Analysis For The Gene Composition And Structure Of 55L9 BAC Clone Located On MHC Class Ⅰ Of Chinese Merino Sheep

)biect:The MHC class I 55L9 BAC clone of Chinese merino genome-wide BAC library was take as the esearch object to study the gene composed and structure character of MHC class I genes in chinses merino heep.using the plaque in situ hybridization method,55L9 BAC clone was prepared probe by 32P labelled 1 terminal to screen one or more genes from the chinese merino sheep pooled cDNA library.These andidates genes screened were analyzed by vrious software,after sequencing and blasting.Methods:(1) After the MHC distract 55L9 BAC of Chinese merino sheep BAC library was sequenced,the mline GeneScan 1.0 software was used to predict the possible existence of gene cloning.Plasmid was ctracted from the 55L9 BAC clone,and digested.The digestion of the plasmid was lablled with 32P on erminal by klenow polymerase enzyme. the hybridization technology to screen the Chinese merino sheep iooled cDNA library.(2) The candidate positive monoclonal gene obtained was spliced, which making the genes transform into PBK-CMV vectors, in order to provide convenience for further preservation and equencing work. The transformed vector plasmid was disgeted by EcoR I and Xho I enzyme to identify 2 objective fragment whether inserted into the vector. These objective cloned fregment were analyzed by orts of bioinformatics information method,after the objective gene was sequenced. thus a preliminary tudy about the 55L9 BAC clones genie composition and structure characteristics start.Lesults:55L9 BAC cloning sequence is approximately 145 kbp afer it was sequenced.The 55L9 BAC clone ave 12 genes,most of them are split genes,inclding 10 complete genes,and 2 incomplete genes which spanher clone. After several times phage in situ hybridization screening test and sequencing,this essay finally et 15 candidate genes, including six genes which are located on the 55L9 BAC clone sequence by using ioinformatics blast software,other genes are not located on the 55L9 BAC clone sequence but they are ossiblly related to immune by blasting with nucleic acid database of NCBI.As cattle and sheep have close elation in species evolutionary,these genes were noted.Finally,this essay have submited 8 sequemces to eneBank including the sequence located on 55L9 BAC sequence.Conclusion:The method of Using pooled cDNA library hybrid with BAC library has great advantage in ;enetic screening, because it main exist two facet reason.First, it can separate the whole gene and can be lunched the research of the gene function. Second it is a quick and effective method to screen the positve ene clone, specially adapted to screen a large amounts of gene cloning...