The Research Of Molecular Cloning And Characterization Of Ig Variable Region From The Spleen Of Sheep Infected With Orf Virus

Contagious pustular dermatitis(CPD),also known as Orf, is an acute, highlycontagious pustular skin lesions disease of sheep, goats, and humans. The incidencerate can be up to90%and the mortality rate can reach to10%. Adult sheep affectedwith ORFV (Orf virus) will be malnourished because of ingestion difficulty. Newbornlambs suffering Orf will be growth slowly and even starvation to death because of thedifficulty to ingest breast milk. Therefore, this disease caused great economic lossto the sheep industry. As an important zoonosis pathogens ORFV can produce avariety of Immunity related proteins and disrupt or block the synthesis of mediumcomponents which play a role in immune protection to escape the host immunesurveillance. ORFV may do harm to sheep and people’s health and it is very special inimmunology, causing the lack of efficient and practical therapeutic agents andvaccines. Pay attention to the research on ORFV immune characteristics makes agreat significance for the formulation of the disease prevention and control measures.Study on the antibody will provide an important means for the immune defense of thedisease.The animal model of ORFV infection was established in the experiment. ThecDNA of the immunoglobulin heavy chain variable regions were amplified byRT-PCR from the spleen of sheep infected with Orf virus, by using the pair ofspecified primers containing the restriction sites were designed and synthesized withzhe software of Primer Premier5.0. Comparing with other IgH sequences inBLAST-N, using ExPASy To translate it into corresponding amino acid sequence andusing CLUSTAL W1.83to make a Multi alignment. Using the software DNAStar andprogram MegAlign and Protean，to make a Analysis of the homology of the sequencesencoding amino acid.IgH sequence alignment according to the results. The49complete sequenceswere obtained without duplicating. The nucleotide identity of49sequences ranged from the lowest84.3%(that of H7and H38)to the highest100%(that of H5and H6).The clustering analysis of aminoacids deduced by V regions of IgH showed that allclones(H1to H49)were classied into two branches. After comparing with other IgHsequences in BLAST, the length of Dregions were different from9to46bases. Thehomology of nucleotide sequences and deduced aminoacid sequences existed obviousvariability. The hydrophilic amino acids mainly distributed in VH regions and Dhypervariable regions combinated with the hydrophilicity profile. The VH regions andD hypervariable regions having stronger hydrophilicity usually formed antigenicdeterminants. IgL sequence alignment according to the results. The nucleotide identityof41sequences ranged from the lowest76.1%(that of L2and L14，L28and L16，L28and L30，L28and L31，L28and L39)to the highest100%(that of L34and L41). Aftercomparing with Kappa immunoglobulin light chain V region gene sequenceinformation from Genbank (Accession number M60441) in BLAST, They havesimilar structural domain. The hydrophilic amino acids mainly distributed in the frontend of the CDR1regions, CDR2regions and CDR3regions combinated with thehydrophilicity profile. The above research not only provides the important referencebasis for the characterization of Ig variable region from the spleen of sheep and makesa broader understanding of the characterization of Ig variable region from the genelevel, but also lay a solid theoretical basis for the diversity of the gene sequences ofvariable region of Ig. The result of this research will provide a experimental basis onpredicting and analysising the targets of the antibody and antigen. Also it will improvethe vaccine and guarantee the clinical medication security.