作　　者： ; ; ; ; ; ;

机构地区： 湛江海洋大学水产学院

出　　处： 《中国病毒学》 2004年第2期137-140,共4页

摘　　要： 根据鳜鱼传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus, ISKNV)主要衣壳蛋白(Major Capsid protein, MCP)基因(mcp)序列设计引物,PCR扩增得到一长约1400bp的DNA片段, 将其克隆到pGEM-T Easy Vector。氨基酸亲水性分析表明,在150-250位氨基酸之间亲水性很高,可构成主要抗原决定簇及形成跨膜区。mcp基因经PCR改造后克隆至原核表达载体pBV220,构建表达MCP的大肠杆菌基因工程菌,该工程菌经42℃诱导,SDS-PAGE检测,在约50kDa处有一特异蛋白带,含量约为菌体总蛋白的23%。用纯化和复性后的蛋白免疫新西兰大白兔制备抗血清,Western-blotting分析显示,重组MCP制备的抗血清能与ISKNV MCP特异结合,说明表达产物具有与ISKNV MCP相似的抗原特性。 A pair of primers was designed according to the sequence of ISKNV mcp gene, and a DNA fragment about 1400bp was amplified by PCR using the designed primers. The amplified product was cloned into the pGEM-T Easy Vector. Amino acid hydrophilic analysis indicated that the amino acids residue between 150 and 250 contained a high hydrophilic region, which is possiblely a trans-membrane region and the main epitope of MCP. In order to construct the MCP recombinant expression bacteria, the mcp gene was modified by introducing EcoRⅠ restriction endonuclease sites at both 5 and 3 ends and cloned into E.coli expression vector pBV220. After temperature induction and SDS-PAGE analysis, a protein of about 50 kDa in molecular weight was detected. The proportion of expressed MCP in total bacterial protein was about 23%. Two New Zealand rabbits were immunized by purified recombinant MCP to produce anti-serum. Westernblotting showed the recombinant Mcp had some epitopes of the native ISKNV MCP.

关 键 词： 鳜鱼传染性脾肾坏死病毒 衣壳蛋白 基因 原核表达 免疫印迹 片段 克隆

领　　域： [农业科学] [农业科学] [农业科学] [生物学]