抗心肌肌凝蛋白重链的单链抗体VH和VL基因的合成

目的为研制心肌显像剂-抗人心肌肌凝蛋白重链（HCMHC）的单链抗体，合成抗HCMHC单链抗体的VH和VL基因。方法用TRIZOL试剂从抗HCMHCMcAb杂交瘤细胞提取总RNA，合成第一链cDNA，以这第一链cDNA为模板，用合成的特异引物、DNA聚合酶和四种单核苷酸，对重链可变区（VH）基因和轻链可变区（VL）基因进行PCR扩增。对各步骤的产物进行鉴定，并做了对RNA的稳定性与贮存温度的关系，MgCl2浓度和循环温度的最优化的选择。结果合成的第一链cDNA纯度达95％；扩增产物的琼脂糖凝胶电泳带呈单一明亮泳带，VH和VL基因分子量分别为340bp和320bp，与文献相一致。结论成功合成了VH和VL基因，为心肌显像及其显像剂抗HCMHC单链可变区抗体的研究打下了基础。

Synthesiaed Genes of VH and VL of Single-Chain Antibody of Human Cardiac Myosin Heavy Chain

ObjectiveTo synthesize genes of the heavy chain variable region(VH) and light chain variable region(VL) of single-chain antibody of human cardiac myosin heavy chain for the development of myocardial imaging agents: single-chain antibody of human cardiac myosin heavy chain.MethodsTo extracted total RNA of the anti-HCMHC McAb hybridoma using TRIZOL reagent,synthesize the first-strand cDNA,using this first-strand cDNA as template,with specific primers,DNA polymerase and four single nucleotides,amplifiy the genes of the heavy chain variable region(VH) and light chain variable region(VL) by PCR.To identifiy products of each step and study relationship between RNA stability and storage temperature and optimize cycle selection temperature with MgCl2 concentration.ResultsThe purity of the first-strand cDNA reached 95%,PCR products by agarose gel electrophoresis showed a single band with a bright swimming,molecular weight of VH and VL genes were 340bp and 320bp,which was consistent with literature reports.ConclusionSynthesized genes of VH and VL,laid the foundation for the development of myocardial imaging agents: single-chain antibody of human cardiac myosin heavy chain.