人Hsp90α基因克隆、中试发酵及生物信息学分析

目的:克隆获得人热休克蛋白90α(Hsp90α)基因,构建其原核表达载体,分离纯化重组蛋白,为Hsp90抑制剂的体外筛选奠定基础.方法:TRIzol Reagent法提取K562细胞总RNA,通过RT-PCR获得Hsp90α-cDNA.以该cDNA为模板PCR扩增Hsp90α基因,目的基因和质粒pET28a(+)经Nco Ⅰ和Xho Ⅰ双酶切后,连接酶切片段,将重组DNA转化DH5α,酶切及测序鉴定重组体.将构建好的重组质粒转化Rosetta(DE3)菌株,IPTG诱导,经条件优化后进行中试发酵、纯化以及Western blot鉴定.结果:原核表达载体pET28a(+)-Hsp90α成功构建,可在Rosetta(DE3)中正确表达,中试发酵收菌574g且表达产物以可溶性形式存在,纯化产物纯度为97%.结论:利用分子克隆技术建立了Hsp90α基因的原核表达和中试发酵的条件,为更深入地研究其在抗肿瘤治疗中的作用打下基础.     

Lys_(10)-PAI-1融合蛋白在大肠杆菌中的表达、纯化和凝胶阻滞实验

用PCR方法构建 10个赖氨酸与纤溶酶原激活剂抑制物 1的融合基因Lys10 PAI 1,并克隆于pET2 8a(+ )和pET32a(+ )原核表达载体 .将重组表达质粒pET32 PAI和pET2 8 PAI转化大肠杆菌BL2 1(DE3) .IPTG诱导后可获得分子量分别为 6 3kD和 4 3kD的目的蛋白 ,表达蛋白占菌体蛋白2 0 %以上 ,大多数重组蛋白以不溶形式存在 .表达产物经变性、复性、超滤、透析和亲和层析等步骤 ,可以得到纯化的Trx·PAI 1和rPAI 1重组蛋白 .Western印迹结果表明 ,目的蛋白具有人PAI 1的抗原性 .凝胶阻滞实验发现 ,纯化的重组蛋白在一定条件下 ,可以与质粒结合 ,使质粒的迁移率明显改变 .研究结果表明 ,Trx·PAI 1和rPAI 1有希望成为受体介导基因转移的配体     

香港海鸥菌OsmC蛋白的原核表达、纯化及活性研究

[目的]原核表达纯化香港海鸥菌OsmC蛋白,并检测其抗氧化功能。[方法]通过PCR法扩增香港海鸥菌OsmC基因,将目的片段进行双酶切后连接到pET28a,构建重组质粒pET28a-OsmC并转化大肠杆菌,诱导OsmC蛋白表达,亲和层析纯化目的蛋白并利用氧化铁二甲酚橙实验检测其过氧化物酶活性。[结果]克隆得到全长基因,大小为441bp,编码147个氨基酸。得到重组表达质粒pET28a-OsmC,表达并纯化获得重组OsmC蛋白,OsmC蛋白能够降解H2O2。[结论]Osm C蛋白具有过氧化物酶活性,为研究香港海鸥菌的抗氧化机制奠定了基础。     

原核表达载体pET28a-EGFP的构建与表达

以质粒PEGFP-N3中增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein,EGFP）基因片段为模板,利用PCR技术扩增得到EGFP基因片段,并设计引物在其2端引入酶切位点EcoRⅠ和HindⅢ,对引入酶切位点的EGFP片段和pET28a质粒进行双酶切处理后,利用T4连接酶连接得到了重组质粒pET28a-EGFP。利用热击法把得到的重组质粒pET28a-EGFP转化至E.coliBL21（Escherichia coliBL21）感受态细胞中,当大肠埃希菌LB（Luria-Bertani）培养液在600 nm下的光密度值OD600=0.4时,通过添加异丙基硫代β-D-半乳糖苷（IPTG）作为诱导剂诱导EGFP表达。结果表明：重组质粒酶切鉴定及测序结果正确。在自然光下,转化子在LB固体培养基（含1 mmol/L的IPTG和50μg/mL的卡那霉素（Kan））中菌落呈绿色。在荧光显微镜下受蓝光激发,可以清楚观察到发绿色荧光的转化子。成功构建的原核表达载体pET28a-EGFP在E.coliBL21中得到了高效表达,为以后作为荧光标记物标记食源性病原菌提供了一定的理论和技术支持。     

