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Construction and Expression of a Recombinant Plasmid Inserted Mycobacterium Tub erculosis HSP65 Gene
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- Author:
- No author available
- Journal Title:
- JOURNAL OF MODERN CLINICAL MEDICAL BIOENGINEERING
- Issue:
- 6
- DOI:
- 10.3760/cma.j.issn.1674-1927.2001.06.004
- Key Word:
- 热休克蛋白-65;结核杆菌;构建;真核表达
Abstract： 目的构建结核杆菌热休克蛋白-65(HSP65)真核表达质粒,并探讨其在人肺癌细胞系A549中的表达. 方法用PCR的方法从结核杆菌H37Rv基因组中扩增出HSP65基因,并定向克隆到真核表达质粒pcDNA3.1(+),构建成重组质粒pcDNA3.1-HSP65;然后用电穿孔的方法将质粒pcDNA3.1-HSP65转染到A549 细胞,经G418筛选后获得抗性细胞克隆;用RT-PCR的方法检抗性细胞中HSP65 mRNA的表达 . 结果经酶切鉴定和DNA序列测定证实重组质粒pcDNA3.1-HSP65构建正确;RT-PCR检测结果发现,重组质粒pcDNA3.1-HSP65转染的A549细胞总RNA中结核杆菌HSP65 mRNA为阳性. 结论真核表达质粒pcDNA3.1-HSP65构建成功 , 并可在真核细胞A549细胞中稳定表达.
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