人BRE1B基因对眼B16黑色素瘤细胞生长的影响

发布时间：2018-05-10 10:32

本文选题：人BREB基因 + 克隆　；参考：《眼科新进展》2017年12期

【摘要】：目的构建人BRE1B基因真核表达载体,并初步探讨该基因对眼B16黑色素瘤细胞生长的影响。方法将传代培养的眼B16黑色素瘤细胞随机分为实验组(转染p CMV-Tag-2B-BRE1B重组质粒)和对照组(转染p CMV质粒)。以乳腺癌文库为模板应用PCR技术扩增出人BRE1B基因的CDS编码区序列,构建p CMV-Tag-2B-BRE1B重组质粒,将p CMV-Tag-2B-BRE1B重组质粒和p CMV质粒瞬时转染眼B16黑色素瘤细胞后,应用Western blot法检测BRE1B蛋白的表达,应用CCK8法和克隆形成法检测该重组质粒对眼B16黑色素瘤细胞生长的影响。结果 PCR技术扩增出人BRE1B基因CDS编码区序列,且条带与预期大小一致;与对照组相比,菌液PCR鉴定结果为阳性,双酶切的条带长度分别为大约4000 bp和3050 bp,测序鉴定人BRE1B基因的编码序列与插入的DNA序列完全一致;Western blot结果表明p CMV-Tag-2B-BRE1B重组质粒在实验组眼B16黑色素瘤细胞成功表达,对照组则未检测到特异条带;CCK8法和克隆形成法结果表明p CMV-Tag-2B-BRE1B重组质粒能够抑制眼B16黑色素瘤细胞的生长。结论本研究成功构建了带有p CMV-Tag-2B标签的人BRE1B基因真核表达载体,并发现该基因抑制眼B16黑色素瘤细胞的生长。
[Abstract]:Objective to construct the eukaryotic expression vector of human BRE1B gene and to investigate the effect of human BRE1B gene on the growth of B16 melanoma cells. Methods B16 melanoma cells were randomly divided into two groups: the experimental group (transfected with p CMV-Tag-2B-BRE1B recombinant plasmid) and the control group (transfected with p CMV plasmid). Using breast cancer library as template, the CDS coding region of human BRE1B gene was amplified by PCR technique, and the recombinant plasmid of p CMV-Tag-2B-BRE1B was constructed. After transient transfection of p CMV-Tag-2B-BRE1B recombinant plasmid and p CMV plasmid into B16 melanoma cells, The expression of BRE1B protein was detected by Western blot assay, and the effect of the recombinant plasmid on the growth of B16 melanoma cells was detected by CCK8 assay and clone formation method. Results the sequence of CDS coding region of human BRE1B gene was amplified by PCR, and the band was the same size as expected. Compared with the control group, the results of PCR identification were positive. The length of the double enzyme digested bands was about 4000 BP and 3050 BP, respectively. The coding sequence of human BRE1B gene was identical with the inserted DNA sequence. The results of Western blot showed that the recombinant plasmid of p CMV-Tag-2B-BRE1B was successfully expressed in B16 melanoma cells of experimental group. In the control group, no specific band CCK8 assay and clone forming method were detected. The results showed that the recombinant plasmid of p CMV-Tag-2B-BRE1B could inhibit the growth of B16 melanoma cells. Conclusion the eukaryotic expression vector of human BRE1B gene with p CMV-Tag-2B tag was successfully constructed, and it was found that the gene inhibited the growth of B16 melanoma cells.
【作者单位】：锦州医科大学附属第一医院;
【基金】：国家自然科学基金资助(编号:81472589、81672602、81402345、81502264)~~
【分类号】：R739.7

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