新疆“生命营养液”体外抗HIV-1活性的分析

[目的]研究新疆"生命营养液"体外抗HIV-1活性,并对其作用机制进行初步探讨。[方法]采用MTT比色法,检测"生命营养液"的细胞毒性;用携带荧光素酶基因的HIV-1假病毒感染体系,检测"生命营养液"体外抗HIV-1活性,分析"生命营养液"体外抗HIV-1机制。[结果]"生命营养液"抑制HIV-1感染活性的半数有效浓度(IC50)为1.784 mg/ml,治疗指数(TI)为7.22,其作用方式更接近于整合酶抑制剂MK-0518。[结论]"生命营养液"具有抗HIV-1活性,其作用机制可能抑制病毒的整合。     

Luciferase preparation,assay conditions and calculated extraction efficiency as factors in the estimation of soil ATP content

Summary Several factors affecting the measurement of soil adenosine 5-triphosphate (ATP) using a crude (macerated firefly tail) preparation of luciferase and a luminometer were investigated. These factors were luciferase preparation purity (crude or purified), use of Tris(hydroxymethyl)methylamine (Tris) or sodium arsenate (arsenate) buffer for the luciferase preparation and the bioluminescent reaction, storage temperature of luciferase throughout the assay (20°C or 1°C), modes of measuring bioluminescence (peak height or integration of bioluminescence decay) and efficiency of extraction (recovery) of ATP from soil.Crude luciferase produced a linear relationship between bioluminescence and ATP concentration very similar to that produced by purified luciferase and could be stored at 20°C for 3 h without deterioration. Arsenate was, overall, the preferred buffer for the assay. Integrating light output within 15 s of mixing luciferase-luciferin and ATP avoided interference from other reactions in the crude extracts. The procedure used to calculate the ATP concentration and the amount of exogenous ATP added to soil both affected the calculated efficiency of ATP recovery. Thus an assay for soil ATP content using crude preparations has been developed which is as sensitive as those using purified preparations.     

海藻糖对虫荧光素酶活性保护作用研究

研究在室温、低温冷藏以及反复冻融等条件下,海藻糖对虫荧光素酶(luciferase)活性的保护作用.结果表明,海藻糖对虫荧光素酶活性有明显的保护作用.反复冻融时,海藻糖的保护作用尤为显著.反复冻融3次时,含海藻糖的酶制剂的保活率为51.8%,不含海藻糖的酶制剂的保活率为19.1%,含糖组保活率是无糖组平均保活率的2.71倍.海藻糖对虫荧光素酶的最适保活浓度约为0.1 mol/L.     

调控民猪ZBED6基因转录元件的筛选与分析

ZBED6是锌指蛋白家族的一员,在胎盘哺乳动物中极其保守,可通过对IGF2的调控参与骨骼肌生长。为进一步探究ZBED6基因自身的表达调控机制,本研究以民猪基因组DNA为模板,通过常规PCR扩增ZBED6基因启动子区系列截短片段,构建克隆质粒,通过双酶切和连接反应定向连入pGL3-basic载体,利用PK15细胞和双荧光素酶检测系统测定重组质粒的相对荧光素酶活性;利用在线软件预测启动子区的转录因子结合位点,使用重叠PCR定点缺失转录因子结合位点,构建突变载体并在PK15细胞中检测突变载体的相对荧光素酶活性。结果表明:ZBED6基因启动子区-2053^-1777 bp存在多个转录因子结合位点,尤其是-1808^-1777 bp,该片段缺失造成启动子活性下降(P<0.01);利用在线软件在该区间预测到3个转录因子HINFP、Adf-1和CREB3,经实验验证后发现这3个转录因子均可调控ZBED6基因的转录,其中Adf-1效果最为明显。据此推测,民猪ZBED6基因的转录调控机制较为复杂,其启动子区存在HINFP、Adf-1和CREB3等多个调控元件的结合位点。     

