论文部分内容阅读
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HCMHC McAb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。
The aim of this study is to develop single-chain anti-human cardiac myosin heavy chain (HCMHC) monoclonal antibody and to synthesize VH and VL genes of anti-HCMHC single chain antibody. Methods The total RNA was extracted from anti-HCMHC McAb hybridoma cells by TRIZOL reagent to synthesize the first-strand cDNA. The first-strand cDNA was used as a template to synthesize specific primers, DNA polymerase and four kinds of single nucleotides. The variable region (VH) gene and the light chain variable region (VL) gene are PCR amplified. The products of each step were identified and the relationship between the stability of RNA and the storage temperature, the optimal choice of MgCl2 concentration and the temperature of the circulation were made. Results The purity of the first-strand cDNA was 95%. The amplified product was single bright band with agarose gel electrophoresis. The molecular weights of VH and VL genes were 340bp and 320bp, respectively, which accorded with the literature. Conclusion The VH and VL genes were successfully synthesized and laid the foundation for the study of myocardial imaging and anti-HCMHC single chain variable region antibody.