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目的:构建非受体蛋白酪氨酸激酶Syk的真核表达载体,转染人乳腺癌细胞,使其在细胞中稳定表达,以研究Syk对人乳腺癌细胞MHC-Ⅰ类分子表达的影响。方法:以RT-PCR法从人乳腺癌细胞株MDA-MB-468扩增出syk编码序列基因片段,将其克隆到真核表达载体pcDNA3.1D/V5-His-TOPO中。对重组质粒进行酶切、PCR及测序鉴定后,以脂质体介导法转染Syk缺失的人乳腺癌细胞MDA-MB-231,经G418筛选,构建syk基因稳定表达的细胞株,通过Western blot、RT-PCR和流式细胞术检测转染乳腺癌细胞Syk、MHC-Ⅰ及ICAM-Ⅰ的表达。结果:构建了hsyk真核表达载体pcDNA3.1D/V5-His-TOPO/hsyk,并在人乳腺癌细胞MDA-MB-231中获得稳定表达。表达Syk的MDA-MB-231细胞同时可以高表达MHC-Ⅰ和ICAM-Ⅰ。结论:成功地构建了syk真核表达载体并在真核细胞中表达,为进一步的肿瘤免疫研究奠定了实验基础。
OBJECTIVE: To construct an eukaryotic expression vector for Syk, a non-receptor protein tyrosine kinase, which was transfected into human breast cancer cells and stably expressed in the cells to study the effect of Syk on the expression of MHC-I molecules in human breast cancer cells. Methods: The syk encoding gene fragment was amplified from human breast cancer cell line MDA-MB-468 by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1D / V5-His-TOPO. Syk-deficient human breast cancer cell line MDA-MB-231 was transfected with Syk-deficient human breast cancer cell line MDA-MB-231 after being identified by restriction endonuclease, PCR and sequencing. The cell lines stably expressing syk gene were constructed. The expression of Syk, MHC-Ⅰand ICAM-Ⅰ in transfected breast cancer cells were detected by RT-PCR and flow cytometry. Results: The hsyk eukaryotic expression vector pcDNA3.1D / V5-His-TOPO / hsyk was constructed and stably expressed in human breast cancer cell line MDA-MB-231. Syk-expressing MDA-MB-231 cells can simultaneously express both MHC-I and ICAM-I. Conclusion: The eukaryotic expression vector syk was successfully constructed and expressed in eukaryotic cells, which laid the experimental foundation for further tumor immunity research.