摘要
目的构建鼠α1-抗胰蛋白酶(AAT)的真核表达载体,观察其在NIH3T3细胞中的表达及定位情况。方法提取BALB/c小鼠肝脏组织的总RNA,通过RT-PCR扩增得到AAT编码序列。然后将该编码序列克隆到带有血凝素(HA)标记的载体pcDNA3-HA上,随后转染NIH3T3细胞,在荧光显微镜下观察结果。结果重组质粒经PCR、酶切和测序鉴定证明构建正确。转染实验发现,该质粒能够在NIH3T3细胞中表达,表达产物主要定位在细胞质中。结论成功构建带HA标签的AAT真核表达载体。该载体能在哺乳动物细胞中有效表达并正确定位,为下一步深入研究AAT在细胞内的相关生物学功能提供了一个重要工具。
Objective To construct a eukaryotic expression vector for alpha-1-antitrypsin （AAT） and detect its expression and localization in NIH 3T3 cells. Methods The total RNA was extracted from the liver tissue of BALB/c mice, and the corresponding coding sequences for mouse AAT （GenBank accession No. NM_009243） were amplified by RT-PCR and cloned into hemagglutinin （HA）-tagged vector pcDNA3-HA. The construct was then transfected into NIH 3T3 cells, which were observed under fluorescence microscope. Results The recombinant plasmid was verified by PCR, enzyme digestion and sequence analysis, and the fusion protein was highly expressed in NIH 3T3 cells. Under fluorescence microscope, the fusion protein was found to distribute mainly in the cytoplasm. Conclusion The expression vector for AAT-HA fusion protein has been successfully constructed and effectively expressed in mammalian cells to allow future functional study of AAT in mammalian cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2009年第3期408-411,共4页
Journal of Southern Medical University
基金
长江学者和创新团队发展计划(IRT0731)
国家自然科学基金委员会-广东省人民政府自然科学联合基金重点项目(U0632004)
国家自然科学基金(30670828
30572151)
广州市科技计划项目(2007J1-C0301)
关键词
Α1-抗胰蛋白酶
血凝素
细胞内定位
载体构建
alpha-1-antitrypsin
hemagglutinin
intracellular localization
vector construction
