| In recent years, environmental estrogens, chemicals are known to pose aninsidious risk to both wildlife and humans, have raised considerable concern. Now,Vitellogenin (Vtg) is widely used as an important biomarker to detect environmentalestrogens. Studies showed that piscine Vtgs are prone to degradation in thepurification process and particular in high temperature. Besides, Vtg may beincreasingly susceptible to proteolysis with prolonged storage at low temperatures,which caused a larger error to quantitative determination of Vtg in plasma. Vtg issequestered by growing oocytes and then cleaved proteolytically into three major yolkproteins, of which lipovitellin(Lv) and β’-component(β’-c) have the sameimmunogenicity with Vtg. Lv, due to their abundance and stable, the researchers takethem as candidate antigens to prepare type-specific antibodies for Vtg detection. Weselected ovarian tissue of goldfish as experimental materials, purified goldfish Lv,study its basic properties, heat-stability and prepared the polyclonal mice antiserumagainst goldfish Lv. Using immunological method analyze immunogenicity betweenthe goldfish Vtg and Lv, and detect the immune cross-reaction between the polyclonalantiserum and other fish Vtg by Western blot. The result shows as follows:Lv was purified by gel filtration and ion-exchange chromatography fromgoldfish ovaries. The Lv was characterized as a phosphoglycoprotein byNative-PAGE, and staining of gels for lipids with sudan black B, and forcarbohydrates with Schiff reagent, and for phosphorus with methy green. The Lvappeared to exist as a homotrimer of approximately385kDa in Native-PAGE, andwas reduced to a single monomer of approximately112kDa in SDS-PAGE. Analysisof the amino acid composition of goldfish Lv revealed the presence of relatively highlevels of Glu(12.28), Ala(10.84%), Val(10.37), Leu(11.02%), Asp(6.56%), accounting for51.07%by weight of the total amino acids. However, the levels of His (2.53) andMet (2.71) were relatively lower. These were closely similar to taimen and rainbowtrout. Compared with goldfish Vtg, the level of Ser reduced by nearly50%, and theyboth didn’t contain Cys. All these results proved that the purified protein was goldfishLv.Purified Lv was used to immunize male mice by intraperitoneal injection. Bydouble immunodiffusion, the effect of the polyclonal mice antiserum against goldfishLv was measured to be at least1:32. Double immunodiffusion assays showed that theplasma and ovary extract from the female goldfish reacted with the antisera, while theplasma from the male goldfish did not react with antisera. This proved that Lv was afemale-specific protein. Immunoelectrophoresis showed that goldfish Vtg reactedwith the antisera against Lv, forming a single immunoprecipitin line, and thisindicated that goldfish Vtg and Lv had common antigenicity. With the western blot forVtg detection, the detection lower limit is1.1μg/mL.The Lv antisera could be further used to detect Vtg from other species. In thispaper, we found the plasma from female common carp and crucian carp could reactwith the antisera, they both had two main bands of118and107kDa. The Vtg fromzebrafish showed high affinity for the antisera. Two marine fish, red drum and stoneflounder could be clearly detected in the low concentration by Western blot.In conclusion, Lv antisera were suitable for detecting environmental estrogens,and it showed good cross-immunity with other fish. |
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