Study On ORFome Of Mycobacterum Tuberculosis H37Rv

Mycobacterium Tuberculosis H37Rv is pathogenic of tuberculosis,Statistics according to World Health Organization,existing tuberculsis patient in the whole world 2,000,000,fall to ill 9,000,000 lately annually,died 300 peoples annually,about 1/3 crowds have the delitescence infection of the tuberculosis.Because of increasing with the population go to tour,immigrating frequently and developing of travels Industry,bearing a medicine resistance,setting down of mankind immunity to virus infect and drug abuse,drinking to excess etc.the tuberculosis presents increasingly and seriously.Therefore,study on genomics,gemomics ect,and namely whole study from a cell life,then can thoroughly of make true that disease mechanism,infection of method, understanding the period of its life,diagnosed in early,vaccine immunity,new medicine development,provided theories foundation.Scientist in British Sanger center French Pasteur,sequenced the whole geneome sequence of Mycobacterium Tuberculosis H37 Rv in 1998,this is provided really fine opportunity.There are 3924 open a reading frames,among them,about 40%have a great achievement an ability,44%may have a great achievement ability,16%is called an orphan sequence,with other microorganism of sequence without likeness.In our study,we use the Invitrogen company provides of Gateway technology, pDONR 221 was provided for body to carry for gene into the pDEST17 expression vector,a pair expressing stains:BL21 and the BL21 Codonplus RP,and on the differend condition,transformat the expression vetor into these stains,we obtain totally to 3056 genes,entry clone 2903,express clone 2798,express protein 1660, among them 66 were purification,there were 16 indificated resolved.The E.coli Expression System with Gateway Technology contains a series of Gateway-adapted destination vectors designed to facilitate high-level,inducible expression of recombinant proteins in E.coli using the pET system.Depending on the vector chosen,the pDEST^TMvectors allow production of native,N-terminal,or C-terminal-tagged recombinant proteins.BL21-CodonPlus(DE3)-RP cells contain extra copies of the argU and proL genes. These genes encode tRNAs that recognize the arginine codons AGA and AGG and the proline codon CCC,respectively.The CodonPlus-RP strains have available the tRNAs that most frequently restrict translation of heterologous proteins of organisms that have GC-rich genomes.The expressed protein according to COG classification,they distribute in the different function of genome,there were 17 belong to putative protein,29 belong to drug resistance protein or related to drug resistance,11 of 33 belong to PE fanmily,20 of 67 belong to PPE faminly,5 of 63 belong to PE-PGRS Family protein 11 of 33 were belong to develop the function egg white chip to provide dependable assurance; They coveraged most ORFs of Mycobacterium Tuberculosis H37Rv.We could proudly say that functional protein chip would be base on the database of expressed protein.There were more than 1THM in the expressed 161 protein analysising by soft ware of bioinformation,and TMH have certain influence to recombination;The gravy didn't influence expression;There were any influence to the expression by isoelectric point,when isoelectric point below 4 the number of expressed protein was below unexpressed,between 4-7 of isoelectric point the number of expressed protein was above unexpressed,and with the isoelectric point increasing the number of expressed protein was below unexpressed again.In our study the Gateway highthroughput have available expressed protein which moleculer weight from 20kDa to 30kDa.Most of the purified protein were cytorrhyctes and protein purify in highthrought is more viable,the data acquires of the Exprion chip automatic electricity is dependable,it wsa the best one of protein quantitation before protein chip.The mixture of peptide fragment after enzymolysis was chromatographic fractionation by liquid chromatography-tandem mass spectrometry,we get a satisfaction result.Above all,the cloning systems with in vitro highthtoughput recombination were feasible with Gateway cloning technology,and Mycobacterium Buberculosis H37Rv adapted also.We would made foundation on study on protein chip and interreaction of protein in vitro.