日本血吸虫单克隆抗独特型抗体NP30重链可变区基因的体外扩增克隆及序列分析

AMPLIFICATION,CLONING AND SEQUENCE ANALYSIS OF THE HEAVY CHAIN VARIABLE REGION GENE OF MONOCLONAL ANTI-IDIOTYPIC ANTIBODY NP30 OF SCHISTOSOMA JAPONICUM

目的 分离日本血吸虫单克隆抗独特型抗体 NP30重链可变区 (VH)基因并测定其序列。方法 根据鼠免疫球蛋白重链可变区基因 FR1和 FR4序列的保守性 ,化学合成体外扩增 Ig重链可变区基因的数对引物。以日本血吸虫单克隆抗独特型抗体 NP30的杂交瘤细胞株基因组 DNA为模板 ,扩增 VH 基因 ,将其克隆入 p U C19载体 ,重组子用 Sanger's双脱氧链终止法测定序列 ,将序列与Gene Bank中已发表的抗体序列比较。结果 VH 基因全长 35 7bp,属鼠免疫球蛋白重链第 亚类 ,由种系基因 V、Dsp2 .8与 JH4重排而来。该 VH 基因序列已被 Gene Bank收录 (accession NoAF2 82 6 2 2 )。结论 该 VH 基因为日本血吸虫单克隆抗独特型抗体

Objective To amplify and sequence the heavy chain of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum. Methods By comparing the conserved regions at each end of the nucleotide sequences of murine germ line genes encoding FR1 and FR4 regions of immunoglobulin heavy chain variable regions, a set of primers for amplification of V H gene were designed. The hybridoma cells secreting anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum were cultured and their genome DNA was extracted and used as template for PCR. The PCR product was then cloned into pUC19 vector. The recombinants were sequenced by Sanger's method. The V H gene was compared with Gene Bank and published mouse V H genes. Results It was proved that a full length V H gene was 357 bp, and a member of mouse Ig heavy chain subgroup Ⅱand originated from rearrangement of germ line V?Dsp2 8 and J H4 genes. The V H gene sequence was registered by Gene Bank (accession No. AF282622). Conclusions It was suggested that obtained V H gene was potentially functional gene of anti idiotypic monoclonal antibody NP30 of Schistosoma japonicum.