Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, Tromsø, Norway.
Fish Shellfish Immunol. 2010 Dec;29(6):1106-9. doi: 10.1016/j.fsi.2010.08.021. Epub 2010 Sep 9.
In this study we describe the production and characterization of an antiserum against recombinant g-type lysozyme derived from Atlantic cod. This is also the first initial analyses of g-type lysozyme protein expression in tissues of Atlantic cod. Recombinant expression and purification of cod g-type lysozyme was used for immunization to rabbit and the rabbit sera were analysed for anti g-type lysozyme antibodies using enzyme-linked immunosorbent assay (ELISA), Western blot and immunohistochemistry. ELISA results showed that antibody titres were mounted between 12,800 and 25,600 as measured at an optical density corresponding to 50% of the maximal level. By Western blot analysis the rabbit immune serum detected a single ∼23 kDa band representing the size of the injected antigen, in both spleen and head kidney homogenates from the Atlantic cod. Immunohistochemisrty detected the native folded g-type lysozyme in tissues and revealed that g-type lysozyme positive cells were observed in haematopoietic tissue of the head kidney and in red pulp of spleen. In conclusion, the rabbit anti g-type lysozyme immune sera was developed and is effectively utilized for ELISA, Western analysis as well as for immunohistochemistry. This has allowed us to obtain new knowledge about this protein regarding localization and distribution in cod tissue.
在这项研究中，我们描述了一种针对源自大西洋鳕鱼的重组 g 型溶菌酶的抗血清的生产和特性。这也是首次对大西洋鳕鱼组织中 g 型溶菌酶蛋白表达进行的初步分析。我们使用重组表达和纯化的鳕鱼 g 型溶菌酶进行免疫接种兔子，并使用酶联免疫吸附测定（ELISA）、Western blot 和免疫组织化学分析兔抗 g 型溶菌酶抗体。ELISA 结果表明，抗体效价在 12800 到 25600 之间，这是在与最大水平的 50%对应的吸光度下测量的。通过 Western blot 分析，兔免疫血清在大西洋鳕鱼的脾脏和头肾匀浆中检测到了单一的约 23 kDa 条带，代表注射抗原的大小。免疫组织化学检测到组织中的天然折叠 g 型溶菌酶，并揭示 g 型溶菌酶阳性细胞存在于头肾的造血组织和脾脏的红髓中。总之，我们开发了兔抗 g 型溶菌酶免疫血清，并有效地用于 ELISA、Western 分析以及免疫组织化学。这使我们获得了有关该蛋白在鳕鱼组织中的定位和分布的新知识。
