目的构建硒蛋白S基因(SELS)真核表达载体pCMV-SELS,转染肝癌SMMC7721细胞,实现SELS在SMMC7721中的成功过表达。方法通过基因克隆技术将SELS基因的完整ORF插入真核表达载体pCMV-3tag-3a(+),得到阳性真核表达质粒pCMV-SELS,将其瞬时转染到肝癌SMMC7721细胞中,利用RT-PCR与Western blot方法验证SELS的过表达水平。结果成功构建了真核表达质粒pCMV-SELS,将其瞬时转染SMMC7721细胞后24 h,收集细胞进行RT-PCR和Western blot检测,结果显示:与阴性对照组pCMV-3tag-3a(+)转染组相比,实验组pCMV-SELS细胞中SELS的mRNA和蛋白表达水平均有显著升高,两组比较具有统计学差异(P0.05)。结论成功构建真核表达载体pCMV-SELS,并实现了SELS基因在SMMC7721细胞中的过表达。

Objective To construction an eukaryotic expression vector pCMV-SELS and observe its expression in liver cancer SMMC7721 cells, and to provide experimental basis for study on the relationship between SELS gene and liver cancer. Methods The liver cancer SMMC7721 cells were transfected transiently with the constructed eukaryotic expression vector pCMV-SELS. The expression of SELS gene in the untransfected SMMC7721 cells, the SMMC7721 cells transfected with pCMV-3tag-3a(+)-SELS and the SMMC7721 cells transfected with pCMV-3tag-3a(+) (control) were detected by RT-PCR and Western blot. Results The eukaryotic expression vector pCMV-3tag-3a(+)-SELS was successfully constructed. The expression of SELS gene in the cells transfected with pCMV-SELS was significantly increased compared with the cells transfected with pCMV-3tag-3a(+) and the untransfected SMMC7721 cells(P<0.05), The Western blot results showed that the expression of SELS gene in the cells transfected with pCMV-SELS was significantly

目的：探讨蛋白酶体激活因子PA28α对食管鳞癌细胞周期和凋亡的影响。方法：扩增PA28α的cDNA全长序列并连接到pCMV-Myc上,构建真核表达载体pCMV-Myc-PA28α,脂质体法转染食管鳞癌细胞系Eca109和EC9706,荧光显微镜观察PA28α蛋白在细胞中的定位;Western blot法鉴定重组载体的表达;流式细胞仪检测PA28α在食管鳞癌EC9706细胞中过表达对其细胞周期和凋亡的影响。结果：转染了重组载体pCMV-Myc-PA28α的食管鳞癌Eca109和EC9706细胞中PA28α蛋白主要存在于细胞质。与空载体转染组（0.342±0.007）相比,pCMV-Myc-PA28α转染组（1.073±0.040）EC9706细胞中有较高的PA28α蛋白表达（F=984.411,P〈0.001）。与空载体转染组（37.642±1.000）相比,pCMV-Myc-PA28α转染组（39.258±0.154）EC9706细胞处于S期细胞比例明显增高（F=24.592,P〈0.001）。pCMV-Myc-PA28α转染组EC9706细胞的凋亡率（14.463%±0.634%）明显低于空载体转染组（43.893%±0.473%）,差异有统计学意义（F=1 448.569,P〈0.001）。结论：PA28α与食管鳞癌的凋亡有密切关系,可能成为治疗食管鳞癌的一个有效靶点。

To investigate the effects of PA 28αon apoptosis and cell cycle of ESCC .Methods:A recombinant vector pCMV-Myc-PA28αwas constructed and then transfected into ESCC cell lines EC 9706 and Eca109 cells using Lipo-fectaminTM2000.ESCC cells transfected with pCMV-Myc were served as control.The subcellular localization of PA28 αwas observed using immunofluorescence .The protein level of PA28αin transfected cells was examined by Western blot .The effects of PA28αon the apoptosis and cell cycle of ESCC were investigated by Annexin V-FITC/PI Flow Cytometry .Re-sults:The fluorescence of PA28αprotein was detected mainly in the cytoplasm of ESCC cells .Compared with the control group, the protein expression of PA 28αin EC9706 cells transfected with pCMV-Myc-PA28αincreased significantly ( F=984.411, P<0.001).Compared with the control group,the numder of EC9706 cells in S phase transfected with pCMV-Myc-PA28αinereased as well(F=24.592, P<0.001).The apoptosis cells proportion of the

