1. 安徽农业大学 生命科学学院, 安徽 合肥 230001;2. 浙江省淡水水产研究所 鱼病室, 浙江 湖州 313001
许娜娜(1986), 女, 硕士研究生, 研究方向为水产病害防治. E-mail：xunana77@yahoo.com.cn
宁波市重大科研项目(2007C10037).
)-IgG对提取组份进行了蛋白浓度测定、间接效价提高A洗脱峰集中在处Goat-IgG 1.4~2.8 mL72.4%效价为2.63%轻链采用鼠抗花鲈经Ig蛋白柱和Ig
The present study aims to establish antibody detection methods for further vaccine research and application. Healthy sea perches () 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 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 1.37per 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 1.39×105 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 effects assessments for sea perch, which would benefit the prevention and control of sea perch diseases.
