The Determination Of The Whole Gene Sequence And Establishment Of The Rapid Diagnosis Method Of Infectious Hypodermal And Hematopoietic Necrosis Virus

Infectious hypodermal and hematopoietic necrosis also called runt-deformity syndrome, sensitive host is Litopenaeus stylirostris and Penaeus vannamei, natural state can affect most of the breeding prawns. IHHNV is the smallest virus of shrimp virus we have known. Nuclear capsid proteins contains at least four peptides, molecular weight are74、47、39and37.5Kd. IHHNV genome is constituted by three open reading frames and two ends of the non-coding sequences, ORF3encodes structural protein of virus, that is capsid protein.This experiment is sequences the whole genome of IHHNV virus:GanYu jiangsu strains, Clones expresses CP gene conservative district, prepares polyclonal antibody, and preliminary studyies on the colloidal gold immunochromatographic assay for rapid detection method.According to the genome sequence of IHHNV Hawaii strains, we designed several pairs of primers. Using PCR technology to amplificate genome segments. Through the sequencing, compare and joining together, get the gene sequence of IHHNV jiangsu GanYu strains, and analysised it.Jiangsu GanYu strains IHHNV gene sequences span3901nt.5’end and Hawaii than plant, at the end of the12to reduce nucleotide, and in70nt-170nt exist a nucleotide difference. And3’end only3790nt place in Hawaii strains, only a nucleotide variation, and Hawaii strains of the similarity as high as98.5%. The genetic sequence of the coding sequence larger variation. Coding sequence variation is small, only a small number of bases mutations, not produce new terminate son makes a protein translation of interrupts coding new protein products, to little effect on the protein synthesis.Based on the genome sequence of GanYu jiangsu strains, primer were designed to amplify CP conservied region.573bp product was got by PCR. The product was inserted to plasmid pET-28a for making recombinant plasmid and then transformed to host strain BL21. The positive clone identified by endonuclease digest were induced by IPTG. Detection target proteins by SDS-PAGE. The recombinant is well expressed in the form of inclusion body with1.0mmol/L IPTG at37℃. An Ni+2affinity column is used to purify the inclusion body protein.The recombinant protein pET28a-CP1was purified with HisTrapTM affinity column. New Zealand rabbits and ICR mice were immunized with the purified protein to produce polyclonal antibody. Detecting the antibody titer by indirect enzyme-linked immunoadsorb assay (ELISA).The optimum coating concentration of CP1recombinant protein are5μg/mL (mouse) and2.5μg/mL (rabbit). The optimum diluting times of rabbit anti-CP1recombinant protein serum and mouse anti-CP1recombinant protein serum were1:6400and1:12800.The mouse anti-CP1recombinant protein and New Zealand rabbits anti-CP1recombinant protein serum titer were about1:12800. Pure IgG was purified from the antiserum by Hitrap Protein G column. Western blot of roughly purified IHHNV polypeptides, rabbit and mouse polyclonal antibodies reacted with55kD IHHNV polypeptide.Two diameter of colloidal gold (20nm and30nm) were used respectively to label two polyclonal antibodies. One is rabbit anti-CP1recombinant polyclonal antibody, the other is mouse anti-CP1recombinant polyclonal antibody. The assay was constructed in the form of sandwich by using two anti-CP1recombinant polyclonal antibodies. Rabbit anti-CP1recombinant polyclonal antibody labeled with20nm colloidal gold was made a conjugated pad. Mouse anti-CP1recombinant polyclonal antibody was immobilized in a defined detection zone on a porous nitrocellulose membrane. Negative control were tested successfully. Detection roughly purified IHHNV with this immunochromatographic test strip, control line is obviously red, while the color of test line is light.