酿酒酵母RAVE复合物的155kD亚基Rav1p的克隆及其在大肠杆菌中的表达纯化

以酿酒酵母基因组DNA为模板,根据CenBank上公布的酿酒酵母Ravlp基因(rav1)序列和表达裁体特性设计 特异性引物,PCR扩增得到4 074 bp的DNA片段,将PCR产物和原核表达栽体pET28a(+)同时进行双酶切;双酶切后的PCR产物和表达栽体进行连接,构建成重组质粒pET28a-ravl.再将pET28a-ravl转化到BL21( DE3)感受态细胞中.经IPTG 16℃低温诱导40h表达His-tag融合的Ravlp.诱导后的菌体进行超声波破碎,然后用GE healthcare公司的AKTA蛋白纯化仪和His Trap HP I mL亲和层析柱纯化目的蛋白.SDS-PAGE电泳分析和Western blot分析显示在155 kD有明显的条带,成功实现了Ravlp在大肠杆菌中的表达纯化.     

结核分枝杆菌H37Ra株HspX蛋白的表达条件优化、纯化和鉴定

以结核分枝杆菌H37Ra菌株基因组DNA为模板,利用PCR技术扩增获得HspX基因,通过DNA无缝克隆技术将其克隆至pET28a质粒中,构建重组表达质粒pET28a-HspX。将pET28a-HspX转化至大肠埃希菌表达菌株BL21(DE3),采用不同温度、IPTG浓度和时间诱导HspX蛋白表达。使用Ni-IDA亲和层析柱纯化目的HspX蛋白,透析去除咪唑,通过Western blot检测HspX抗原特异性。最终确定表达重组蛋白HspX的最佳诱导条件为：诱导温度37℃、IPTG浓度0.2 mmol/L、诱导时间8 h。结果表明,获得了高纯度和被特异性识别的可溶性Hsp X蛋白,为未来Hsp X蛋白用于结核病诊断试剂和疫苗奠定基础。     

人vasorin蛋白的基因克隆、表达及纯化

目的:克隆、表达人vasorin(VASN)蛋白。方法:利用PCR方法从HepG2细胞的cDNA中扩增获得目的基因,并插入带有6×His标签的原核高效可溶性表达载体pET28a中,构建重组表达质粒pET28a-VASN,将重组表达质粒转化大肠杆菌BL21(DE3),经IPTG诱导后目的基因获得表达,对融合目的蛋白进行Ni2+金属螯合柱纯化。结果:内切酶鉴定及基因序列测定证实重组表达质粒构建成功;对目的蛋白进行了原核表达,SDS-PAGE显示相对分子质量为61×103的特异表达条带;Western印迹证实目的蛋白为VASN,且主要以包涵体形式存在;对经尿素变性的表达产物进行了亲和层析纯化,有利于以后的变性、复性过程。结论:获得了人VASN融合蛋白,为其进一步的生物学功能研究奠定了基础。     

大黄鱼MSTN成熟肽区基因的原核表达

根据本实验室克隆的大黄鱼肌肉生长抑制素(MSTN)基因编码序列设计引物,通过RT-PCR法扩增大黄鱼MSTN成熟肽区域的基因,将目的片段插入pMD18-T克隆质粒,并测序验证。验证后用BamHⅠ和HindⅢ限制性内切酶酶切含目的片段的pMD18-T克隆质粒和pET-28 a表达质粒,构建MSTN-pET-28 a重组表达质粒。将重组表达质粒转化至BL21菌中,经IPTG诱导后表达蛋白的大小约17 kD。     

