克氏原螯虾早期发育及卵黄蛋白含量的研究
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摘要
对克氏原螯虾早期发育进行了取样观察,参考相关文献并根据胚胎发育各个时期的特点将胚胎发育划分为了8个时期,分别为：受精卵期、卵裂期、囊胚期、原肠期、前无节幼体期、后无节幼体期、蚤状幼体期、孵化准备期。对克氏原螯虾胚胎和幼体发育的不同时期的形态特征分别进行了详细的描述。
采用电泳法从雌性克氏原螯虾成熟卵巢中纯化得到了卵黄蛋白,并利用卵黄蛋白作为抗原免疫新西兰大白兔,制备了卵黄蛋白抗血清,免疫扩散检测显示得到的抗血清是高效价高特异性的。
利用纯化的Vn和抗血清建立了间接竞争免疫吸附反应(IC-ELISA)测定克氏原螯虾卵黄蛋白的方法,抗血清与包被抗原的最佳工作浓度分别为1：40000和31.25 ng/ml卵黄蛋白；检测范围是10～1000 ng/ml,在这个区间内标准曲线有良好的线性和重复性,批间批内误差都小于10%,使对卵黄蛋白的测定能够快速准确的进行。
应用IC-ELISA方法定量测定了克氏原螯虾早期发育阶段卵黄蛋白的含量变化,发现卵黄蛋白的含量呈一定规律性变化,在前无节幼体期卵黄蛋白含量有所增加,达到0.1888 mg/ml。之后呈下降趋势,到第三龄幼体时期几乎消耗殆尽,仅存0.0022mg/ml,此时期幼体已可以开口摄食。
The early development of crayfish was observed. Refering to existing documents and according to their characteristics embryonic development can be divided in to eight period: fertilized egg stage, cleavage stage, blastula stage, gastrul stage, early stage nauplius, late stage of nauplius, the zoea stage and incubative prepare stage. The morphological characteristics of embryos and larva in different developmental periods were studied respectively in detail.
Vitellin was purified from mature ovary of female crayfish via electrophoresis method. Specific antiserum against purified vitellin was raised from rabbit. It was proved that the antiserum was specific by means of immunoprecipitation reaction.
An indirect competitive enzyme-linked immunosorbent assay (ELISA) for detecting vitellin content of Procambius clarkii was established. The optimum concentration of antiserum and antigen coating were 1:40000 and 31.25 ng/ml vitellin respectively. The linear detection ranged from10ng/ml to 1000ng/ml, which has good linearity and repeatability, with mean intra-and inter-assay coefficients of variation less than 10%. The content of vitellin was determined rapidly and accurately.
ELISA was used to detect the change of vitellin in the crayfish early developmental stage. It was found that nauplius vitellin was increased in early stage. The content can reach 0.1888 mg/ml. Then it declined. Vitellin only left 0.0022 mg/ml in the third instar. The larval already can feed in this period.
采用电泳法从雌性克氏原螯虾成熟卵巢中纯化得到了卵黄蛋白,并利用卵黄蛋白作为抗原免疫新西兰大白兔,制备了卵黄蛋白抗血清,免疫扩散检测显示得到的抗血清是高效价高特异性的。
利用纯化的Vn和抗血清建立了间接竞争免疫吸附反应(IC-ELISA)测定克氏原螯虾卵黄蛋白的方法,抗血清与包被抗原的最佳工作浓度分别为1：40000和31.25 ng/ml卵黄蛋白；检测范围是10～1000 ng/ml,在这个区间内标准曲线有良好的线性和重复性,批间批内误差都小于10%,使对卵黄蛋白的测定能够快速准确的进行。
应用IC-ELISA方法定量测定了克氏原螯虾早期发育阶段卵黄蛋白的含量变化,发现卵黄蛋白的含量呈一定规律性变化,在前无节幼体期卵黄蛋白含量有所增加,达到0.1888 mg/ml。之后呈下降趋势,到第三龄幼体时期几乎消耗殆尽,仅存0.0022mg/ml,此时期幼体已可以开口摄食。
The early development of crayfish was observed. Refering to existing documents and according to their characteristics embryonic development can be divided in to eight period: fertilized egg stage, cleavage stage, blastula stage, gastrul stage, early stage nauplius, late stage of nauplius, the zoea stage and incubative prepare stage. The morphological characteristics of embryos and larva in different developmental periods were studied respectively in detail.
Vitellin was purified from mature ovary of female crayfish via electrophoresis method. Specific antiserum against purified vitellin was raised from rabbit. It was proved that the antiserum was specific by means of immunoprecipitation reaction.
An indirect competitive enzyme-linked immunosorbent assay (ELISA) for detecting vitellin content of Procambius clarkii was established. The optimum concentration of antiserum and antigen coating were 1:40000 and 31.25 ng/ml vitellin respectively. The linear detection ranged from10ng/ml to 1000ng/ml, which has good linearity and repeatability, with mean intra-and inter-assay coefficients of variation less than 10%. The content of vitellin was determined rapidly and accurately.
ELISA was used to detect the change of vitellin in the crayfish early developmental stage. It was found that nauplius vitellin was increased in early stage. The content can reach 0.1888 mg/ml. Then it declined. Vitellin only left 0.0022 mg/ml in the third instar. The larval already can feed in this period.
引文
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