【摘要】 目的:对小鼠β防御素-2(mBD2)基因进行克隆,构建其真核表达载体,筛选出稳定表达细胞株,并研究mBD2的生物学特性及其抗肿瘤机制。方法:通过向BALB/c小鼠腹腔注射内毒素(LPS),建立小鼠急性时相反应,取其肺组织提取总RNA,采用RT-PCR方法扩增小鼠mBD2基因,经EcoRⅠ和XhoⅠ双酶切后插入相同酶切的pcDNA3.1(+)真核表达载体,对其进行酶切和测序鉴定。将构建好的真核表达质粒pcDNA3.1(+)/mBD2转染SiHa细胞,采用G418进行稳定表达株的筛选,用免疫荧光染色和RT-PCR鉴定细胞内mBD2蛋白表达情况。结果:提取小鼠肺组织总RNA,采用RT-PCR方法扩增了250bp左右的产物,通过EcoRⅠ和XhoⅠ双酶切,构建了真核表达质粒pcDNA3.1(+)/mBD2。SiHa被该质粒转染后,在100mg/LG418浓度下筛选20d,得到了稳定表达mBD2的细胞株,用免疫荧光染色显示mBD2蛋白在胞质中有大量表达,RT-PCR反应扩增到了mBD2的mRNA。结论:成功地构建了pcDNA3.1(+)/mBD2真核表达质粒,mBD2蛋白在SiHa细胞中能稳定表达,这些结果为进一步深入研究mBD2的生物学特性及其抗肿瘤的作用奠定了基础。更多还原

【Abstract】 AIM: To clone murine beta defensin-2 gene (mBD2) and to express the mBD2 protein eukaryotically. METHODS: Total RNA was isolated from the lungs of BALB/c mice which were injected with LPS in advance. The DNA fragment encoding mBD2 was amplified by RT-PCR and inserted into the plasmid pcDNA3.1(+), which was then digested with EcoRⅠand Xho I to construct the recombinant plasmid, pcDNA3.1(+)/mBD2. The pcDNA3.1(+)/mBD2 was identified by endonuclease digestion, PCR, and sequencing analysis. The SiHa cells were transfected with pcDNA3.1(+)/mBD2 plasmid and screened by G418 of 100 mg/L over 20 days. Steady expression of mBD-2 was confirmed by immunofluorescent staining and RT-PCR. RESULTS: About 250 bp DNA fragment was amplified by RT-PCR from lung total RNA of the mice injected with LPS. The eukaryotic expression vector, pcDNA3.1(+)/mBD2, was successfully constructed after inserting the mBD-2 fragment into pcDNA3.1(+). Most of SiHa cells transfected with pcDNA3.1(+)/mBD2 and screened by G418 could express the mBD2 protein, confirmed by immunofluorescent staining and RT-PCR. CONCLUSION: The eukaryotic vector of pcDNA3.1(+)/mBD2 was successfully constructed and transfected into SiHa cells, which established a solid foundation for further study on the biological characteristics and anti-tumor mechanisms of the mBD2 protein.更多还原