果蝇Hsp83多克隆抗体的制备及鉴定

目的制备和鉴定抗Hsp83蛋白的多克隆抗体。方法利用PCR技术从果蝇cDNA中获得hsp83基因片段,构建重组质粒;将其转化到BL21(DE3)菌株中诱导蛋白表达,利用Ni-NTA亲和法纯化重组蛋白;再将纯化的蛋白免疫BALB/C小鼠制备多克隆抗体;利用免疫印迹法(Western blot)和免疫荧光染色法检测多克隆抗体的特异性。结果构建的pET28ahsp83质粒在大肠杆菌中成功表达了Hsp83融合蛋白,蛋白纯化后作为抗原免疫小鼠,获得了抗Hsp83的多克隆抗体。免疫印迹法和免疫荧光染色法检测显示,抗果蝇Hsp83多克隆抗体具有较高的特异性,并能检测出内源性Hsp83蛋白。果蝇卵巢免疫荧光染色显示,Hsp83蛋白定位在卵巢细胞的细胞质中。结论成功制备了小鼠抗Hsp83蛋白的特异性抗体,此工作为深入研究Hsp83蛋白的功能奠定了基础。     

人早胚发育相关蛋白ZNF268的抗体制备

目的:获得纯化抗原用于制备ZNF268的多克隆抗体。方法:PCR扩增目的基因片Spacer区序列,亚克隆入融合蛋白表达载体pET28a+,构建了重组质粒pET28a+/SPA。然后将该重组质粒转化E.coliDE3(Rosseta),IPTG诱导表达,SDS-PAGE分离,获纯化融合蛋白6His-SPA。用6His-SPA免疫家兔,颈动脉取血,分离血清,从血清中获得ZNF268特异的多克隆抗体。结论:通过构建融合蛋白重组表达质粒pET28a+/SPA,用获得的初步纯化融合蛋白6His-SPA,制备了特异的ZNF268的多克隆抗体6His-SPA Ab。     

Crystal structure, detonation performance, and thermal stability of a new polynitro cage compound: 2, 4, 6, 8, 10, 12, 13, 14, 15-nonanitro-2, 4, 6, 8, 10, 12, 13, 14, 15-nonaazaheptacyclo [5.5.1.13, 11. 15, 9] pentadecane

A new polynitro cage compound 2, 4, 6, 8, 10, 12, 13, 14, 15-nonanitro-2, 4, 6, 8, 10, 12, 13, 14, 15-nonaazaheptcyclo [5.5.1.1(3,11).1(5,9)] pentadecane (NNNAHP) was designed in the present work. Its molecular structure was optimized at the B3LYP/6-31 G(d,p) level of density functional theory (DFT) and crystal structure was predicted using the Compass and Dreiding force fields and refined by DFT GGA-RPBE method. The obtained crystal structure of NNNAHP belongs to the P-1 space group and the lattice parameters are a = 9.99 ?, b = 10.78 ?, c = 9.99 ?, α = 90.01°, β = 120.01°, γ = 90.00°, and Z = 2, respectively. Based on the optimized crystal structure, the band gap, density of state, thermodynamic properties, infrared spectrum, strain energy, detonation characteristics, and thermal stability were predicted. Calculation results show that NNNAHP has detonation properties close to those of CL-20 and is a high energy density compound with moderate stability.     

Distribution of N,P, K,Ca, Mg,S, Zn,Fe, Mn and Cu in groundnut

Summary Concentration of N, P, K, Ca, Mg and S in summer groundnut crop was higher than in kharif while Zn, Fe, Mn and Cu contents were higher in summer crop. Kernel's N, P and Zn; Leaflet's Ca and Mn; Stem's K and Fe; Root's S and Cu and Petiole's Mg contents were highest. Shell's N, P, K, Mg, S, Zn and Cu; Kernel's Ca, Fe and Mn contents were the least. N, P, K, S, Zn and Cu concentrations decreased linearly as the crop grew. Ca, Mg, Fe and Mn concentrations did not display any distinct pattern. Ca concentration was positively correlated with pod yield in both the seasons.     

