Prokaryotic Expression, Antibody Preparation And Prelimary Application Of Extracellular Region Of CD8α Gene Of Cherry Valley Duck

In this experiment, through cloning and sequencing Cherry valley duck and Sheldrake CD8a ORF,it was a prediction result to the great difference CD8a extracellular region between Cherry valley duck and homo sapien using molecular biology software. Prokaryotic expression of extracellular region of Cherry valley duck CD8αby cloning technology, preparation of polyclonal antibody rabbit against Cherry valley duck CD8a, and observation of changed regularity of CD8+T in peripheral blood applying SABC immunohistochemistry which providing methods for research of cellular immunity.Mainly complete following research procedures:1 Cherry valley duck CD8a ORF gene cloned, sequenced and sequence analysisAccording to public pekin CD8a gene sequence, specific primers were synthesized. Cherry valley duck and sheldrake CD8a ORF genes were cloned and sequenced, determinate sequences were loged on to Genbank (the accession number: FJ527828/FJ527912). Sequence analysis results by related software alignment showed that Cherry Valley duck CD8a ORF deduced amino acid sequence similarity is about 91% compared with pekin and sheldrack, and about 61% and 32% respectively compared with chicken and homo sapiens. CD8αORF deduced amino acid disparity analysis among different animals by related software showed that the most conservative position is cytoplasmic and the most unstale is the extracellular domain. The results which analyzed by related software showed that there were marked differences of deduced amino acid sequences extracellular region of CD8a between cherry valley duck and homo sapiens.2 Prokaryotic expression of Cherry Valley CD8a extracellular domainAccording to Cherry Valley CD8a ORF sequence, specific primers were designed, and cloned CD8a extracellular region. Prokaryotic expression plasmid (pGEX-KG-Du CD8a) was constructed.The expressed product which was induced by IPTG in E.coli BL21 (DE3) were analyzed on SDS-PAGE electrophoresis. The molecular size of expressed protein was about 43.8KDa, in concordance with the expected molecular weight of the protein.The Western-blotting analysis showed that the protein had good biological activity.These results confirmed that Cherry Valley CD8a extracellular domain was successfully expressed in E.coli BL21 (DE3).3 Antibody preparatiom and prelimary application of extracellular region of CD8αgene of cherry valley duckThe purified recombinant protein was used as immunogen. The research smoothly prepared the rabbit anti-recombinant protein serum according to designing immune program. The serum titer is 1: 51200 which was measured by indirect ELISA. During different phases of post-infected duck plague virus, the percentage of CD8 positve lymphocyte concerning the all lymphocyte in control group was 29-32%, yet the infected group was 10-21%. The rate of peripheral blood CD8 positve lymphocyte decreased from the first day to the third day after infected, but the rate got to raise regularly after the third day. But the expression level of CD8+T lymphocyte was lower than control group.On the other hand, there was positive sigals on partial neutrophil surface after immunohistochemical method dyeing.The result showed successful expression of the extracellular domain of CD8a, preparation of polyclonal antibody of rabbit anti-Cherry valley duck CD8a which providing methods for detection of duck CD8+T lymphocytes in peripheral blood.