IL-1Ra和IL-10真核表达质粒载体的构建及其表达检测

目的:构建人IL-1Ra和人IL-10真核表达质粒载体并检测其表达。方法:用双酶切方法切取PCDI-IL-1Ra和PCDI-IL-10质粒中包含人IL-1Ra和人IL-10 CDS全长序列的cD-NA片段,并分别连接到真核表达质粒pcDNA3.1上,然后用壳聚糖转染上述质粒到原代软骨细胞,RT-PCR检测其mRNA水平的表达。结果:成功将人IL-1Ra和IL-10 CDS全长序列的cDNA片段克隆到真核表达载体,mRNA水平检测到目的基因的表达明显提高。结论:真核表达质粒可以用于外源基因的原代软骨细胞导入和表达,为进一步的基因治疗研究提供依据。     

HBV转录激活蛋白HBx、MHBst78MHBst155真核重组载体构建

目的分别构建人乙型肝炎病毒(HBV)X蛋白、羧基端截断的中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,以便进一步研究其转录激活功能及对宿主细胞信号传导通路的影响。方法设计合成3对寡核苷酸引物,以adr亚型HBV质粒pHBVDNA为模板,采用PCR法分别扩增HBVX基因、MHBst78与MHBst155编码基因片段;用HindⅢ,KpnⅠ双酶切HBVⅩ基因;用HindⅢ和BamHⅠ双酶切MHBst78与MHBst155编码基因片段后,分别定向插入到真核表达载体pcDNA3.1相应酶切位点,转化宿主菌JM109,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。结果酶切重组体显示所切下的片段大小均与预计相符,测序结果与文献报道序列及预计结果一致。结论成功构建了HBVX基因、羧基端截断的HBV中分子表面蛋白MHBst78、MHBst155编码基因的真核重组表达载体,为进一步研究HBV转录激活蛋白HBx、MHBst78MHBst155对宿主细胞信号转导通路的影响奠定基础。     

人白细胞介素12双亚基共表达真核载体的构建

目的 :为满足基因治疗的需要构建人白细胞介素 12 (hIL 12 )双亚基共表达质粒。方法 :先从人胚肾组织的逆转录产物中用PCR方法扩增出它的两个亚基P4 0 和P3 5的cDNA全长 ,分别克隆入真核表达载体pcDNA3.1(+/ )中构建单亚基质粒———P(+) /P4 0 和P( ) /P3 5,然后再将二者串联克隆入真核表达载体 pcDNA3.1(+)中得到P(+) /IL 12质粒。脂质体转染HepG2后 2 4,48hELISA检测细胞培养上清内hIL 12蛋白质表达。结果 :P(+) /IL 12质粒经酶切和测序证明两亚基连接方向正确 ,序列无突变 ;ELISA检测细胞上清结果证实可表达hIL 12蛋白质。结论 :成功构建P4 0 和P3 5双亚基真核共表达质粒———P(+) /IL 12 ,为模拟hIL 12生理表达方式 ,简化hIL 12基因治疗操作奠定了基础。     

具有靶向免疫抑制作用新型重组融合蛋白B7-2-PE40KDEL真核表达载体的构建及其生物效应

目的:构建B7-2-PE40KDEL的真核表达载体,探讨通过体内表达重组融合蛋白,特异性杀伤高表达CD28 T细胞,阻断B7∶CD28/CTLA4共刺激信号途径诱导免疫耐受的可能性.方法:以原核表达载体pRSETA-B7-2-PE40KDEL质粒为模板,采用PCR及酶切连接的方法构建真核表达载体pcDNA3.1/Zeo( )-B7-2-PE40KDEL.利用RT-PCR、Western 印迹及ELISA法从mRNA、蛋白质水平检测了B7-2-PE40KDEL在RPE-CHO真核细胞中的表达情况.采用MTT法对其特异性杀伤高表达CD28 T细胞的生物活性进行测定.结果:成功构建了pcDNA3.1/Zeo( )-B7-2-PE40KDEL真核表达载体,转染入RPE-CHO细胞后可转录翻译为重组融合毒素蛋白,相对分子质量约为71×103,平均106个转染细胞24 h表达0.23 μg/L.活性测定显示真核载体所表达的B7-2-PE40KDEL可特异性地抑制高表达CD28的人T淋巴细胞系,而对CD28阴性的人白血病细胞系的抑制率较低.结论:真核载体表达的B7-2-PE40KDEL具有良好的靶向性免疫抑制活性,为进一步开展其体内特异性防治移植排斥反应及自身免疫性疾病奠定了基础.     