Simultaneous measurement of the trace elements Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum and their reference ranges by ICP-MS

The goal of this article was to establish reference ranges of the concentration of trace elements in human serum and to compare these results with those reported by other authors. We describe the sample preparation and measurement conditions that allow the rapid, precise, and accurate determination of Al, As, B, Be, Cd, Co, Cu, Fe, Li, Mn, Mo, Ni, Rb, Se, Sr, and Zn in human serum samples (n=110) by inductively coupled plasma-mass spectrometry (ICP-MS). Accuracy and precision were determined by analyzing three reconstituted reference serum samples by comparison with other methods and by the standard addition procedure. The advantages of the ICP-MS method include short time of analysis of the elements mentioned, low detection limit, high precision, and high accuracy. Disadventages include a high risk of contamination due to the presence of some of the elements of interest in the environment, the relatively delicate sample handling, and the high cost of the equipment.     

Reference values for the concentrations of As,Cd, Co,Cr, Cu,Fe, I,Hg, Mn,Mo, Ni,Pb, Se,and Zn in selected human tissues and body fluids

This report attempts to formulate reference ranges of elemental concentrations for 15 trace elements in selected human tissues and body fluids. A set of samples consisting of whole blood, blood serum, urine, milk, liver, and hair were chosen and considered for 15 elements of biological significance: As, Cd, Co, Cr, Cu, F, Fe, I, Hg, Mn, Mo, Ni, Pb, Se, and Zn. The results represent wholly or partially data received from 40 countries of the global regions of Africa, Asia, Europe, North, South, and Central America, Australia, and New Zealand. This survey, even if qualitative, has been useful in demonstrating certain trends of trace-element scenarios around the world. It is of course recognized that both diet and environment exert a strong influence on the distribution pattern of several elements, such as As, Cd, Mn, Pb, Se, and Zn. A limited comparison of the available information on soil status of different countries reflected some interesting associations for elements, such as Mn and Zn. Importantly, this study revealed that only a few countries were in a position to identify a reasonable amount of data on samples requested for this project. Regretably, for a number of countries, any dependable data for even such essential elements as Cu, Fe, and Zn were not available. In view of the nutritional importance of many elements, the time is ripe for concerted efforts by intergovernmental agencies to initiate investigations or commission task forces/projects to generate reliable reference data for selected global regions, which sadly lack data of any kind at present.     

Relations of the Al,B, Ba,Br, Ca,Cl, Cu,Fe, K,Li, Mg,Mn, Na,P, S,Si, Sr,and Zn mass fractions to morphometric parameters in pediatric and nonhyperplastic young adult prostate glands

The variation with age of the 18 trace element mass fractions and some histological characteristics of intact prostate glands of 50 subjects aged 0–30 years was investigated by instrumental neutron activation analysis, inductively coupled plasma atomic emission spectrometry, and a quantitative morphometric analysis. Mean values ± standard error of the mean (M ± SΕΜ) for the mass fractions (in milligrams per kilogram wet tissue) of these trace elements in pre-puberty were: Al 28.5 ± 9.0, B 0.40 ± 0.11, Ba 1.48 ± 0.44, Br 10.5 ± 1.5, Ca 241 ± 30, Cl 3,203 ± 278, Cu 3.51 ± 0.89, Fe 33.7 ± 4.1, K 2,364 ± 145, Li 0.020 ± 0.004, Mg 153 ± 23, Mn 0.46 ± 0.06, Na 2,286 ± 130, P 1,391 ± 100, S 1,698 ± 132, Si 62 ± 11, Sr 0.38 ± 0.08, and Zn 27.6 ± 2.3. During puberty and postpuberty, when there is a significant increase in circulating androgens, the mean values were: Al 7.2 ± 1.4, B 0.21 ± 0.05, Ba 0.25 ± 0.06, Br 5.8 ± 1.0, Ca 433 ± 81, Cl 2,314 ± 201, Cu 1.77 ± 0.13, Fe 20.9 ± 1.6, K 2,585 ± 118, Li 0.0088 ± 0.0014, Mg 232 ± 27, Mn 0.34 ± 0.04, Na 1,875 ± 107, P 1,403 ± 98, S 1,673 ± 73, Si 22.2 ± 3.1, Sr 0.22 ± 0.03, and Zn 93.3 ± 8.9. Mean values (M ± SΕΜ) of percent volumes (%) of the stroma, epithelium and lumen in the prostate before puberty were 73.4 ± 2.6, 20.4 ± 1.7, and 4.45 ± 0.94, respectively, versus 46.5 ± 2.5, 38.5 ± 1.9, and 14.9 ± 1.2 during puberty and postpuberty. This work’s results confirm that the Zn mass fraction in prostate tissue is an androgen-dependent parameter. For the first time it has been demonstrated that the glandular lumen is a main pool of Ca, Mg, and Zn accumulation and that the stroma is a main pool of Al, B, Ba, Br, Cl, Cu, Fe, Mn, Na, and Si accumulation in the normal human prostate, for the age range 0–30 years. It was concluded that the Ca, Mg, and Zn binds tightly within the prostatic fluid, because the volume of glandular lumen reflects the volume of prostatic fluid.     

