Induction Of Gambusia Affinis Vitellogenin And Its Application In The Detection Of Environmental Endorine-Disrupting Chemicals

At present,the research using fish to detect the aquatic environmental endocrine disrupting chemicals(ECDs) has been focus in the golbe.The vitellogenin in the plasma of male fish can be used as a biomark and combined with an enzyme-linked immunosorbent assay.The existence of aquatic ECDs can be rapidly and efficiently detected in this method.This method is widely used in the open field detection,owing to its special and sensitive quality.This research used two steps of chromatography elution method to purify the Lipovitellin(Lv)from the Gambusia affinis. The first step of purification was to use Sephacryl s-300 column on FPLC.There were all 5 elution peaks existed.After concentrating and PAGE analysis of the sample,we found the 3th elution peak contained high concentration of protein.The second step of purification was to use DEAE-cellulose column in ion-exchange chromatography elution.The Nacl gradient ranged from 0.1¹M to get purifed Lv. And polyclonal antibodies against the purified Lv were generated in rabbits.The ELISA for measuring vitellogenin in male fish was developed and validated by injection immunization.After PAGE and Western blotting analysis ,the antibodies showed respectively special quality. The minimum detectable vtg is 62.5ng/ml.The effective detection ranges from 62.5ng/ml to 1000ng/ml.In the laboratory experiment of toxicological induction of E₂ and NP,fish were exposed in aquatic environment to detect the . relation among reacting concentration-time-dose. E₂ and NP were divided into 5 different dose groups to measure the concentration of Vtg over a duration 14 days post-exposure.The treatment groups of lower concentration showed no detectable rising concentration of vtg during the exposed 14 days.Groups of 500ng/L E₂ and 1000ug/L NP showed reaction on 5th day,200ng/L group of E₂ on 8th day,500ug/L NP on 14th day. In treatment groups the concentration of vtg increased according to the dose.Compared with the control groups, high dose treatment groups showed significant variation(P<0.05), and the concentration of vtg increased until the end of the exposure.During the 14d expoure time,the lowest valid concentration of E₂ was200ng/L,the highest invalid concentration was 100ng/L;the lowest valid concentration of NP was 500ug/L,the highest invalid concentration was 200ug/L.In the open field detection experiment,five spots of suzhou river in shanghai city were selected (Beixingjing Rd spot,Kaixuan Rd spot,Wuning Rd spot,Zhongtan Rd spot and Zhejiang Rd spot).Vtg concentration of male fish was detected in the 5 spots above during August and December.Lower congcentration of Vtg has been detected in the fish of the zone of Kaixuan Rd spot and Wuning Rd spot, the male fish Vtg concentration of both above spots separately attained to the rate of 50% and 25% in all the detected samples.Single factor variance analysis and SPSS12.0 software were used to detect the difference between the treated and the control groups.Through the comparison method of Duncans,we found the male fish Vtg concentration of Kaixuan Rd showed great difference compared with the control groups(P<0.05).And the Vtg concentration were 12.8% highter than the control groups.At the same time other spots showed no significent difference.The result of this study show that,through comparing the different exposure effect between the laboratory and open field experiment, vtg of Gambusia affinis together with the detection method of ELISA can be used as a sensitive and efficient measurement of EDCs,which also can be applied in the investigation and preliminary analysis of aquatic environment pollution.