Identification Of The Hemocyanin’s Functional Peptides In Abalone(Haliotis Diversicolor) And The Differential Proteome Of Blood Plasma Under AbSV Infection

The taxonomic position of abalone: Mollusca, Gastropoda, Prosobranchia, Haliotiidae.Abalone is called the best seafood, sweet-meated, eutrophy, which is the traditionaldelicacies of China, furthermore, it has very high medicinal value. Haliotis diversicoloris the important breeding stock at the south of China. In recent years, abalone bleedingrapidly expanding, breeding environment increasingly become deteriorated, the diseaseof abalone increase at the trend of explosive growth,and cause great damage to theabalone breeding. However, at the molecular level of the innate immune system andimmune factor is still very limited, which limits the disease prevention and control thedevelopment of the technology to abalone. In the present study for material bloodlymph of abalone utilize two-dimensional electrophoresis and Western research forprotein group and hemocyanin, to carry out in-depth study of abalone molecularimmunity and lay a good foundation.For the materials with abalone blood lymphatic establish two-dimensionalelectrophoresis system and compatible with mass spectrometry analysis: first, identifiesacetone precipitation and RC DC protein quantitative kit on the basis of proteinpreparation and quantitative methods, secondly, explore the sample pretreatmentmethods, Through the500r/min of low speed centrifugal separate the blood cells andplasma components, through the35000r/min speeding centrifugal2h to away the someof the abundant mrnas blood blue protein, and to reduce the high molecular weight ofblue blood protein regional background. Further use18cm pH3-10nonlinear blockgine and standard electrophoretic procedures to different sample weight on the silverstain electrophoresis result is compared, can obtain good two-way electrophoresispattern. Eventually select four protein point to mass spectrometry appraisal, respectivelyactin, original myosin, collagen and blood blue protein. Abalone blood lymphatictwo-dimensional electrophoresis provides a subsequent abalone differences and functionproteomics research provides technical support.Blood blue protein has variety of immune function, complete protein molecules playimmune action, at the same time, it still can degrade into smaller function of peptides,this experiment using two-dimensional electrophoresis and Western combining to get abundant blood protein of features blue peptides. After two-dimensional electrophoresis,using the laboratory have preparation good blood blue protein purification forWestern polyclonal antibody detection, blood lymphatic total protein samples on120μg,the first hybrid of scale is1:20000, the second is1:5000. get a clear hybrid spotsfigure, blue blood protein hybrid article mainly concentrated in figure of signal centralpartial on regional. Get ten hybrid spots are mass spectrum identification, successfullyidentified24protein, which are7different function peptides.Since it was discovered, abalone shriveling syndrome-associated virus (AbSV) wasrepeated outbreak in every winter and spring, and every time was devastating. The virusis not known to the virus types, virus form has not yet been established, geneticstructure special, not the protein coding, moreover some scholars doubt host blood blueprotein may provide protection for virus nucleic acid. This experiment from the point ofview of proteomics before and after infection by AbSV Haliotis diversicolor plasmatotal proteins at different time points difference,0h,6h,12h and24h4time points ofthe continuous variation are relatively obvious have20protein points, includingcontinued to rise, first increased and then drop, first drop and then increased, and specialpoint, infected72h later, there are63differences protein points. Mass spectrumidentification results including actin, original myosin, collagen protein, fructose enzymeand blue blood protein, including the structure and function of the protein and part ofthe protein involved in the immune disease resistance. These proteins information willhelp us to master pathogenesis mechanism and the immune mechanism, obtain theimmune related proteins, for providing favorable basis of disease control.