Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells

Molecular Biology Reports - Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small,...     

Nucleotide sequence and expression of the 14-3-3 from the halotolerant alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Dunaliella salina, however, the nucleotide sequence of this gene have not been reported so far. In the present study, the cloning and characterization of the nucleotide sequence, the gene copy and expression were undertaken. The coding sequence of the gene was found to be interrupted by five introns of 132, 266, 153, 152 and 625 bp, respectively. Introns 3-5 were found in conserved positions as compared to the Chlamydomonas reinhardtii 14-3-3 gene. D. salina 14-3-3 cDNA was inserted into the prokaryotic expression plasmid pET-28 and transformed into E. coli BL21, and the recombinant expressed 14-3-3 protein was purified from E. coli and immunized the rabbit. Indirect ELISA coated with 14-3-3 illustrated that the rabbit antisera titration was 1:1.00E + 06. Western blotting assays confirmed that prepared rabbit antibodies could recognize the recombinant 14-3-3 protein. Southern blotting results showed that there was only one copy of the 14-3-3 present in the genome of D. salina and 14-3-3 expression did not change throughout the Dnualiella cell cycle.     

人14-3-3蛋白家族的生物信息学分析

目的:分析人14-3-3蛋白家族的同源性及分子进化关系。方法:利用已公布的人基因组数据库,采用BLASTN程序检索人14-3-3蛋白家族各成员的编码基因和假基因,并利用DNAMAN软件进行序列联配,绘制其分子进化树。结果:该家族半数成员具有多个假基因序列,为返转座类型假基因。进化分析表明该家族有共同的祖先,可归为3个亚类。结论:人14-3-3蛋白家族每个成员长期进化所形成的多样性提示其功能具有独特性。     

长期施肥对黄土高原旱地黑垆土水稳性团聚体的影响

通过27年的长期定位试验,研究了长期施肥对黄土高原旱地黑垆土水稳性团聚体的影响.结果表明：与自然土壤相比,黑垆土开垦后,0～10 cm和10～20 cm土层＞0.25 mm的水稳性团聚体含量分别下降了21.35%和38.82%;0.5 mm以上各粒级的水稳性团聚体含量均呈下降趋势,其中>5 mm和5～2 mm大粒径团聚体的下降幅度最大;而0.5～0.25 mm的水稳性团聚体含量却增加了104.75%和23.13%.0～10 cm土层各粒级水稳性团聚体含量均高于10～20 cm土层.单施有机肥和有机无机肥配施处理5～2 mm粒级的水稳性团聚体含量增加最多,>5 mm粒级次之;增施有机肥、有机无机肥配合施用和秸秆还田处理能显著增加大粒径水稳性团聚体含量,改善土壤团聚体的结构.各处理0～10 cm土壤水稳性团聚体平均质量直径（MMD）均大于10～20 cm土层;自然土壤的团聚体MMD显著高于同层次耕作土壤,长期施肥处理MMD大于无肥对照,有机物质培肥处理明显高于化肥处理.土壤团聚体MMD与＞0,25 mm水稳性团聚体含量呈显著正相关.     

新型生物反应器——杜氏盐藻研究进展

转基因植物作为生物反应器生产外源物质已成为基因工程领域的研究热点之一 ,而杜氏盐藻 (Dunaliellasalina)作为新型生物反应器生产外源蛋白具有独特的优点 ,就盐藻这一生物反应器的特点、存在问题和开发应用前景等的最近研究进展作一简要综述。     

Artificially lipid-anchored proteins can elicit clustering-induced intracellular signaling events in Jurkat T-lymphocytes independent of lipid raft association

