柑桔溃疡病菌重组单链抗体的高效表达及活性检测

运用PCR方法扩增利用核糖体展示技术筛选的抗柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,XAC)的单链抗体(ScFv95)基因片段,将单链抗体基因重组到原核表达载体pET30a( )中,构建单链抗体高效表达载体pET30a( )-XAC-ScFv。再将pET30a( )-XAC-ScFv质粒转化进大肠杆菌BL21(DE3)后诱导表达,并对表达产物进行纯化、复性及活性检测。获得了抗XAC单链抗体的高效表达蛋白,以包涵体形式存在的表达蛋白大小约32kDa。包涵体蛋白经过变性、纯化和复性后,初步获得有功能的单链抗体。同时用Biacore分析XAC-ScFv-95与XAC LPS作用,结果表明复性后的XAC-ScFv-95具有较高的亲和力,从而为柑桔溃疡病菌XAC的免疫诊断和防治研究提供了新的工具和途径。     

乙肝表面抗原a表位粘膜疫苗的构建及免疫原性检测

利用DNA重组技术 ,构建霍乱毒素B亚单位与乙肝病毒表面抗原a表位的融合基因 ,并在大肠杆菌表达体系pET 30a BL2 1 (DE3)中获得以包涵体形式的高效表达。免疫印迹表明表达产物具有免疫学活性 ,包涵体复性后 ,表达产物能够象CTB那样形成五体 ,以腹腔注射、灌胃、鼻饲形式免疫小鼠均能产生抗HBsAg抗体     

人分泌型磷脂酶A2(hsPLA2-IIA)基因的克隆、表达及活性鉴定

目的为了构建人分泌型磷脂酶A2(secretaryphospholipaseA2,sPLA2-IIA)的有效表达系统,从胎脾中提取总RNA。方法采用RT-PCR方法扩增出编码sPLA2-IIA的基因定向地克隆于硫氧环蛋白基因融合表达载体pET32a的TrxA基因3′末端,构建符合读码框的融合表达载体pET32a-sPLA2-IIA。37℃下经IPTG诱导,hsPLA2-IIA融合蛋白在大肠杆菌BL21(DE3)中获得高效表达,表达产物以包涵体的形式存在。包涵体经8mol/L尿素溶解、复性后检测结果显示具有较高的催化活性并呈现剂量依赖关系。结论以大肠杆菌为宿主,成功表达了hsPLA2-IIA蛋白,为进一步进行hsPLA2-IIA的大量生产和功能研究奠定了基础。     

人分泌型磷脂酶A2(hsPLA2-IIA)基因的克隆、表达及活性鉴定

目的 为了构建人分泌型磷脂酶A2(secretary phospholipase A2, sPLA2-IIA) 的有效表达系统,本文从胎脾中提取总RNA,采用RT-PCR方法扩增出编码sPLA2-IIA的基因定向地克隆于硫氧环蛋白基因融合表达载体pET32a的TrxA基因3’末端,构建符合读码框的融合表达载体pET32a-sPLA2-IIA。37℃下经IPTG诱导,hsPLA2-IIA融合蛋白在大肠杆菌BL21(DE3)中获得高效表达,表达产物以包涵体的形式存在。包涵体经8M尿素溶解、复性后检测结果显示具有较高的催化活性并呈现剂量依赖关系。结论：以大肠杆菌为宿主,成功表达了hsPLA2-IIA蛋白,为进一步进行hsPLA2-IIA的大量生产和功能研究奠定了基础。     

MMP-9-PEX原核表达、纯化及其对结肠癌细胞侵袭能力的影响

用RT-PCR法扩增出基质金属蛋白酶-9(MMP-9)C末端血红素结合蛋白样结构域(PEX),克隆至表达载体pET32a中并转化至E.coilBL21(DE3),经IPTG诱导后在约为42kDa处发现有外源基因的表达,密度扫描显示表达蛋白含量占菌体总蛋白的50%左右。重组蛋白质pET32a/PEX主要以包涵体形式表达,其在上清液中的含量极微。含8mol/L尿素和10mmol/LDTT的裂解缓冲液溶解的包涵体采用金属整合层析有效分离出目标蛋白,纯化后pET32a/PEX蛋白质的纯度大于90%。在Transwell实验中发现复性纯化后的重组蛋白质pET32a/PEX能抑制结肠癌细胞的侵袭,且呈剂量依赖性。     

EGF-SEA融合蛋白在大肠杆菌中的表达和纯化

根据基因库中查到的金黄色葡萄球菌肠毒素A(SEA)基因序列和人体表皮生长因子(EGF)基因序列进行密码子优化,以适于大肠杆菌表达.人工合成SEA基因与EGF基因.将两目的基因克隆至原核表达栽体pFT22b中,经测序验证表明成功构建了重组表达质粒pET22b-EGF-SEA.将构建好的pET22b-EGF-SEA质粒转化大肠杆菌BL21(DE3),经IPTG诱导进行表达;SDS-PAGE分析表明融合基因EGF-SEA在大肠杆菌BL21(DE3)中以包涵体的形式得到了高效表达,产物相对分子质量约为44kDa,与理论值大小一致.包涵体经洗涤,变性、复性后用His Bind Kit进行分离纯化,所得蛋白纯度≥95%.高纯度EGF-SEA融合蛋白的获得为进一步研究其生物学活性及肿瘤治疗奠定了基础.     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

