中国水产科学2011，Vol.18Issue(1)：103-109,7.DOI:10.3724/SP.J.1118.2011.00103
花鲈免疫球蛋白的分离纯化及部分特性分析
Purification and partial characterization of serum immunoglobulin in Lateolabrax japonicus
摘要
Abstract
The present study aims to establish antibody detection methods for further vaccine research and application. Healthy sea perches (Lateolabrax japonicus) were injected with commercial goat IgG emulsified with Freund's adjuvant four times in two months to develop good immune response. The sera of immunized sea perches were collected for immunoglobulin (Ig) isolation with saturated ammonium sulfate (SAS) precipitation, protein A-sepharose affinity chromatography and goat IgG-immobilized sepharose 4B affinity chromatography. It was shown that Ig mainly distributed in 30％-50％ SAS precipitation, with the maximum ELISA titer per mg protein being 45％ SAS, 2.2 times higher than that of original serum. Protein A column produced single elution peak with the elution volume of 0.8-1.6 mL, accounting for 76.2％ of total collected protein, with the ELISA titer being 1.37× l05 per mg protein, 15 times higher than that of original serum. Goat IgG-immobilized sepharose 4B affinity chromatography produced single elution peak with the elution volume of 1.4-2.8 mL, accounting for 72.4％ of total collected protein, with the ELISA titer being 1.39× l05 per mg protein. SDS-PAGE showed that Ig eluted by both protein A column and goat IgG-immobilized sepharose 4B affinity chromatography yielded two protein bands of 79 kD and 29 kD, which could be considered as H chain and L chain of sea perch Ig, while Ig precipitated with 45％ SAS yielded more bands besides H and L chains. Protein bands of 79 kD and 29 kD were confirmed to be H and L chains by western-blotting with the antiserum and monoclonal antibody AA5. The Ig yield coefficients of protein A and goat-IgG methods was calculated as 2.57％ and 2.63％, respectively. These results indicated that both protein A column and IgG-immobilized sepharose 4B affinity chromatography could successfully isolate Ig from sea perch serum with one step of absorption and elution. Protein A column method could be considered as a more effective and less time consuming method for sea perch Ig isolation, yielding narrower elution volume and higher antibody titer, without long period of vaccination with goat IgG to fish. The current research provide a useful technical tool for further studies on humoral immune response for vaccine development and vaccine effectsassessments for sea perch, which would benefit the prevention and control of sea perch diseases.关键词
花鲈/免疫球蛋白/纯化/亲和层析分类
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许娜娜,钱冬,赵海泉..花鲈免疫球蛋白的分离纯化及部分特性分析[J].中国水产科学,2011,18(1):103-109,7.基金项目
宁波市重大科研项目(2007C10037). （2007C10037）
