三疣梭子蟹卵黄磷蛋白纯化及其ELISA测定方法

Purification of vitellin and ELISA determination of vitellin of swimming crab(Portunus trituberculatus)

采用凝胶过滤层析法纯化了三疣梭子蟹成熟卵巢的卵黄磷蛋白(vitellin,Vn),采用变性凝胶电泳(SDS-PAGE)确定了Vn亚基数量和分子量,以纯化的Vn为抗原,制备了三疣梭子蟹Vn多克隆抗血清,纯化后得到Vn抗体,在此基础上比较和优化了三疣梭子蟹Vn测定的酶联免疫吸附法(ELISA)参数,建立了稳定的三疣梭子蟹Vn含量测定的ELISA方法。结果表明:(1)SDS-PAGE显示三疣梭子蟹成熟卵巢中的Vn含有分子量为100、75和66 ku的3个亚基,Western-blotting检测表明,这3个亚基均具有较强的免疫特异性;(2)样品直接包被法比双抗体夹心法具有更高的线性相关性,Vn抗体最佳稀释倍数为1∶90 000,最佳包被时间为8 h,一抗和二抗反应时间分别为2 h和1 h,显色时间为20 min;(3)该方法定性检测的灵敏度为14.9 ng/mL左右,标准曲线方程为y=0.000 9x++0.399 1(R2=0.986 1),其中x和y分别代表Vn浓度和OD450值,工作范围为200～900 ng/mL;(4)应用该方法测定三疣梭子蟹卵巢中Vn含量,结果表明,批次内和批次间平均变异系数分别为3.59%和3.10%,重复性良好。

Vitellin is the most important nutrient and energy source for the development of oocytes as well as developing embryos.The quality and quantity of vitellin in the mature ovary play a vital role in the crustacean reproduction and offspring quality.The swimming crab,Portunus trituberculatus,is a commercially important fisheries resource and mariculture species in East Asian countries.Because of overfishing,destruction of coastal spawning and nursery grounds and marine pollution,the landing of this crab has shown a declined trend in East China Sea since 1990s.The decline in natural stock and increase in market demands have driven substantial aquaculture interests for the crab species.However,the seed production of the crab is dependent on wild-caught broodstock,which could become a constraint to the sustainable development of this crab aquaculture.Our previous studies have shown pond-reared female broodstock have poor ovarian development and worse reproductive performance compared to wild females.Therefore,it is very urgent to understand the mechanism of vitellin synthesis and accumulation for female P.trituberculatus.The present study was conducted to purify vitellin from mature ovary of female P.trituberculatus by gel filtration chromatography,and the elution process was monitored at wavelength of 280 nm and 470 nm,respectively.The number and molecular weight of subunits were identified by sodium dodecyl sulphate-PAGE（SDS-PAGE）and marker protein,respectively.Then,the rabbit polyclonal vitellin anti-serum was prepared and purified.Based on the vitellin anti-body,we optimized the reaction parameters of Enzyme-Linked Immunoabsorbent Assay（ELISA）,and then the stable and standard ELISA was developed for female P.trituberculatus.The results showed that vitellin was divided into three major polypeptides with molecular weight of 102,75,and 66 ku,respectively by the denatured SDS-PAGE.The Western-blotting confirmed all three major polypeptides had specific reactivity with vitellin anti-body.The higher linear correlationship could be found on the sample direct coating ELISA than the antibody sandwich coating ELISA.Then,the optimal dilution rate of purified vitellin antibody and coating period were shown to be close to 1∶90 000 and 8 hours at 4 ℃,respectively.At 37 ℃,the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour,respectively,while the best color development solution was around 20 minutes.Furthermore,based on these parameters,the standard linear equation,y=0.000 9x＋0.399 1（R2=0.986 1,x and y represent Vn concentration and OD450 value,respectively）,was established for the determination of female P.trituberculatus vitellin concentration with the valid range of 200-900 ng/mL.The susceptibility of this ELISA was around 14.9 ng/mL for Vn contents.Finally,the ELISA was used to determine ovarian Vn contents for the identification of validity of this assay.The results demonstrated the mean coefficients of variation of intra-assay and inter-assay were 3.59% and 3.10%,respectively.In conclusion,the developed ELISA of this study is precise,stable and repeatable.