目的针对人SND1基因2个蛋白翻译起始密码子AUG构建真核表达质粒pCMV-N-Flag-SND1-No1/2,并分析2个AUG在SND1应激颗粒形成中的作用。方法以SND1全长转录本为模板,PCR法扩增含BamHⅠ和EcoRⅠ酶切位点的目的基因SND1-No1/2,双酶切法分别酶切目的基因片段和线性pCMV-N-Flag,以T4-DNA连接酶将两者连接成pCMV-N-Flag-SND1-No1/2重组质粒,然后将构建的重组质粒转染入HeLa细胞内,以Western印迹法检测Flag标签(DYKDDDDK)与SND1-No1/2的融合表达,最后以细胞免疫荧光实验检测在氧化应激状态下Flag-SND1-No1/2融合蛋白与内源性SND1应激颗粒的胞内共定位情况。结果以单/双酶切及基因测序法鉴定构建的重组质粒无误,Western印迹结果检测到融合蛋白Flag-SND1-No1/2的表达;细胞免疫荧光结果显示Flag-SND1-No1/2均可与内源性SND1应激颗粒共定位。结论重组pCMV-N-Flag-SND1-No1/2质粒构建成功,SND1基因第1个AUG的缺失并不影响SND1应激颗粒的形成。

Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1(from 1st AUG)or SND1-No2 (from 2nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en-zyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected in-to HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enz

目的 明确肝癌细胞系HepG2中前S2区突变乙型肝炎表面抗原大蛋白(hepatitis B virus large surface protein,LHBs)与C53蛋白相互作用对细胞生物学功能的影响.方法 分别构建pCMV-5a-pre S2 LHBs和pCDNA-4-C53质粒,共同转染肝癌细胞系HepG2后,分别检测其相互作用对Chk1活性的影响；BrdU法细胞增殖检测明确其对细胞有丝分裂及增殖的影响.结果 成功构建pCMV-5a-pre S2 LHBs和pCDNA-4-C53质粒；其在细胞内的相互作用增加细胞Cdk1活性并促进细胞的增殖和有丝分裂过程.结论 肝癌细胞系HepG2内pre S2 LHBs及C53的相互作用对细胞生物学功能存在影响,提示可能与HBV后肝癌的发病机制相关.

Objective To identify the effects on cell biology functions of HepG2 cells by interaction between pre-S2 LHBs and C53.Methods The plasmids pCMV-5a-pre-S2 LHBs and pCDNA-4-C53 were reconstructed.The plasmids pCMV-5a-LHBs and pCDNA-4-C53 were cotransfected into the hepatoma cell line HepG2,the roles of the binding of pre-S2 LHBs with C53 on Cdk1,Chk1 activation and mitotic entry were studied.Results The plasmids pCMV-5a-LHBs and pCDNA-4-C53 were reconstructed successfully.The binding of pre S2 LHBs with C53 increased Cdk1 activation and mitotic entry.Conclusions By counteracting C53,pre-S2 LHBs promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and delayed the function of Chk1,which may be a novel potential mechanism for HBV-induced hepatocellular carcinoma (HCC).

目的：构建携带 IL-2/ NK4双基因真核表达载体的减毒沙门菌菌株。方法利用分子克隆技术将 IL-2及 NK4基因分别插入 PIRES-SEQ 质粒 IRES 序列的上下游中,构建为双基因真核共表达载体 pCMV-IL-2-IRES-NK4,将该质粒利用脂质体转染入细胞,采用 ELISA 及 PCR 法检测 pCMV-IL-2-IRES-NK4转染细胞后 IL-2/ NK4基因及蛋白表达情况。将所构建质粒以电转化法转化减毒沙门菌菌株 Ty21a,从而得到能够表达 IL-2/ NK4双基因的重组减毒沙门菌菌株。结果 ELISA 及 PCR 法检测该质粒可在细胞中以较高的效率稳定表达 IL-2及 NK4基因和蛋白；同时表达 IL-2/ NK4双基因的减毒沙门菌菌株 TPIN 构建成功。结论本研究成功构建了携带 IL-2/ NK4双基因的减毒沙门菌菌株。

Objective To construct an attenuated Salmonella typhimurium strain (TPIN) containing IL-2 and NK4 co-expressed eukaryotic vector. Methods A Eukaryotic expression vector (pCMV-IL-2-IRES-NK4) was construc-ted which contained IL-2 and NK4 genes by genetic engineering technology. The vector was transfected into cells using li-pofectamin and detected the expression levels of IL-2 and NK4 by enzyme linked immunosorbent assay (ELISA) and pol-ymerase chain reaction (PCR) methods. And then, the vector pCMV-IL-2-IRES-NK4 was conducted into an attenuated Salmonella typhimurium Ty21a by electrotransformation to build TPIN. Results The IL-2 / NK4 co-express vector steadi-ly expressed the IL-2 and NK4 genes and albumen with high levels by ELISA and PCR detection; the attenuated Salmo-nella typhimurium DNA vaccine with IL-2 / NK4 genes (TPIN) was constructed successfully. Conclusion An attenuated Salmonella typhimurium strain carrying IL-2 / NK4 genes has been successfully constructed.