中文摘要：

目的利用基因定点突变技术构建RHD变异型克隆,克服RHD变异型克隆难以获得的问题。方法提取总RNA并反转录后,扩增获得RHD和RHCE混合c DNA,从克隆质粒中筛选RHD;根据弱D12、弱D25和弱D33的特异性突变位点设计引物,分别进行PCR扩增反应,诱导目的基因突变,Dpn I酶处理PCR产物,转化后测序验证。结果克隆测序的结果显示,质粒中分别存在830A、341A和520A的突变,证实已经通过基因定点突变技术成功获得了弱D12、弱D25和弱D33的克隆。结论通过基因定点突变可以改造野生型RHD-c DNA,获得变异型的RHDc DNA,为血型研究打开新的路径。

英文摘要：

Objective To construct RHD variant clone by site-directed mutagenesis, and to resolve the difficulty in ac- quiring RHD variant clone, Methods Total RNA was extracted and eDNA was obtained by reverse transcription PCR. RHD and RHCE genes were amplified and RHD gene was screened from plasmid. The primers of site-directed mutagenesis were designed according to the special mutation sites at weak D12, weak D25 and weak D33. Target gene mutations were induced by PCR. PCR products were digested by DpnI enzymes. The mutations were verified by clone sequencing. Results Sequen- cing results showed that there were 830A, 341A and 520A mutants in plasmid while weak D12, weak D25 and weak D33 clones were successfully constructed by site-directed mutagenesis. Conclusion Variant RHD-cDNA can be obtained from wild-type RHD-cDNA by site-directed mutagenesis, which could be a new pathway for blood group research.