Germline Gene Locus Structure And Diversity Mechanism Analysis Of Goat Immunoglobulin Heavy Chain Variable Region

The immunologlobulins are important effectors in the adaptive immune systems of all jawed veterbrates, being found in fish, amphibian, reptiles, birds and mammals. Nowadays,information about immunoglobulin structure and types is obtained from human and mice,while limited knowledge is available for domesticated animals, excepcially in goat. To study the immunoglobulin gene structure and expression mechanism will not only improve the understanding of the disease-resistant mechanism, but also provide theoretial foundations to promote the disease-resistant breeding work.Based on the recently released goat genome, we have characterized the genomic organization and diversity mechanism of the Xinong Saanen Dairy Goats immunoglobulin heavy chain variable region. Results were shown as follows:A BLAST search with goat immunoglobulin rearranged variable region clones(NCBI accession No. EU344746-EU344750) as the queries revealed that goat eight immunoglobulin VH genes locus on five genome scaffolds. They could be classified into two gene families based on nucleotide identity. FamilyⅠcontained three potentially functional VHs, 1 ORF and2 pseudogenes, homologous to sheep IGHV1 and mice IGHV2. Family Ⅱ contained two pseudogenes. Three DH and six JH germline genes were located on scaffold1847. All three DHs, arranging in 5’-DH1-DH2-DH3-3’, were potentially functional genes. Six JHs were arranged in 5’-ψJH1-ψJH2-ψJH3-JH1-ψJH4-JH2-3’.A total of 238 unique cDNA sequences of immunoglobulin heavy chain rearranged variable were cloned and sequenced from three goat spleens by RT-PCR. Several VH, DH and JH segments were found to be preferentially utilized in the expressed variable region repertoire. IGHV1-2 was most frequently used in all clones. More than two-thirds in percentage clones were encoded by JH1. 225 clones were identity to DH segments. The majority of clones were expressed in reading frame Ⅲ. DH1 had an apparently strong expression preference, and 28 clones seemed to contain two rearranged DH gene segments.The length of CDR3 varied from 5-27 amino acids(averaged in 11.33).Alignment between all cDNA sequences and three potentially functional VH genes were done to analysis the base substitutions and insertions/deletions. Data revealed that a bias for transtions over transversions in rearranged variable region. A-to-G transtions were the most subtitutions and followed by G-to-A. Subtitutions in purnines were two times over pyrimidines. The mutations located in CDR2 and FR3 were obviously more than other immunoglobulin domains. A higher frequency of insertions/deletions was found in CDRs with theee or multiples of three bases.In conclusion, this study revealed the germline gene structure of goat immunoglobulin heavy chain variable region, and described the important roles of VDJ recombination and somatic hypermutation mechanisms to diversity the expression repertoire.