Cloning And Expression Research And Its Polyclonal Antibody Preparation From Carassius Auratus In Qihe River

Interleukin-8(IL-8) is a CXC chemokine, which is closely related to innate immunity offish. In this study, Carassius auratus in Qihe river was the research object.Using homologycloning and RACE technology, we cloned the full sequence of IL-8cDNA,and theimmunogenic protein of IL-8by pET-32a (+) expression plasmid expressing, and IL-8wereprepared polyclonal antibody. Secondly, using real-time fluorescent quantitative PCRtechnology,we detected the expression of IL-8gene in different tissues of healthy crucian carp,as well as after intraperitoneal injection of LPS, IL-8gene expression levels were exmined inmuscle, spleen, kidney, tissue.1. The cloning and sequence analysis of IL-8in crucian carpThe full length sequence of IL-8cDNA in crucian carp is641bp, including102bp5’UTR,242bp3’ UTR and297bp open reading frame, which encodes98amino acids. Peptidestructure contains four conservative cysteine, the first two were separated by a argininebetween cysteine; It lacked the classic ELR (glutamic acid leucine-arginine) structure beforethe first cysteinecystine; Prediction of the spatial structure contains threeβ sheet and an αhelix; The IL-8of crucian carp compared a higher amino acid homology with cyprinidae fishsuch as carp, bighead carp, grass carp, silver carp.2.The constitutive and inducible expression of IL-8in crucian carpThe result of Quantitative PCR displayed that IL-8gene was the constitutive expression in head kidney, muscle, spleen, liver and pancreas, intestines, gills, eart, brain.The highestexpression was in muscle.The expression of IL-8mRNA in the muscle,spleen, kidney, intestinal were upregulatedafter intraperitoneal injection of LPS. The expression of IL-8mRNA in muscle after injection3days was the highest;The expression of IL-8mRNA in spleen reached a high level in4hour,and then stabilized;The kidney IL-8mRNA reached maximum after injection4hour, thendecreased gradually, expression levels after3days stabilized;Intestinal expression of IL-8mRNA showed the first increased and then decreased over time, and teached the maximumafter12hour. Saline may also upregulate the expression of IL-8in the four tissues.3. Prokaryotic expression and preparation of polyclonal antibody of IL-8in cruciancarpWe selected the coding sequence of IL-8without the signal peptide, introducing EcoR Iand Hind III restriction enzyme site, constructed pET-32a (+)-IL-8recombinant expressionvector, the E. coli Rosetta prokaryotic expression after IPTG induction, won the IL-8fusionprotein. We induced the engineering bacteria to produce large amounts of recombinantproteins and selected the purified IL-8protein as antigen. We adopted the method ofsubcutaneous multi-point injection to immune the New Zealand white rabbit to obtain theantiserum of IL-8. The titers was1:100000by ELISA.