Immunoglobulin mimicry by Hepatitis C Virus envelope protein E2

Hepatitis C virus (HCV) establishes persistent infection in the majority of infected individuals. The currently accepted hypothesis of immune evasion by antigenic variation in hypervariable region 1 (HVR1) of glycoprotein E2 does not however, explain the lack of subsequent immune recognition. Here, we show that the N-terminal region of E2 is antigenically and structurally similar to human immunoglobulin (Ig) variable domains. E2 is recognized by anti-human IgG antibodies and also possesses common amino acid (aa) sequence features of the conserved v-gene framework regions of human Ig light chains in particular but also heavy chains and T cell receptors. Using a position specific scoring system, the degree of similarity of HVR1 to Ig types correlated with immune escape and persistence in humans and experimentally infected chimpanzees. We propose a unique role for threshold levels of Ig molecular mimicry in HCV biology that not only advances our concept of viral immune escape and persistent infection but also provides insight into host-dependent disease patterns.     

Effect of pancreatic lipase on supramolecular DNA complexes in eukaryotic cellsin vivo andin situ

Effect of pancreatic lipase on supramolecular DNA complexes, nuclear matrix, and 70% ethanol-fixed cells is studied by elastoviscosimetry. It is shown that lipase induces DNA degradation not onlyin vitro but also in whole cells. Possible role of neutral lipids, in particular, DNA-bound diglycerides, in the arrangement of chromosomal DNA is discussed. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 12, pp. 635–639, December, 1997     

Specificity and mechanism of the histone methyltransferase Pr-Set7

Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.     

人Nanog基因的克隆及其在COS-7L细胞中的表达

目的 :克隆人Nanog基因 ,构建其真核表达载体 ,并观察其在哺乳动物细胞COS 7L中的表达。方法 :利用HE2 93细胞的人基因组DNA为模板 ,以LA PCR技术 ,扩增Nango的基因 ,定向克隆到带Flag标签的pCMV载体中 ,测序后 ,挑选序列正确的真核表达质粒pFlag Nanog转染COS 7L细胞。用抗Flag标签的抗体 ,进行Westernblot和间接免疫荧光染色法检测Nango蛋白的表达。结果 :从人基因组DNA中克隆到序列正确的Nanog全长编码序列。所构建的Nanog质粒在COS 7L细胞中获得高效表达。结论 :人Nanog基因的克隆、真核表达载体的构建及在COS 7L中的表达均获得成功 ,为进一步研究其功能 ,尤其是探讨其在神经干细胞中的作用奠定了基础。     

携带增强绿色荧光蛋白基因的Id2真核表达载体构建

目的：构建大鼠Id2基因真核荧光表达载体，为骨骼肌的组织工程研究提供有效的分子工具。方法：利用RT-PCR的方法扩增出Id2全长cDNA，利用T4 DNA连接酶将载体pGEM-T和Id2 cDNA进行连接，构建克隆载体，经限制性内切酶EcoR1酶切pGEM-Id2克隆载体和pEGF-C2真核表达载体，构建出重组真核表达载体pEGFP-C2-Id2，经酶切分析、PCR鉴定及DNA测序证实cDNA片段大小和序列的正确性；并通过细胞转染技术将Id2基因导入L6成肌细胞中。结果：经酶切分析和序列测定证实pEGFP-C2-Id2含大小正确的正向Id2 cDNA片段，获取了转染外源性Id2基因的L6细胞。结论：我们成功构建了同时携带有G418筛选位点和增强绿色荧光蛋白的Id2真核表达载体？     

针对S基因的siRNA或shRNA表达框对HepG2.2.15细胞中HBV基因表达和病毒复制的抑制作用比较

目的 化学合成针对乙型肝炎病毒（HBV）S基因的siRNA，同时制备针对相同区段的shRNA表达框，并比较二者对HepG2．2．15细胞中HBV基因表达和病毒复制的抑制作用。方法 化学合成针对HBVS基因的带有FITC标记的siRNA，设计合成带有FTrc标记的引物，PCR扩增含有RNA聚合酶Ⅲ启动子H1序列和shRNA编码序列的表达框，并对扩增产物进行纯化，将等摩尔数siRNA和shRNA表达框分别用脂质体转染稳定表达HBV的HepG2．2．15细胞，转染1d后流式细胞仪检测转染效率，转染3d后RT-PCR检测靶基因mRNA的水平，用SDS-PAGE、Westem blot及间接免疫荧光染色检测HBsAg的表达。收集转染前和转染后1、3、5和7d的细胞培养上清，检测HBsAg水平，同时用荧光定量PCR方法检测HBV　DNA的含量。结果成功制备了针对HBVS基因的shRNA表达框，将其与等摩尔数siRNA分别转染HepG2．2．15细胞后，RT-PCR证实细胞中HBV　mRNA水平降低，SDS-PAGE、Westem blot及间接免疫荧光染色检测到HBsAg的表达受到抑制，细胞培养上清中HBsAg和HBV DNA含量下降。与siRNA相比，shRNA表达框对HBV的抑制作用虽然起效较慢，但持续时间更长。结论shRNA表达框和siRNA均可明显抑制HBV的转录和表达，与siRNA相比，shRNA表达框能够更持久地抑制靶基因的表达。