Construction of eukaryotic recombinant vector of renalase and its expression as a eukaryotic protein

Renalase is a secreted amine oxidase that metabolizes catecholamines. It has been proposed to modulate blood pressure and heart rate and its downregulation might result in hypertension. Despite its potential relevance for human health, the biochemical characterization of renalase is still scarce. The aim of this study is to synthesize the human renalase eukaryotic protein by genetic engineering. The human renalase gene was amplified by polymerase chian reaction (PCR). After digestion by BamH I and Xho I enzymes, the DNA fragments were cloned into the transfer vector, pFastBacHTb-Fc, to generate the pFastBacHTb-renalase expression vector. The ligation products were transformed into E. coli DH10Bac to obtain recombinant transposon rBacmid-renalase. The recombinant transposon was further transferred into insect high-V cells, and the recombinant human renalase eukaryotic protein was expressed successfully.     

Assessment of levator hiatus by 3D ultrasound volume contrast imaging in normal nulliparas

The ability of 3D ultrasound volume contrast imaging in C-plane (VCI-C) in the assessment of female levator hiatus was analyzed in eighty normal nulliparas. Interoperator variability in levator hiatus measurements was analyzed. The comparison of the axial image of magnetic resonance imaging (MRI) and C-plane image of volume contrast imaging (VCI) in thirty normal nulliparas was also done. It shows that VCI-C can clearly demonstrate the structure and accurately quantitates the size of the levator hiatus. It is reliable, convenient and without contraindication in assessment of female levator hiatus. The technology opens up entirely new modality for assessing female pelvic floor. Foundation item: the Scientific Research Project of Shanghai Municipal Health Bureau (No. 2008083)     

Codes From Strongly Regular （α,β）-reguli

Strongly regular (α, β)-reguli are a class of incidence structures with given conditions which were introduced by Hamilton and Mathon. We introduce two classes of codes constructed from strongly regular (α, β)-reguli within PG(k − 1, q). The codes are related with two-weight codes intimately. Foundation item: the Scientific Research Start-up Foundation of Qingdao University of Science and Technology in China (No. 0022327)     

A method to improve first order approximation of smoothed particle hydrodynamics

Smoothed particle hydrodynamics (SPH) is a useful meshless method. The first and second orders are the most popular derivatives of the field function in the mechanical governing equations. New methods were proposed to improve accuracy of SPH approximation by the lemma proved. The lemma describes the relationship of functions and their SPH approximation. Finally, the error comparison of SPH method with or without our improvement was carried out. Foundation item: The National Natural Science Foundation of China (No. 50778111); The Key Project of Fund of Science and Technology Development of Shanghai (No. 07JC14023)     

抗人γ-精浆蛋白V_H单域抗体基因的构建、表达及空间构象分析

目的　扩增出抗人γ 精浆蛋白 (γ Sm)单克隆抗体 (mAb)的重链可变区 (VH)单域抗体基因 ,并进行原核表达及空间构象分析。方法　从分泌抗γ SmmAb的杂交瘤细胞系E4B7中提取总RNA ,经RT PCR扩增VH 单域抗体基因 ,将其克隆到融合蛋白表达载体 pGEX 4T 1中进行表达 ,并利用计算机软件对表达产物进行空间构象分析。结果　VH 单域抗体基因全长 363bp ,含起始码和终止码。将其克隆到 pGEX 4T 1内 ,转化大肠杆菌DH5α ,获得高效表达 ,表达量占全菌总蛋白质的 38% ,以包涵体形式存在。经初步纯化和复性后 ,用谷胱甘肽 (GST)亲和色谱纯化 ,再经凝血酶水解获得抗γ SmVH 单域抗体。竞争结合抑制实验证明 ,该表达产物具有前列腺癌细胞亲和活性。空间构象分析结果显示 ,该抗体具有完整的抗原结合位点。结论　构建成功抗γ Sm的VH 单域抗体 ,为进一步临床应用奠定基础。     

表皮生长因子及其受体在培养的人脑膜瘤细胞中的表达和作用

目的　探讨表皮生长因子 (EGF)在脑膜瘤细胞中的表达和作用 ,以及体外针对该生长调节环路抑制脑膜瘤细胞增殖的可能性。方法　通过对 2 1例脑膜瘤原代培养 ,采用ABC免疫细胞化学法检测EGF和EGF受体 (EGFR)在脑膜瘤细胞中的表达 ,并选用四唑盐 (MTT)比色法观察EGF、EGF单抗和EGFR单抗对脑膜瘤细胞的增殖调控作用。结果　脑膜瘤细胞可表达EGF和EGFR ;EGF以剂量依赖关系刺激培养脑膜瘤细胞增殖 ,而EGF单抗和EGFR单抗皆以剂量依赖关系抑制培养脑膜瘤细胞 ,且EGFR单抗的抑制效果明显强于EGF单抗 (P <0 .0 1)。结论　EGFR单抗较EGF单抗是更为有效的脑膜瘤增殖抑制单抗 ,为脑膜瘤的生物治疗提供新的实验基础     

