小鼠BMP-McAb可变区基因的体外扩增、克隆与序列分析  

Amplification, cloning and sequence analysis of the variable region of an antiBMP monoclonal antibody in vitro

作　　者：孙远[1] 杨连甲[1] 高玉好[1] 金岩[1] 

机构地区：[1]第四军医大学口腔医学院基础教研室

出　　处：《细胞与分子免疫学杂志》1997年第4期12-16,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：自然科学基金

摘　　要：从体外培养的ＢＭＰＭｃＡｂ杂交瘤中提取细胞总ＲＮＡ，反转录成ｃＤＮＡ。以ｃＤＮＡ为模板，利用两对根据免疫球蛋白重链与轻链可变区５′端序列和Ｊ区序列设计合成的引物，通过ＰＣＲ方法，扩增出抗体重链、轻链可变区基因片段（ＶＨ，ＶＬ）。将扩增产物分别克隆入ｐＵＣ１８，ｐＵＣ１９质粒，筛选出阳性克隆，利用双脱氧链终止法进行序列测定，经计算机分析，ＶＨ基因全长为３４５ｂｐ，编码１１５个氨基酸；ＶＬ基因全长为３１５ｂｐ，编码１０５个氨基酸。AntiBMP monoclonal hybridoma cells were cultured and the total RNA of the cells was extracted. By reverse transcription, the cDNA firststrand was synthesized. Using two sets of oligonucleotide primers that were designed according to V and J region gene sequences of mouse immunoglobulin heavy chain and κ light chain, We amplified the VH and VL gene fragments from the cDNA by polymerase chain reaction(PCR). The PCR products then were cloned into pUC18/19 vectors, respectively. The recombinant vectors containing VH and VL gene inserts were identified by sequencing with Sanger′s method and computerassisted comparative sequence analysis. The total length of VH gene was 345 bp, encoding 115 amino acids, and that of VL gene was 315 bp, encoding 105 amino acids.

关 键 词：BMP PCR 可变区基因 序列分析 

分 类 号：R392.12[医药卫生—免疫学] R392－33[医药卫生—基础医学]