鼠抗HFRSV衣壳蛋白McAb F3株可变区基因的获取及特性分析

培养鼠抗肾综合征出血热病毒衣壳蛋白Ｆ３杂交瘤细胞株，提取总ＲＮＡ，根据鼠源ＩｇＧ抗体基因家族可变区基因碱基序列的特点，设计简并引物，通过逆转录聚合酶链反应，获得抗体轻链可变区和重链可变区基因。分别将其克隆入载体ＰＴ７ＢｌｕｅＴ Ｖｅｃｔｏｒ，选取阳性重组克隆各两个，分别测定了所载重链可变区和轻链可变区基因的碱基序列，比较了不同克隆轻链可变区基因之间和重链可变区基因之间碱基序列的差异；分析了各自的氨基酸框架及其对应蛋白的亲水性。结果显示，两个重链可变区基因碱基序列有４处不同，同源性为９７９％；其中重组克隆ＺＧ３６４ ５Ｆ所载重链可变区基因有完整的开放阅读框架，对应的蛋白含有丰富的亲水基因，第１１２氨基酸处亲水性最高；另一重组克隆ＺＧ３６４ ４Ｆ所载重链可变区基因不能通读。两个轻链基因碱基序列有４处不同，同源性为９９１％，重组克隆ＺＧ３６５ ５Ｆ和ＺＧ３６５ ７Ｆ所载轻链可变区基因均有完整的开放阅读框架，对应的蛋白均含有丰富的亲水基因，ＺＧ３６５ ５Ｆ所载基因对应蛋白第６７氨基酸亲水性最高，ＺＧ３６５ ７Ｆ所载基因对应蛋白第３４氨基酸亲水性最高。

Generation and charicteristic analysis of mouse monoclonal antibody line F3 variable region(Fv)fragment to HFRSV capsid protein

Total RNAs were extracted from mouse anti HFRSV hybridoma cells;the variable region fragments of heavey and light chain were generated using primers designed according to Fv nucleotide sequence characteristic of mouse IgG gene family by RT PCR,and cloned into vector PT7Blue T vector.Two samples of heavy and light chain recombinant clones were selected respectively,the corresponding heavy and light chain Fv were sequenced,the differences of different heavy and light chain Fv nucleotide sequence and hydrophilicity of the corresponding protein were analysed.The results demonstrated there are eight different nucleotides between two heavy chain genes,there is a nucleotide homology of 97.9%,the heavy chain gene of recombinant clone ZG365 5F has a complete open reading frame,the corresponding protein is full of hydrophilic groups,there is the highest hydrophilic site in the 112 th amino acid,the heavy chain gene of another heavy recombinant clone is not reading through.There are four different nucleotides between two light chain genes,there is a nucleotide homology of 99.1%,both of them have a complete open reading frame,two corresponding proteins are full of hydrophilic groups,there is the highest hydrophlic site in the 67 th amino acid of light chain of ZG365 5F and in the 34 th amino acid of light chain of ZG365 7F.