We have incorporated artificial lipid-anchored streptavidin conjugates with fully saturated or polyunsaturated lipid anchors into the plasma membranes of Jurkat T-lymphocytes to assess previous conclusions that the activation of signaling processes induced in these cells by clustering of endogenous glycosylphosphatidylinositol-anchored proteins or ganglioside GM1 depends specifically on the association of these membrane components with lipid rafts. Lipid-anchored streptavidin conjugates could be incorporated into Jurkat or other mammalian cell surfaces by inserting biotinylated phosphatidylethanolamine-polyethyleneglycols (PE-PEGs) and subsequently binding streptavidin to the cell-incorporated PE-PEGs. Saturated dipalmitoyl-PE-PEG-streptavidin conjugates prepared in this manner partitioned substantially into the detergent-insoluble membrane fraction isolated from Jurkat or fibroblast cells, whereas polyunsaturated dilinoleoyl-PE-PEG-anchored conjugates were wholly excluded from this fraction, consistent with the differences in the affinities of the two types of lipid anchors for liquid-ordered membrane domains. Remarkably, however, antibody-mediated cross-linking of either dipalmitoyl- or dilinoleoyl-PE-PEG-anchored streptavidin conjugates in Jurkat cells induced elevation of cytoplasmic calcium levels and tyrosine phosphorylation of the scaf-folding protein linker of T-cell activation in a manner similar to that observed upon cross-linking of endogenous CD59 or ganglioside GM1. The amplitude of the cross-linking-stimulated elevation of cytoplasmic calcium moreover showed an essentially identical dependence on the level of incorporated streptavidin conjugate for either type of lipid anchor. Confocal fluorescence microscopy revealed that PE-PEG-streptavidin conjugates with saturated versus polyunsaturated anchors showed very similar surface distributions vis à vis GM1 or CD59 under conditions where one or both species were cross-linked. These results indicate that cross-linking of diverse proteins anchored only to the outer leaflet of the plasma membrane can induce activation of Jurkat T-cell-signaling responses, but they appear to contradict previous suggestions that this phenomenon rests specifically on the association of such species with lipid rafts.     

小鼠canstatin C端片段基因的克隆及其在大肠杆菌中的表达

为了构建小鼠canstatinC端片段的原核表达载体并在大肠杆菌中表达。以小鼠肝脏组织总RNA为模板,通过RT-PCR扩增小鼠canstatinC端片段(mCan-C)基因,克隆到pMD18-T载体中并进行序列分析。将mCan-C基因定向克隆于原核表达载体pET30a(+)中,构建表达载体pET／mCan-C,转化大肠杆菌BL21(DE3),IPTG诱导表达。结果表明,小鼠canstatinC端片段的cDNA长度为399bp,含有1个终止密码,编码132个氨基酸,与已知的人canstatinC端片段氨基酸的同源性为61％。IPTG诱导mCan-C在大肠杆菌E．coliBL21中表达,表达量约占菌体总蛋白量的28％,重组蛋白主要以包涵体形式存在。首次克隆了小鼠canstatinC端片段的cDNA,IPTG诱导mCan-C在大肠杆菌E．coliBL21中高效表达。小鼠canstatinC端片段的cDNA序列已收入GenBank,接受号为:AY502947。     

一种通用的基因组DNA提取方法

目的:探讨一种通厢的基因组DNA提取方法.方法:采用改良的膜法分别从动植物组织、外周血、细菌、细胞等标本提取基因组DNA,DNA样品经紫外吸收、琼脂糖凝胶电泳、PCR扩增和酶切进行榆测.结果:该方法提取的基因组DNA纯度较高,电泳条带清晰,DNA质量能满足下游分子生物学研究的需要.结论:该方法简便快速、适用范围广,是提取基因组DNA的一种有效方法.     

单核苷酸突变基因型PCR - SSP快速检测方法的建立

目的:探讨多样本、多基因的单核苷酸突变基因分型操作的PCR - SSP的最优参数,建立最佳反应体系.方法:优化PCR - SSP反应中扩增体系参数,选取优化后的参数做为反应体系;在反应体系已优化的条件下,分别优化解链温度、循环参数.结果:优化后Mg2+、dNTPs、Taq酶、序列特异性引物、内对照引物、DNA模板在20μL反应体系中的终浓度分别为:3.75 μmol/L、0.5m mol/L、2.5U、0.5μmol/L、0.2μmol/L、0.15μg;采用改良的TouchDown做为循环参数,其中DNA变性时间至15min,增加5个起始循环.结论:成功建立了PCR - SSP反应的快速操作体系,扩增条带清晰,普通、琼脂糖凝胶电泳即可检测单核苷酸突变基因型.优化后的体系适合对人、小鼠等各类型DNA样本进行快速多基因单核苷酸多态性分型.