Gooses MHC Class Ⅰ CDNA Cloning, Genomic Structure, Analysis Of Alles Polymorphism

Major histocompatibility complex (MHC) antigen was such gene cluster that linked with a certain chromosome encoding a group of cell membrane glycoprotein that correlated with immune response. Its function is not only confined in rejection of transplant but also correlated with immune response for extra-antigen. This finding make researchers highly interested in MHC.MHC Sequences of chicken, other birds, amphibian, crawl species were derived from DDBJ/EMBL/GenBank and analyzed by alignment. Degenerated primers were designed at conserved domains external between two Cysteines of MHC class I a 2 domain. According with TRIZOL kit using statement to abstract the total RNA from goose spleen, MHC I a 2 gene was cloned by RT-PCR method from goose spleen fcDNA and then sequenced. According with the sequenced result to design a new sense primer, the gene from MHC I a 2 conserved domain to 3'-terminate PolyA tail was cloned by 3'-RACE method and sequenced. Similiarly designing a anti-sense primer, gene from S'-UT to MHC I a 2 domain was cloned, in term of the standard protocol of PCR Library kit, from cDNA double chain synthesized by the second-band cDNA synthesized kit, after linked with T-easy vector, and sequenced. Finally, the sequences of 3'-terminate and S'-treminate were overlapped together a full length sequence to design primer pairs by this sequence, then full length gene was cloned by RT-PCR with these primer pairs, after linked with T-easy vector, and sequenced. Moreover, expression of MHC I in goose different organs was tested by RT-PCR method. And employing LA-PCR method cloned MHC IDNA gene from goose genomic DNA and analyzed the structure of MHC I genomic DNA. About the alleles polymorphism of MHC I, after obtaining fcDNA from additional 4 gooses, full length genes of MHC I were cloned by RT-PCR method and sequenced, then using bioinformation method to analyze the alleles polymorphism.Genomic structure, alleles polymorphism, antigen polypeptide binding domain (PBD) amino acid replacement rate and tertiary structure were quantitatively analyzed. Results indicated that mature peptides of Ancy-MHC I was composed of 348 amino acids, which have 14 amino acids in Ancy-MHC I a1 and a2 domains that are highly substitue rate sites, and existed recombinational crossover .Genomic DNA consisted of 8 extrons and 7 introns. According to genetic distance, Ancy-MHC I alleles may be classified 3 types (-UA~ -UC). Ancy-MHC amino acid sequence maintained 8 critical amino acids of HLA-A2 which binding with antigen polypeptide, having 85.1~98.9% amino acids homologous rate among goose alleles, having 61.0-66.7% amino acids homologous rate with chicken MHC class I (B21.B2, Y), and 40.8-43.4% amino acids homologous rate with human and mouse MHC I. We discovered by RT-PCR method that Ancy-MHC I can be expressedin heart, brain, liver, spleen, kidney and skin. Compared with human HLA-A2 crystal structure, Ancy-MHC I has variety of 2 amino acid absence in a1 domains, but a -helix and B-sheet domains were relatively conserved. The Ancy-MHC class I molecular phylogenetic tree farther revealed evolutionary relation between goose and chicken, duck, amphibian, mammalian and human.