The Effect of Age and Gender on Al, B, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Sr, V, and Zn Contents in Rib Bone of Healthy Humans

The effect of age and gender on major, minor, and trace element contents in the intact rib bone of 80 relatively healthy 15–55-year-old women and men was investigated. Contents or upper limit of contents of 16 chemical elements in the rib bone were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Mean values (M?±?SΕΜ) for the mass fraction of Ba, Ca, Cu, Fe, K, Li, Mg, Na, P, S, Sr, and Zn (milligram per kilogram of dry bone) were as follows: 2.54?±?0.16, 171,400?±?4,050, 1.35?±?0.22, 140?±?11, 1,874?±?71, 0.049?±?0.011, 2,139?±?38, 5,378?±?88, 75,140?±?1,660, 1,881?±?51, 291?±?20, and 92.8?±?1.5, respectively. The upper limits of contents of Al, B, Mn, and V were <7.20, <0.65, <0.36, and <0.03, respectively. Statistically significant tendency for the Ca, Mg, and P content to decrease with age was found in the human rib bone, regardless of gender. The mass fraction of Fe in the male rib bone increases with age. It was shown that higher Ca, Mg, Na, P, and Sr mass fractions as well as lower Fe content were typical of female ribs as compared to those in male ribs.     

Potential Compounds for Oral Cancer Treatment: Resveratrol,Nimbolide, Lovastatin,Bortezomib, Vorinostat,Berberine, Pterostilbene,Deguelin, Andrographolide,and Colchicine

Oral cancer is one of the main causes of cancer-related deaths in South-Asian countries. There are very limited treatment options available for oral cancer. Research endeavors focused on discovery and development of novel therapies for oral cancer, is necessary to control the ever rising oral cancer related mortalities. We mined the large pool of compounds from the publicly available compound databases, to identify potential therapeutic compounds for oral cancer. Over 84 million compounds were screened for the possible anti-cancer activity by custom build SVM classifier. The molecular targets of the predicted anti-cancer compounds were mined from reliable sources like experimental bioassays studies associated with the compound, and from protein-compound interaction databases. Therapeutic compounds from DrugBank, and a list of natural anti-cancer compounds derived from literature mining of published studies, were used for building partial least squares regression model. The regression model thus built, was used for the estimation of oral cancer specific weights based on the molecular targets. These weights were used to compute scores for screening the predicted anti-cancer compounds for their potential to treat oral cancer. The list of potential compounds was annotated with corresponding physicochemical properties, cancer specific bioactivity evidences, and literature evidences. In all, 288 compounds with the potential to treat oral cancer were identified in the current study. The majority of the compounds in this list are natural products, which are well-tolerated and have minimal side-effects compared to the synthetic counterparts. Some of the potential therapeutic compounds identified in the current study are resveratrol, nimbolide, lovastatin, bortezomib, vorinostat, berberine, pterostilbene, deguelin, andrographolide, and colchicine.