Molecular Cloning, Expression And Characterization Of Wulong Goose MHC Class Ⅰ

The major histocompatibility complex (MHC) is an extended cluster of genes withextraordinary polymorphism and close linkage in the chromosomes of all vertebrates. MHCmolecules are also considered as the primary determinants in the transplant rejection.According to their structures and functions, MHC molecules could be divided into class I,IIand III. MHC class I consists of peptide-binding region, immunoglobulin-like region,transmenbrane region and cytoplasmic region. Until now, few results about goose molecularimmunology have been published. As a Chinese indigenous breed,Wulong goose breed waschoosed as model animal for studying the properties of MHC molecules.1 According to the mRNA sequence of MHC I molecules of chicken(XM₄24372),mouse(AY064389), Zebrafish(BC066745) registered in GenBank, the Primer Premier5.0 andOligo6.0 were adopted to design a pair of primers, the RT-PCR procedure was taken toamplify the target gene from Wulong goose spleen cells that was stimulated by the ConA.After being identified by restriction and PCR analysis, The recombinant plasmids were ligatedto the pAT2 and transformed into the DH5α competent cells, distilled and sequenced. thetarget gene involved 1062bp and was submitted to GenBank(accession: AM114925).2 According to the cDNA sequence already determined, new primers were designed,MHC class I gene was cloned by LA-PCR method and analyzed with restriction andPCR.The results showed that the sequence of MHC class I of Wulong goose obtained wasabout 2594bp and has been submitted to GenBank(accession: AM114924).In addition, theDNA and cDNA sequences of MHC class I of Wulong goose were compared by using theContigExpress software and the VecScreen programe, the results showed that 8 exons and 7introns were contained in Wulong goose MHC class I.3 With the technique of TaqMan,a new method of (Real-time) RT-PCR wasdeveloped to detect MHC class I gene of the Wulong goose. The heart, liver, spleen,lung, kidney, thymus gland, brain, muscle and embryo tissues were used to fix thequantity of PCR through Fluorescent method which showed the virtues of better sensitivityand specialty. The results suggested that the order of expression level of MHC class I genes intissues listed above was bursa>spleen>thymus> brain > lung > muscle > kidney >heart>embryo > liver.4 DNA sequence and the amino-acid sequence of MHC class I of Wulong goose wereanalysed by the methods of Bioinformation. There are 21 common restriction sites throughnuclear acid DNA sequence. the melecular weight of WL MHC class I is 39074.69m.w, PI is5.53, the hydrophobic property is 3.433, there is a strong hydrophobic area at the C-terminateof 300-320 amino acid, two protein superfamilies were indicated. Sub-cell orientationanalysis suggested that the possibility of WL MHC existing in the plasmids is 44.4%. Thethere-dimensinal structure of MHC class I of Wulong goose was made up with two long α-helix, three β-sheet, and a spacial hollow among the α-helixes. The phylogenetic tree isformed based on the sequence alignment of MHC class I of Wulong goose and other 16species which showed the similarity between Wulong goose and chicken, mice and humanbeing was 64.1%, 43.5-44.4% and 42.9% ,respectively.