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。     

Flt3配体胞外域的原核表达纯化及活性鉴定

目的:构建pET32a(+)-hFLext原核表达载体,诱导hFLext蛋白表达、纯化及活性鉴定.方法:以人淋巴细胞cDNA文库为模板,克隆hFlext,构建pET32a(+)-hFLext重组表达载体.转化大肠杆菌BL21,IPTG诱导蛋白表达,镍珠亲合层析纯化蛋白,SDS-PAGE及Western blot鉴定.细胞增殖实验检测其生物学活性.结果:成功克隆获得hFLext,并构建了pET32a(+)-hFLext重组表达载体.在大肠杆菌BL21,经1 mM IPTG 30℃诱导12 h,成功表达Trx-hFLext融合蛋白,主要以包涵体形式存在.经8M尿素变性包涵体蛋白,逐步透析复性,镍珠亲合层析纯化蛋白,SDS-PAGE及Western blot鉴定,成功获得高纯度的Trx-hFLext融合蛋白.细胞增殖实验证实其具有生物学活性,能够有效刺激脐血细胞增殖.结论:成功构建了pET32a(+)-hFLext重组表达载体,表达、纯化了具有生物学活性的Trx-hFLext融合蛋白,为造血干/祖细胞的体外扩增研究奠定了基础.     

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.     

Monovalent Cation Permeation through the Connexin40 Gap Junction Channel : Cs,Rb, K,Na, Li,TEA, TMA,TBA, and Effects of Anions Br,Cl, F,Acetate, Aspartate,Glutamate, and NO3

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (γ_j in pS): CsCl (153), RbCl (148), KCl (142), NaCl (115), LiCl (86), TMACl (71), TEACl (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt γ_j are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO₃ (157), KF (148), KCl (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li⁺ are: Cs⁺ (1.38), Rb⁺ (1.32), K⁺ (1.31), Na⁺ (1.16), TMA⁺ (0.53), TEA⁺ (0.45), TBA⁺ (0.03), Cl⁻ (0.19), glutamate⁻ (0.04), and NO₃− (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 Å from a theoretical fit of the relationship of relative permeability and cation radius.     

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.     

Reliability of Analyses of Hg,Fe, Ca,K, P,pH, Alkalinity,Conductivity, Hardness and Colour from Lakes

The aim of this report has been to present results concerning analytical quality controls of Hg analysis of fish and sediment, analyses of Fe, Ca, total-P, K, pH, alkalinity, conductivity, colour and hardness (Ca + Mg) of lake water samples. Despite the fact that these are standard parameters in many regular water control programs, there are major differences in the reliability with which these parameters can be determined. The focus here is on an overall inter-laboratory comparison between the parameters. Six laboratories have been involved in the analysis. Selected results: pH gives the lowest (average) relative standard deviation (error), about 2 %; conductivity gives an error of about 5–7 %; alkalinity yields an average error of as much as 13–25 %, which is the largest among the parameters studied here; colour also gives a high error, 9–15 %; hardness gives a relative standard deviation of about 6–7 %. Of the other parameters (i. e., Hg, Fe, Ca and P), Hg gives the best reliability and Fe and P the lowest. To have knowledge of the reliability of the analytical data is of paramount importance in most control programs and research projects.     

Competitive inhibition and selectivity enhancement by Ca in the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, Hf) by carrot (Daucus carota cv. U.S. harumakigosun)

We investigated the uptake of inorganic elements (Be, Na, Mg, K, Ca, Sc, Mn, Co, Zn, Se, Rb, Sr, Y, Zr, Ce, Pm, Gd, and Hf) and the effect of Ca on their uptake in carrots (Daucus carota cv. U.S. harumakigosun) by the radioactive multitracer technique. The experimental results suggested that Na, Mg, K, and Rb competed for the functional groups outside the cells in roots with Ca but not for the transporter-binding sites on the plasma membrrane of the root cortex cells. In contrast, Y, Ce, Pm, and Gd competed with Ca for the transporters on the plasma membrane. The selectivity, which was defined as the value obtained by dividing the concentration ratio of an elemental pair, K/Na, Rb/Na, Be/Sr, and Mg/Sr, in the presence of 0.2 and 2 ppm Ca by that of the corresponding elemental pair in the absence of Ca in the solution was estimated. The selectivity of K and Rb in roots was increased in the presence of Ca. The selectivity of Be in roots was not affected, whereas the selectivity of Mg was increased by Ca. These observations suggest that the presence of Ca in the uptake solution enhances the selectivity in the uptake of metabolically important elements against unwanted elements.     

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.