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目的构建人乳腺癌易感基因BRCA1的真核表达载体,并对其生物学功能进行初步检测。方法利用PCR技术从人乳腺文库中扩增出人BRCA1基因,将其克隆到p XJ-40-myc载体中,酶切和测序验证后转染到乳腺癌ZR75-1细胞中,通过Western印迹检测其表达情况,并用CCK8法测定细胞生长曲线。结果从人乳腺文库中扩增获得大小为5600 bp的DNA片段,并成功克隆至p XJ-40-myc载体上,经测序与目的序列完全一致;转染乳腺癌ZR75-1细胞后目的基因成功表达;细胞生长曲线结果显示,转染myc-BRCA1的乳腺癌细胞较空载体细胞生长缓慢;划痕实验说明,转染myc-BRCA1的乳腺癌细胞较空载体细胞迁袭缓慢。结论成功构建了带myc标签的人BRCA1真核表达载体,为进一步研究BRCA1在乳腺癌发生发展中的功能奠定了基础。
Objective To construct a eukaryotic expression vector of human breast cancer susceptibility gene BRCA1 and to detect its biological function. Methods Human BRCA1 gene was amplified from human mammary gland by PCR and cloned into pXJ-40-myc vector. The BRCA1 gene was transfected into ZR75-1 breast cancer cells by digestion and sequencing. The expression of BRCA1 gene was detected by Western blot Its expression, and use CCK8 method to measure cell growth curve. Results The DNA fragment of 5600 bp was amplified from the human breast cDNA library and cloned into pXJ-40-myc vector. The sequence was exactly the same as the target sequence. The target gene was successfully transfected into ZR75-1 breast cancer cells The results of cell growth curve showed that the growth of breast cancer cells transfected with myc-BRCA1 was slower than that of empty vector. Scratch experiments showed that the breast cancer cells transfected with myc-BRCA1 migrated slowly compared with empty vector. Conclusion The human BRCA1 eukaryotic expression vector with myc tag was successfully constructed, which laid the foundation for further study on the function of BRCA1 in the development of breast cancer.