Purification And Characterization Identification Of Female Protein From Pelteobagrus Vachelli And Established Enzyme-Linked Immunosorbent Assay

After a 7-day intraperitoneal injection of 17β-estradiol(E₂),the male Pelteobagrus vachelli will produce the Vitellogenin(Vtg).Vtg from E₂ treated P.vachelli plasma was isolated and purified by gel filtration and ion-exchange chromatography;Mg²⁺—EDTA and gel filtration;Mg²⁺—EDTA and electronic elution.By the phosphor-,lipo- and glycol-protein staining methods,we can verify this protein as Vtg.The molecular weight of Vtg is about 240kDa detected by Native-PAGE.In SDS-PAGE the Vtg broke into 2 same subunits,each with a molecular weight of 140kDa.The purified P.vachelli Vtg may contain carotenoid,but no disulfide bond.It is relatively stable to heat.Making use of purified Vtg,we prepared the polyclonal antiserum against P.vachelli Vtg.Double immunodiffusion showed determination of the titre for Vtg antisera was 1:32, Western-blotting analyses demonstrate that polyclonal antiserum has preferably specific effect.An indirect competitive enzyme-linked immunosorbent assay(ELISA) is then established for detecting Vtg of P.vachelli.The technique is developed with antiserum of Vtg resistance as the antibody and Vtg as the antigen.The results indicate that the working range is 31.2—4000 ng/ml,and the sensitivity 15.6ng/ml.The equation linear part of a typical ELISA calibration curve is y=0.099x+0.4529(R²=0.9327),which shows good linearity in the working range.Lv from P.vachelli stageâ…£of ovarian extract was isolated and purified by gel filtration and ion-exchange chromatography.By the phosphor-,lipo- and glycol-protein staining methods,we can verify this protein as Lv.The molecular weight of Lv is about 230kDa detected by Native-PAGE.In SDS-PAGE the Lv broke into 2 same subunits,each with a molecular weight of 106kDa.The purified P.vachelli Lv may contain carotenoid,but no disulfide bond,it is relatively stable to heat.Making use of purified Lv,we prepared the polyclonal antiserum against P.vachelli Vtg.Double immunodiffusion showed determination of the titre for Lv antisera was 1:32 and has clearly immunity cross-reacted between Vtg and Lv.Western-blotting analyses demonstrate that Lv polyclonal antiserum has preferably specific effect,its can specific reactivity with Vtg.