Construction Of Subtracted Gene Library For Deer Tuberculosis Wild Strain And BCG

Deer TB bacteria is caused by Mycobacterium tuberculosis.It may be cited of deer with chronic consumption and transmitted diseases.Tuberculosis is a wide range of popular in Deer farm in the various countries, almost every country in his Deer farm are detected TB, or tuberculosis bacteria. The pathology characterized is granulomas and cheese necrosis become in tissues and organs.In recent years,the deer TB is widespread in all over the word,it lead to huge economic losses and seriously affecting the deer industry developing.Many countries depending on the different situation of the deer TB,take the whole group of checks,remove sick animals,and other measures to prevention the deer TB.China use BCG to prevent the deer tuberculosis.But BCG extended from the Mycobacterium bovis. Therefore,often interfere with diagnosis of TB,in particular,tuberculin diagnosis can not accurately identify the vaccine strainsand natural epidemic strains,vaccination of animals and naturally infected animals, lead to many countries do not immune heir animals,hinder TB prevention and control process.Practice has proved that,BCG immunization is still the best means of prevention of deer tuberculosis.If we could selected differentially expressed genes,we accurately distinguish between vaccine strains and natural pandemic strain,vaccine immunized animals and naturally infected animals,which will undoubtedly promote the development of the Deer Industry,has real economic significance.1996,Diatchenko establish the Suppression Subtractive Hybridization.The method us es the hybridsecond-order kinetics, when annealing the homologous hybridization rate of the a bundance of single-stranded DNA is faster than the low abundance of single-stranded DNA. Basically the same of relative content of single-stranded DNA abundance.Suppression PCR use the chain annealing is better than chain annealing,so that at both ends of nontarg-et sequence fragments generate similar to issuing the complementary structure in the annealing,could not pairs with primer,selectively inhibit non-target gene fragment amplifying.Compared with other selecting differentially expressed genes methods,this method have the advantage of sensitivity,efficiency,high specificity,easier to operate,good versatility and low false-positive.We isolate deer tuberculosis wild strain and BCG genes especially,and to construct two DNA libraries by using suppression subtractive hybridizat-ion. We used the company of clontech’PCR-Select TM Bacterial Genome Subtraction Kit to subtractive hybridization to clone the differential genomic genes between Deer Tuberculosis wild strain and BCG.After two times of subtractive hybridization and two times of PCR,the products of secondary PCR amplification were inserted into pMD18-T vector and be transformed into the JM109,selected of blue and white clones of the transformants,construction of subtracted gene library for Deer Tuberculosis wild strain and BCG.Select white colony,using Nester primer1and Nester primer2R amplification insert fragments,208clones obtained by single and more than100bp fragment.The PCR products corresponding point in the two nylon membranes, tag the deer tuberculosis wild strain and BCG DNA fragments as probes,doing the dot hybridization with the probe,60positive clones obtained.Sequencing the positive clones.Using the BLASTN and BLASTX search analysis the sequencing results.In the nucleotide level’s fragment analysis,we coul-d find that the number sequencing results located in RD1is13.Though analysis of sequencing results and predict the partial results’function,we hope to lay a good foun-dation in deer TB control,differential diagnosis and development the new vaccines...