Separation,Identification And Environment Influence Of Norovirus Receptor Hbgas-like In Crassostrea Gigas

Food borne diseases caused by biological pollution has become a prominent issue affecting food safety,and is increasingly concerned by the world.As the leading causative agents of epidemic and sporadic viral gastroenteritis worldwide,human noroviruses have been popular in all over the word,which can lead patients to death,and posed a great threat to the consumers' health.Oyster is delicious,nutritious and huge production.Oyster farming area has reached 140,000 hectares in 2015,an increase of 6.1% over 2014.The total output more than 4.57 million tons,accounting for China's total aquaculture production 33.03%.However,as an important communication media of No Vs,oysters are recognized as high risk foods that always cause the viral gastroenteritis outbreaks.Studies have shown that most of the shellfish-related No Vs epidemic associated with edible oysters,and conventional shellfish purification processing method is difficult to effectively remove No Vs from the oyster.Being the receptor of No Vs,Human blood group antigen plays an important role in the susceptibility of diffident person to No Vs.Tian has found that HBGAs-like are also existing in oysters to specificity combine with No Vs,which is the probably reason that the oysters enriched No Vs.Extraction method of HBGAs-like molecules was respectively established from gut and gill by comparing 5 glycoprotein extraction methods.Distribution and classification of HBGAs-like were analyzed by the established extraction and ELISA method.The expression of HBGAs-like and FUT2 genes in the gut and gill of Crassostrea gigas were analyzed by changing the temperature and salinity of oyster culture conditions.The A type HBGA-like was separated and purified by DEAE Sepharose FF and SepharcrylTM S-300 gel;The structure of A type HBGA-like was studied by MALDI-TOF-TOF-MS,IR,GC/MS and monosaccharide composition analysis.This study provided a theoretical basis for further study on the mechanism of No Vs concentration in Crassostrea gigas.The main results of this study were as follows:1.The extraction method and classification of HBGAs-like in gut and gill of oysters was established.The extraction of oyster HBGA by using water as a leaching solution is superior to that of using PBS as extract,and the extraction methods of HBGAs in different tissues are different.Twenty-four oyster samples were used for HBGA genotype detection,and the polymorphic characteristics of HBGAs-like in oyster was found;The A type HBGA-like was stably expressed in gut with detection rate 100%,while type A and Leb in gill with detection rate 83.3% and 91.7%,respectively.2.Temperature and salinity effection of HBGA-like and its key genes in oyster were studied.The analysis of temperature effection of HBGA-like and FUT2 genes in oyster found that the expression of A type HBGA-like and FUT2 gene in gut were significantly increased under low temperature and high salt.Those results were consistent with the season of norovirus outbreak,which showed that there was a certain correlation between the seasonality of norovirus outbreak and the expression of HBGAs-like and FUT2 gene in Crassostrea gigas.3.A type HBGA-like was separated and purified.The extractive of A type HBGAs-like was separated and purified by DEAE-sepharose fast flow,and the separated component F1 and F2 were obtained.F2 was further purified by Sepharcryl S-300 gel,and the purified component YY1 was obtained.4.The structure of A type HBGA-like was primarily explored.Separated by SDS-PAGE and Verified by Western Blot for F1 and F2,this study found that the molecular weight of A type HBGA-like was longer than 180 k Da,and it was significantly different from that of human.These results indicated that there could be some different in the structure between A type HBGA-like and human A type HBGA;Two kinds of matching protein and 20 peptide sequences were searched by MALDI-TOFTOF-MS and analysis software Mascot2.3 analyzing.Among them,the credibility of 3 peptide sequences,that polypeptide sequences are VYDLEFVFDYRDDYAR,DNPEQPDIPELPTETFVVNR and AQSIEDTNSEGPVVQDTALAVLR,are alone exceeding the value of scores.Analyzing by IR,GC/MS and monosaccharide composition,YY1 may contain a same type of polysaccharide structure as that of human A type HBGA.However,kinds of monosaccharide composition andmiscellaneous of components,YY1 needs to be further separated and purified.