Establishment And Application Of Two ELISA Methods For MPⅡ In Perinereis Aibuhitensis

In this research,a fusion protein metalloproteinⅡwas obtained on account of therecombinant expression srtain was activated, enlarge cultivated and induced by IPTGand further purified with His-Bind purification kit,the MPⅡ concentration wasdetected by Coomassie brilliant blue G-250;the high drops of rabbit anti-serum wasprepared using the MPⅡ as an antigen, then the titer was evaluated by Dot-ELISAthe MP Ⅱ was used as a coating antigen after it was diluted200,400,800,1600,3200,6400times with carbonate buffer solution (pH9.6),2μL perhole,the anti-serum was diluted200,400,800,1600,3200,6400and12800times withphosphate-buffered saline(PBS)at pH7.4,10μL per hole, PBS(pH7.4) was used asnegative control, the saturation was realized for45minutes at37℃with10·ml of a3%bovine serum albumin (BSA)in PBS;the reaction conditions of indirectenzyme-linked immunosorbent assay were optimized; different tissues of Perinereisaibuhitensis were detected and the target tissue was determined using the optimizedindirect ELISA;then,the Perinereis aibuhitensis were acute exposed to the followingconcentrations of cadmium40,80,120mg/L,lead10,15,20mg/L,and of zinc30,60,90mg/L for5days and chronically exposed to the concentrations of cadmium0.01,0.5,5mg/L,lead0.05mg/L,0.1mg/L,0.5mg/L,and of zinc0.5mg/L,1mg/L,5mg/L for15days.The indirect ELISA was used to detect the concentration of MPⅡ. Theexperimental results showed that the molecular weight of metalloprotein Ⅱ with Histag was about32KDa;the titer of the anti-serum was1:512000; in Dot-ELISA,theLOD of envelope antigen was0.03μg/mL，dilution of anti-serum was1︰6400；theoptimal detection conditions of indirect ELISA were as following: the optimalconcentration of envelop antigen was0.24μg/mL,the optimal enveloping conditionwas4℃over night;2hours’ closing,the optimal dilution of anti-serum was1:200,30minutes’incubation;45minutes’ incubation of enzyme-labeled antibody;10minutes’development.The senstivity of indirect ELISA were as followings:the concentration ofantigen was0.06μg/mL,dilution of anti-serum was1:3200;the maximum P/N was atthe concentration of antigen was0.24μg/mL,and the dilution of anti-serum was1:200.On the basis,a standard protein curve regression equation wasy=0.5596x+2.3332;its linear detection was from300to4800ng/mL(R2=0.9993);thetarget issue was digestive tract,followed by the head, body wall and parapodum werethe least and with no significant difference between them.In acute exposure to heavymetal,three kinds of heavy metal all increased the expression of MPⅡ,which was significant in the first3days,and it was positively related to the concentrations ofheavy metal, cadmium standing out significantly among all the metal;in chronicalexposure,the expression of MPⅡ also increased but not as significant as acuteexposure,among which the high concentrations’ exposure standing out,however,thelow concentration group were not significant and irregular.