红笛鲷仔鱼体内特异性母源抗体降解动力学研究
DEGRADATI ON KINETI CS OF LARVAL SPEC IFIC MATERNAL ANTIBODY FROM CRIM SON SNAPPER (LUTJANUS ERYTHOPTERUS)
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摘要: 应用山羊IgG免疫红笛鲷亲鱼,人工催产孵化出仔鱼后,用双抗体夹心法检测仔鱼特异性抗体的降解过程,发现抗体水平随时间变化呈下降趋势且可以持续7d,每尾仔鱼特异性抗体降解量约0.001μg,最低检测限为0.482μg/mL.采用饱和硫酸铵分步盐析与亲和层析等方法纯化红笛鲷仔鱼特异性抗体,结果显示,盐析仔鱼特异性抗体最理想的硫酸铵饱和度为40%-50%;亲和层析分离到两个明显独立的层析峰.SDS-PAGE证明纯化的抗体具有极高纯度,其L、H链分子量分别为29kD和77.5kD,抗体分子量约为852kD.该抗体不仅能够与山羊IgG产生特异性免疫反应,而且能与兔抗红笛鲷IgM抗血清产生免疫反应,因而认为红笛鲷母源抗体可以垂直转移至仔鱼.Abstract: The mature female crimson snapper (Lutjanus erythopterus) which was immunized with goat IgG propagated the larvae1Degradation kinetics of the larval specific antibody was analysed by using the double antibody sandwich ELISA1Purification and partial characterization of the specific antibody of larvae was studied1The purposes above were to elucidate the transfermechanism of fish maternal antibody and increase the resistance diseases of larvae1The female fish were immunized by intraperitoneal (i.p.) injection with the mixture of the goat IgG added equal volumes of FCA or FI A adjuvant four times interval two week during the period of egg development1After twenty days of final immunization, the female fish were i1p1injected with the mixture of LRH-A3 + HCG, then propagated the larvae1The results showed that the optimal concentration of the coating antigen, rabbit polyclonal antibody against crimson snapper Ig M and horseradish perox-idase-labelled goat anti-rabbit IgG were 11563 μg/mL, 1:800 and 1:2000 respectively1The standard curve indicated that the detection limitwas01482 μg/mL1The specific antibody of larvae degraded gradually and dropped to the lowest levels at 7 day after hatching1Meanwhile, ELISA showed that the specific antibody of larvae reacted with goat IgG and the serum of rabbit anti fish serum Ig M,which suggested that the specific antibody of larvae had some homologywith the serum Ig M1The specific antibody of larvae was purified from the larval homogenization using ammonium sulfate precipitation and affinity chromatography1The results showed that the larval antibody was salted out with 40%-50% saturation of ammonium sul-fate1Two independent peakswere obtained with the affinity chromatographymethod1SDS-PAGE showed that other proteins were not found in the purified larval antibody1The molecularweights of light and heavy chain of the antibodywere 29 kD and 7715 kD respectively, and grossmolecularweightwas calculated to be 852 kD1It should play an important role in enhancing resistance disease and viability of the larval fishes that the passively immunized mechanism through acquiring specialmaternal antibody via the yolk of the female fish immunized with antigen
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Keywords:
- Lutjanus erythopterus /
- Larval antibody /
- Degradation kinetics /
- Purification
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