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目的构建人源性肿瘤干细胞标记物AC133-1基因的真核表达载体pcDNA4/TO-AC133-1,并进行真核细胞的表达。方法通过RT-PCR从人急性髓性白血病(AML)骨髓样品中获得编码AC133-1基因的cDNA,定向克隆到真核表达载体pcDNA4/TO载体中,进行酶切鉴定并测序来验证目的基因的正确性。然后在293T细胞中进行转染48h后,采用细胞免疫荧光法和蛋白印迹法来检测AC133-1的表达。结果成功克隆AC133-1全长基因并构建pcDNA4/TO-AC133-1真核表达载体。结论人源性AC133-1的真核表达载体的克隆和构建的成功,为进一步制备其相关抗原、抗体及其生物学功能研究打下基础。
Objective To construct eukaryotic expression vector pcDNA4 / TO-AC133-1 of human tumor stem cell marker AC133-1 and to express it in eukaryotic cells. Methods The cDNA encoding AC133-1 gene was obtained from human acute myeloid leukemia (AML) bone marrow by RT-PCR and cloned into the eukaryotic expression vector pcDNA4 / TO vector for enzyme digestion and sequencing to verify the target gene Correctness After 48 h of transfection in 293T cells, the expression of AC133-1 was detected by immunofluorescence and Western blotting. Results The full-length AC133-1 gene was successfully cloned and the eukaryotic expression vector pcDNA4 / TO-AC133-1 was constructed. Conclusion The successful cloning and construction of human AC133-1 eukaryotic expression vector lay the foundation for the further preparation of its related antigens, antibodies and their biological functions.