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目的:构建人CD34真核表达载体,转染到3T3细胞获得稳定表达CD34的细胞株。方法:在人KG1a细胞中提取细胞总RNA,运用RT-PCR方法扩增CD34基因,定向克隆入真核表达载体pCI-neo,通过电转仪转染3T3细胞,流式细胞仪分选建立稳定转染的3T3细胞系,用RT-PCR、FACS检测目的蛋白的表达。结果:构建了重组载体pCI-neo/CD34,建立了稳定表达CD34的3T3细胞系。结论:重组载体pCI-neo/CD34构建成功和稳定转染3T3-CD34细胞系的建立为制备抗人CD34单克隆抗体(mAb)做了准备,为进一步研究CD34分子的功能奠定了基础。
OBJECTIVE: To construct eukaryotic expression vector of human CD34 and transfect it into 3T3 cells to obtain a cell line stably expressing CD34. Methods: Total RNA was extracted from human KG1a cells. CD34 gene was amplified by RT-PCR and cloned into eukaryotic expression vector pCI-neo. The 3T3 cells were transfected by electrotransducer. Stained 3T3 cell line, and the expression of the target protein was detected by RT-PCR and FACS. Results: The recombinant vector pCI-neo / CD34 was constructed and a 3T3 cell line stably expressing CD34 was established. CONCLUSION: The establishment of recombinant vector pCI-neo / CD34 successfully and stably transfected 3T3-CD34 cell line prepared for the preparation of anti-human CD34 monoclonal antibody (mAb), and laid the foundation for further study on the function of CD34 molecule.