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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。
OBJECTIVE: To clone the mouse β defensin-2 (mBD2) gene and construct its eukaryotic expression vector. The stable cell line was screened and the biological characteristics of mBD2 and its anti-tumor mechanism were studied. Methods: Acute phase reaction was established in mice by intraperitoneal injection of endotoxin (LPS) into BALB / c mice. The total RNA was extracted from the lung tissue and the mBD2 gene was amplified by RT-PCR. The mBD2 gene was double-digested with EcoRⅠ and XhoⅠ After digested, the same digested pcDNA3.1 (+) eukaryotic expression vector was digested and sequenced. The constructed eukaryotic expression plasmid pcDNA3.1 (+) / mBD2 was transfected into SiHa cells, and the stable expression strains were screened by G418. The expression of mBD2 protein was identified by immunofluorescence staining and RT-PCR. Results: The total RNA was extracted from the lungs of mice. The products of about 250bp were amplified by RT-PCR. The recombinant plasmid pcDNA3.1 (+) / mBD2 was constructed by digestion with EcoRI and XhoI. After transfection with SiHa, the cell lines stably expressing mBD2 were screened for 20 days at a concentration of 100 mg / LG418. The mBD2 protein was abundantly expressed in the cytoplasm by immunofluorescence staining and amplified by RT-PCR to mBD2 mRNA. CONCLUSION: The eukaryotic expression plasmid pcDNA3.1 (+) / mBD2 was successfully constructed and mBD2 protein was stably expressed in SiHa cells. These results lay the foundation for further study on the biological characteristics of mBD2 and its anti-tumor effect.