细毛羊皮肤毛囊miR-1298-5p的靶基因预测及验证

为探讨miR-1298-5p及其靶基因与细毛羊毛囊生长发育的关系,本实验以乾华肉用美利奴羊不同发育时期皮肤毛囊组织为材料,通过生物信息学方法预测miR-1298-5p的靶基因,采用RT-PCR和Western Blot对miR-1298-5p及其靶基因进行定量检测;扩增靶基因3'UTR区,构建野生型和突变型靶基因表达载体后与miR-1298-5p mimics共同转染到HEK293T/17细胞中,利用双荧光素酶报告基因实验验证miR-1298-5p与靶基因的靶向关系。结果表明:FGF_2基因3'UTR区含有miR-1298-5p结合位点,FGF_2为miR-1298-5p潜在靶基因,通过核酸水平和蛋白水平定量表达规律发现miR-1298-5p与靶基因FGF_2呈负相关,初步确定FGF_2为miR-1298-5p的靶基因。双荧光素酶报告基因实验显示,miR-1298-5p mimics能够抑制野生型FGF_2的靶位点报告载体的荧光素酶活性;但当靶基因结合位点突变后,FGF_2被抑制的荧光素酶活性能够得到恢复。综上,miR-1298-5p与FGF_2有靶向关系,miR-1298-5p能通过负调控FGF_2的表达调控羊毛囊发育。     

三种植物甾醇调控SRE元件和3T3-L1细胞脂肪化活性的作用

[目的]为了研究植物甾醇对脂质代谢的调控作用。[方法]利用所构建的SRE荧光素酶活性测定系统,体外考察了豆甾醇、β-谷甾醇和菜油甾醇3种常见的植物甾醇对SRE元件的调控活性。[结果]植物甾醇能够增强SRE元件活性。植物甾醇也可以增加3T3-L1细胞的脂肪化作用。[结论]植物甾醇可能正调控脂质的合成。     

Expression of GFP and Luc reporter genes driven by human myocardial myosin light chain gene promoter in human cardiomyocytes

AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFP_C) or red fluorescent protein(RFP_C) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.     

角蛋白启动子荧光素酶表达载体的构建及其表达活性分析

角蛋白14(K14)和角蛋白5(K5)主要在毛囊基质和复层鳞状上皮基底层的角质细胞里表达。这两个启动子已被广泛应用于毛囊特异表达外源基因的转基因小鼠模型的研究中,为了能将其应用于转基因绵羊生产,探讨基因对绵羊毛囊发育的影响,从人基因组中克隆得到K14和K5启动子,利用不同的报告基因在细胞水平分析各启动子的表达活性。结果表明,这两个启动子在不同细胞系中都能驱动外源基因的表达,而且K14启动子在小鼠皮肤细胞系JB6-C41中的表达活性高于其他细胞。     

chi-miR-4110对奶山羊 Smad2基因的靶向调控作用

为揭示chi-miR-4110对奶山羊繁殖机能的调控作用,在前期构建的多羔(1胎3~5羔)和单羔(1胎1羔)关中奶山羊发情期卵巢差异表达miRNA文库的基础上,利用生物信息学、qRT-PCR和Western blot等技术验证chi-miR-4110的靶基因及其对靶基因的调控作用。荧光素酶检测结果表明,chi-miR-4110通过与Smad2基因的3′UTR相互作用导致荧光素酶活性降低,初步鉴定Smad2为chi-miR-4110的靶基因。将chimiR-4110mimics和阴性对照分别转染到奶山羊卵巢颗粒细胞中,qRT-PCR和Western blotting结果表明,在奶山羊卵巢颗粒细胞中,过表达chi-miR-4110使Smad2 mRNA以及Smad2蛋白的表达量均显著降低,表明chi-miR-4110在转录后水平对Smad2起负调控作用。表明Smad2是chi-miR-4110的靶基因,在奶山羊卵巢颗粒细胞中,chi-miR-4110靶向调控Smad2的表达。