国际呼吸杂志
國際呼吸雜誌
국제호흡잡지
INTERNATIONAL JOURNAL OF RESPIRATION
2010年
24期
1490-1494
,共5页
刘忠泉%邢爱英%贾红彦%李自慧%郑晓静%曹庭明%杜风娇%杜博平%古淑香%张宗德
劉忠泉%邢愛英%賈紅彥%李自慧%鄭曉靜%曹庭明%杜風嬌%杜博平%古淑香%張宗德
류충천%형애영%가홍언%리자혜%정효정%조정명%두풍교%두박평%고숙향%장종덕
真核表达%复苏因子E%中国仓鼠卵巢细胞
真覈錶達%複囌因子E%中國倉鼠卵巢細胞
진핵표체%복소인자E%중국창서란소세포
Eukaryotic expression%Resuscitation promoting factor E%China hamster ovary cells
目的 构建结核分枝杆菌复苏因子E基因的真核表达质粒.方法 PCR扩增结核分枝杆菌复苏因子E基因片段.将片段克隆至PcDNA 3.1(一)载体,重组质粒测序正确后,转染至CHO细胞中,表达复苏因子E蛋白质.RT-PCR检测克隆基因mRNA在真核细胞中的表达,收集并纯化表达的蛋白质,SDS-PAGE分析和Western blot分析检测目的 蛋白的表达,淋巴细胞增殖实验(CCK-8)鉴定表达蛋白的免疫原性.结果 成功构建了结核分枝杆菌复苏因子E基因的真核表达质粒.RT-PCR证实克隆基因mRNA在真核细胞中表达,SDS-PAGE分析和Western blot分析均证实目的 蛋白在真核细胞中成功表达,淋巴细胞增殖实验(CCK-8)进一步证实了表达蛋白具有免疫原性.结论 我们成功构建了复苏因子E真核表达系统.
目的 構建結覈分枝桿菌複囌因子E基因的真覈錶達質粒.方法 PCR擴增結覈分枝桿菌複囌因子E基因片段.將片段剋隆至PcDNA 3.1(一)載體,重組質粒測序正確後,轉染至CHO細胞中,錶達複囌因子E蛋白質.RT-PCR檢測剋隆基因mRNA在真覈細胞中的錶達,收集併純化錶達的蛋白質,SDS-PAGE分析和Western blot分析檢測目的 蛋白的錶達,淋巴細胞增殖實驗(CCK-8)鑒定錶達蛋白的免疫原性.結果 成功構建瞭結覈分枝桿菌複囌因子E基因的真覈錶達質粒.RT-PCR證實剋隆基因mRNA在真覈細胞中錶達,SDS-PAGE分析和Western blot分析均證實目的 蛋白在真覈細胞中成功錶達,淋巴細胞增殖實驗(CCK-8)進一步證實瞭錶達蛋白具有免疫原性.結論 我們成功構建瞭複囌因子E真覈錶達繫統.
목적 구건결핵분지간균복소인자E기인적진핵표체질립.방법 PCR확증결핵분지간균복소인자E기인편단.장편단극륭지PcDNA 3.1(일)재체,중조질립측서정학후,전염지CHO세포중,표체복소인자E단백질.RT-PCR검측극륭기인mRNA재진핵세포중적표체,수집병순화표체적단백질,SDS-PAGE분석화Western blot분석검측목적 단백적표체,림파세포증식실험(CCK-8)감정표체단백적면역원성.결과 성공구건료결핵분지간균복소인자E기인적진핵표체질립.RT-PCR증실극륭기인mRNA재진핵세포중표체,SDS-PAGE분석화Western blot분석균증실목적 단백재진핵세포중성공표체,림파세포증식실험(CCK-8)진일보증실료표체단백구유면역원성.결론 아문성공구건료복소인자E진핵표체계통.
Objective To construct the eukaryotic expression plasmid of resuscitation promoting factor E of Mycobacterium tuberculosis. Methods Resuscitation promoting factor E gene fragment of Mycobacterium tuberculosis was amplified by PCR. The fragment was cloned into PcDNA 3.1 (-)vector. After sequencing the recombinant plasmid, it was transfected into CHO cells to express resuscitation promoting factor E protein. The expression of cloned gene mRNA in eukaryotic cells was detected by RT-PCR. The protein was collected and purified, and the targeted protein expression was detected by SDS-PAGE analysis and Western blot analysis. The immunogenicity of protein was identified by lymphocyte proliferation assay (CCK-8). Results The eukaryotic expression plasmid of resuscitation promoting factor E of Mycobacterium tuberculosis was successfully constructed. It was confirmed that the cloned gene mRNA was expressed in the eukaryotic cells,the targeted protein was successfully expressed in the eukaryotic cells, and the expressed protein had immunogenicity. Conclusions The eukaryotic expression system of resuscitation promoting factor E was successfully constructed.
