鼠β.docVIP

鼠β 作者：魏晓丽, 施桥发, 李虹, 李婉宜, 蒋忠华, 李明远 【关键词】 mBD2;,真核表达;,免疫荧光;,RT 　　Cloning and eukaryotic expression of murine betadefensin2(mBD2) 　　; AIM: To clone murine beta defensin2 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 RTPCR and inserted into the plasmid pcDNA31(+), which was then digested with EcoRⅠand Xho I to construct the recombinant plasmid, pcDNA31(+)/mBD2. The pcDNA31(+)/mBD2 was identified by endonuclease digestion, PCR, and sequencing analysis. The SiHa cells were transfected with pcDNA31(+)/mBD2 plasmid and screened by G418 of 100 mg/L over 20 days. Steady expression of mBD2 was confirmed by immunofluorescent staining and RTPCR. RESULTS: About 250 bp DNA fragment was amplified by RTPCR from lung total RNA of the mice injected with LPS. The eukaryotic expression vector, pcDNA3.1(+)/mBD2, was successfully constructed after inserting the mBD2 fragment into pcDNA31(+). Most of SiHa cells transfected with pcDNA31(+)/mBD2 and screened by G418 could express the mBD2 protein, confirmed by immunofluorescent staining and RTPCR. CONCLUSION: The eukaryotic vector of pcDNA31(+)/mBD2 was successfully constructed and transfected into SiHa cells, which established a solid foundation for further study on the biological characteristics and antitumor mechanisms of the mBD2 protein. 　　; 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RTPCR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RTPCR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RTPCR方法扩增了250 bp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100 mg/L G418浓度下筛选20 d, 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, RTPCR反应扩增到了mBD2的mRNA。结论: 成功地构建了pcDNA3.1(+)/mBD