摘要
目的 :获得抗人红细胞单链抗体 (single chainantibody ,ScFv)基因 ,并在大肠杆菌中表达具有能与人红细胞特异性结合的ScFv。方法 :以从分泌抗人红细胞单克隆抗体细胞株中提取细胞的总RNA为模板 ,利用扩增鼠源性抗体可变区基因的通用引物 ,通过RT PCR分别获得轻链 (VL)、重链 (VH)可变区基因 ,并通过 15个氨基酸的连接肽 (Gly4Ser) 3 按VL 接头 VH 的顺序连接起来 ,组建成ScFv基因。结果 :核苷酸序列分析证明 ,获得的单链抗体基因VH 基因属于小鼠抗体重链可变区基因家族的V(A)组 ,VL 基因属于小鼠抗体κ轻链可变区基因家族的IV组。与pGEX 5X 3表达载体上的GST融合蛋白表达后 ,在相对分子质量 5 4 0 0 0处有一明显的空载体对照没有的蛋白带 (ScFv2 80 0 0 ,GST2 60 0 0 )。间接免疫荧光证明该表达产物能与人红细胞特异性结合。结论 :获得了抗人红细胞ScFv基因和具有抗体活性的抗人红细胞ScFv 。
ObjectiveTo obtain antihuman red blood cell s in glechain antibody gene and to express singlechain antibody which can combin e with human red blood cells. Methods The gene fragments encodi ng variable regions of immunoglobulin light and heavy chain were obtained by rev ersetranscription polymerase chain re a ction (RTPCR) using the universal primers for amplifying mouse immunoglobulin v ariable region gene from the hybridoma cell 2A~{*-~}4 secreting antihuman red bloo d cell monoclonal antibodies. The two variable region gene fragments were linked by a linker of encoding peptide (Gly~{*-~}4Ser)~{*-~}3 to assemble a singlechain Fv(S cFv) gene.Results The sequence analysis demonstrated that the S cFv of V~{*-~}H and V~{*-~}L chains belong to heavy chain group V(A) and kappa light cha in group IV of mouse immunoglobulin variable region,respectively. ScFv Gene was expressed as a fusion protein with the glutathioneStransferase. The relativ e molecular mass of expression products was 54?000 (ScFv 28?000, GST 26?000) shown by SDSPAGE analysis, which was consistent with expected molecular weigh t.The activity of the expressed singlechain antibodies was determined by indir ec t immunofluorescence assay using human red blood cell as antigen. The results sh owed that the expressed ScFv could react with human red blood cell.Concl usionsAntiHRC ScFv gene and expressed ScFv which can combine with HRC by indirect immunofluorescence assay were successfully got.
出处
《军事医学科学院院刊》
CSCD
北大核心
2001年第3期180-185,共6页
Bulletin of the Academy of Military Medical Sciences
