein Brandt discovered ESAT6 is the person or the animal immunity reply process which the branching bacillus infects one of T cell recognition main target antigen.Simultaneously Skjot discovered ESAT6 can induce the organism to produce high level IFN- gamma,it plays the quite vital role in tuberculosis preventing and controlling.Therefore the ESAT6 protein may as well as becomes the DNA vaccine and the Asian unit vaccine component the candidate antigen,thus achieves long-term to the tuberculosis,the effective prevention.At present the overseas scholar has carried on many research works in the mycobacterium tuberculosis ESAT6 highly effective expression and the vaccine development aspect and has got a series of achievement.The protein expresses many kinds of systems at present,among them Escherichia coli is the mainest expression system.Its major advantage is that it costs low,the output is high,easy to operate.But Escherichia coli original nuclear living beings,have real core biological gene expression regulation and control mechanism and processing of protein modify ability,and the expression amount of the protein is low,the activity is bad,so people turn to very nuclear expression system.At present eukaryon expression system a most frequently used one to finish Pichia Pastoris express system,it has already expressed many,kinds of other source proteins successfully.Other source proteins expression of finishing Pichia Pastoris divide afterbirth express and secrete,reach outside afterbirth two ways.Afterbirth express,suitable for common to express or include not only S-S but also key the candy base albumen among afterbirth thick liquid,but the result purification is more complicated.Or have toxin activity and have albumen to poison function as to easy unstability albumen that degrade those to host fungus,afterbirth express,can store in exist,separate on methanol Pichia Pastoris specific cell in the cell extensively them-of the body peroxide enzyme.Because Pasteur finish Piehia Pastoris secrete very low-level endogenous albumen only,secrete other source that express albumen purification very much convenient,so secrete and express it for prior choice way.This research mainly is constructs the mycobaeterium tuberculosis ESAT6 gene eukaryon expresses vector and to change over to finishes in the Pichia Pastoris to carry on the expression.H37Rv genome DNA as the template,amplify the tubercle bacillus secreting albumen ESAT6 gene,connect to PGEM-T carriers. Connect ESAT6 gene to the expression carriers of Pichia Pastoris after constructing on PpIcza-A.After the linearization its,punch with electricity law channel Pichia Pastoris Gs115 into.Adopt Zeocin go on gradient screen high transformation son of resisting etc..Use methanol as,induce thing,fetch table ferment,train clear liquid at 4d,carry on SDS-PAGE.In order to improve the expression amount of the albumen at the same time, the fermented condition of Pichia Pastoris after adopting the orthogonal experiment to optimize,The main factor that its influence ferments is as follows,pH of the solution,joining amount,temperature,number that ferment of methanol。Have got and recombinated and finished the optimum value of several influence factors in the fermented course of Pichia Pastoris finally,i.e.PH5.5,the joining amount of the methanol is 1%,temperature is 30℃,time is 4d.In sum,research this realize ESAT6 gene secretion of finishing Pichia Pastoris express,have got ESAT6 with biological activity,secrete the expressing amount and reach 30mg/L.It is it ESAT6 original in gene to simplify at the same time want core after expressing sex change replying issue.Simultaneously the ESAT6 gene's has not only solved the nucleus expression after eukaryon system success expression must pass through denatured complex question,moreover also has laid the foundation for the new vaccine's development and tuberculosis's prevention.