The MHC Class Ⅰ Gene Polymorphism Analysis Of Varies Sheep In Xinjiang

Molecules expressed by genes of Major histocompatibility complex in ovine are known as OLA, which are divided into three zones, class Ⅰ, class Ⅱ and class Ⅲ. The production of class Ⅰ and class Ⅱ genes that are MHC class Ⅰ and class Ⅱ molecule plays extremely crucial roles in stimulating organism to produce immunological responses against the invade of pathogens in the process of antigen presentation. Due to the close association between OLA genetic polymorphism and their disease resistance, many scientists focus their attention on the ovine MHC polymorphism. Currently the researches on ovine MHC are mainly focus on the DQ and DR locus and their polymorphism but class Ⅰ gene is less researched. Research on the polymorphisms of these regions has significance in understanding sheep disease resistance and susceptibility and should be informative and supportive to breeding new sheep species with stronger disease resistance and also will enrich OLA class Ⅰ sequence data.In order to obtain rich sequence data and well understand the ovine MHC class Ⅰ genetic characteristics and polymorphisms, we selected four breeds of sheep, Chinese Merino sheep, Hazakh sheep, Suffolk sheep and Chinese Meat-Type Merino, cloned and sequenced fragments of whole length of OLA class Ⅰ cDNA isolated from156experimental sheep in total. The blood samples were taken for total RNA extraction, and then the first-strand cDNA was synthesized using total RNA as templates. cDNA of both OLA class Ⅰ heavy and light chains was amplified using high fidelity PCR. Electrophoresis results displayed that the heavy chain cDNA is about1.1kb. After PCR selection with OLA class Ⅰ specific primers and nucleic restriction endonuclease digestion, double positive clones were sequenced by biotech companies. Sequence analysis showed that ovine MHC class Ⅰ gene heavy chain length is1107bp, encoding a protein of368amino acids and the base composition has a certain imbalance:A is19.26%, G is33.15%, T is18.26%and C is29.34%. GC content (62.49%) was significantly higher than the AT content (37.51%). The heavy chain has a signal peptide of28amino acids in length and is a soluble protein. It contains a-helical secondary structure:29.51%, stretchers:29.23%β-turn:9.84%random coil:31.42%. They have high sequence homology with the sequences in NCBI database, the nucleotide homology is from89.87to99.91%. The phylogenetic analysis showed ovine MHC class Ⅰ gene sequence of heavy chain is divided into two large branches, and are well separated from the sequences published the database, which are clustered independently. A eukaryotic expression system of OLA class Ⅰ molecules was also constructed by inserting cDNA into pCNDA3.1vector using T4ligase, and the contraction was verified by both nucleic restriction endonuclease digestion and PCR with OLA class Ⅰ specific primers, which will be supportive near future research on functional analysis of different allelic OLA class Ⅰ molecules.The length of light chain is357bp, encoding119amino acids, base composition is balance. Nucleotide homology analysis results the homology between ovine sequence in datebase is93.28%, while the bovine sequence in database homology reached98.88%, phylogenetic analysis had got the same result.As the most polymorphism region of ovine MHCI gene is theal and a2coding region, which is the2nd and3rd exons region, they comprise the MHC I molecules and peptide binding region, substantially can reflect polymorphisms of MHC I gene. The sheep blood genomic DNA was extracted as templates PCR amplified the second and third exons of the MHC class Ⅰ gene heavy chain genes of different sheep breeds. Results showed that the sequences of the second and third exons of OLA I genes are highly polymorphic, particularly in the peptide binding region and region interaction with the TCRs. Phylogenetic analysis using bioinformatics software was carried out on base of base replacement, synonymous, substitutions, nonsynonymous substitutions and nucleotide differences in the average nucleotide and polymorphism. The results showed that the grouping of the2nd and3rd exons of OLA class Ⅰ genes are not very clearly, and a cross-species polymorphism phenomenon also exist. The results produced in my thesis will enrich OLA class I sequence data and be supportive to our understanding of genetic polymorphism of ovine MHC genes and their relationship between disease resistance or susceptibility, and also provide information for antigenic epitope vaccine research.