Characteristic Analysis Of Population Polymorphism Of B-F Gene And The Antigenicity Of Their Expressed Products In Huainan Partridge Chicken

Major histocompatibility complex (MHC) is a gene group of high degree of polymorphism in vertebrate genomes, which code immune molecules. These molecules are divided into calss ?, ? and ?molecules. Chicken class ?I molecules are B-F, which composed of ? chain and ?2 microglobulin. The ? chain showed high polymorphism, including extracellular domain (?1, ?2, ?3), transmembrane (TM) and cytoplasmic (Cyt) domains, in ?1 and ?2 regions there is a peptide binding region (PBR), which is capable of combining antigen peptide and inducing specific immune response. The polymorphic MHC molecules result in the diversity in disease resistance and therefrom influence development and production. The different populations of chicken have diversity in morphological feature and production capacity because their area distribution and response to environmental pressures such as infectious disease in the long term evolution. Bird Mhc as resistant genes has been influenced and formed high polymorphism. About the characters of chicken MHC molecules have been reported, but the research about MHC of Huainan Mahuang, as a local population in Anhui province, have reported. Therefor, in this study we cloned its gene whole sequences and analyzed their molecular structure to know the MHC Evolution characteristics in this population.Firstly, total RNA of blood cells from 50 individuals was extracted, and then the cDNAs of B-FA were achieved by RT-PCR method. These gene segments were cloned into pMD18-T vector and transformed into E. coli DH5arespectively. The positive clones of each individual were sequenced respectively. We obtained 34 sequences of B-FA, which had 1136bp, and 15 sequences of B-FB, which had 396bp, respectively. Then, the molecular characteristics of polymorphic B-F gene were analyzed by bioinformatics software. The results showed that there two ORFs within B-FA, namely 1068bp and 1035bp segments, coding 356 and 345aa respectively. The both had a signal peptide (21aa), al (88aa), a2 (91aa) and a3 (91aa), their difference was in TM/CY, one of the both lack an 7th exon. In contrast, B-FB appeared conservative.Secondly, the prokaryotic recombinant plasmids having B-F genes were structured and then they were transfected into E. coli and induced expression. The results of SDS-PAGE identification indicated that B-FA was expressed in inclusion body and B-FB was expressed in a soluble protein. The MWs of B-FA and B-FB were 52.1 kDa and 33.3 kDa respectively. The purified proteins were also injected into mice to produce specific antibody. The result of western blot showed that this antibody could bind the object protein.Finally, the eukaryotic recombinant plasmids containing B-F genes were structured. After transfection of 293T cells their transient expression was observed. Both B-FA and B-FB were expressed and localized membranae serosa of cells.All of these results provide an experimental basis for further study MHC class I molecules carrying antigenic peptides mechanisms, maintaining and promoting the stability of its function.