THE EUKARYOTIC EXPRESSION OF HUMAN PERFORIN PROTEIN AND THE ESTABLISHMENT OF HYBRIDOMAS PRODUCING ANTI-HUMAN PERFORIN PROTEIN

Perforin is expressed mainly in activated cyto-toxic T lymphocytes (CTLs ) and natural killer(NK) cells. The human perforin gene has beencloned. The full length of its cDNA is l668bp, andthe mature HP protein is made up of 534 aminoacid residues with a molecular weight of 66Kd~75Kd. In CTLs and NK cells, perforin is stored incytoplasmic granules and is a major effector of cy-tolysis by these cells. Upon granules releasing,perforin monomers insert into the plasma mem-branes of target ce…     

葎草花粉致敏蛋白多克隆抗体的制备及其在筛选致敏蛋白组分的应用

目的制备、鉴定兔抗葎草花粉蛋白多克隆抗体,并初步应用于葎草花粉致敏蛋白组分的筛选。方法用葎草花粉变应原浸液免疫兔,获得高效价抗葎草花粉蛋白抗血清,应用琼脂免疫双扩散、ELISA法以及Western blotting鉴定产生的抗体。同时对葎草花粉过敏性哮喘患者血清进行Western blotting检测。结果兔抗血清效价经ELISA检测,效价>1∶10000,免疫双扩散结果表明抗血清只与葎草花粉变应原形成特异性IgG沉淀线,Western blotting检测兔抗血清IgG可以特异性识别葎草花粉8种蛋白组分,分子质量分别为100、88、76、69、65、55、49、38 ku,患者血清IgG可以识别5种蛋白组分,分子质量分别为100、76、55、493、8 ku。结论制备的兔抗血清具有较高效价和较好的特异性,应用该血清筛选出的葎草花粉致敏蛋白组分与患者血清筛选的组分相似,因此可进一步应用于初筛cDNA表达文库。     

大鼠激活转录因子3单克隆抗体的制备和鉴定

目的制备特异性识别大鼠激活转录因子3(ATF3)的单克隆抗体(mAb)。方法根据软件预测的结果合成偶联有匙孔绒血蓝蛋白(key holeli mpet hemocyanin,KLH)的大鼠ATF3第168-181位氨基酸多肽。随后免疫BALB/c小鼠,运用杂交瘤技术制备抗大鼠ATF3的mAb。采用快速定性试纸法鉴定该单克隆抗体的亚型和亚类;通过ELISA、Western blot和免疫组织化学法鉴定单克隆抗体的特性。结果获得一株可稳定分泌抗大鼠ATF3 mAb的杂交瘤细胞,其分泌的ATF3单抗的亚型是IgG1,轻链为κ链。Western blot结果显示,该单抗能够特异性识别大鼠细胞内表达的ATF3蛋白。免疫组织化学检测结果表明,在Thy-1肾炎(Thy-1N)大鼠肾细胞胞质及细胞核内均有ATF3表达,但细胞核内的表达量较为显著。结论成功制备了一株抗大鼠ATF3的mAb,为进一步研究ATF3的生物学功能提供了可用的实验材料。     

A mechanical and experimental study on the heat loss of solar evacuated tube

The experimental system of heat loss of all-glass evacuated solar collector tubes (evacuated tube) is firstly designed and constructed, which uses electric heater as thermal resource. The equilibrium temperatures are less than ±1℃ during the test, and the temperature differences of up/middle/low node in the tube are less than 1 ℃, 3 ℃, and 7℃ respectively. The heat loss of evacuated tube increases about 2.7% with vacuum state of 0.01-1 mPa, and it has the best performance at tube temperature of 20-280℃. The invalidation tube (> 200 mPa) has the biggest heat loss that increases linearly with the tube temperature. The evacuated tubes with the vacuum of 0.01-1 mPa are suitable for most solar adsorption refrigeration.     

β淀粉样肽1～42单克隆抗体的制备

目的　制备稳定分泌β -淀粉样肽 1～ 4 2 (Aβ1-42 )单克隆抗体的杂交瘤细胞 ,产生高效价抗体。方法　通过基因工程技术 ,将Aβ1-42 基因融合于乙肝核心抗原 (HBcAg)的主要免疫优势区 (MIR) ,制备Aβ和HBcAg的融合蛋白 ,免疫BALB/c小鼠 ,取脾细胞与SP2 / 0细胞融合 ,筛选能稳定分泌Aβ1-42 单克隆抗体的杂交瘤细胞。结果　得到两株能稳定分泌Aβ1-42 单克隆抗体的杂交瘤细胞 ,Aβ1-42 单克隆抗体的Ig亚类为IgG3 。结论　制备得到的Aβ1-42 单克隆抗体特异性强 ,效价高     

Synchronization research of a modified time-delayed multi-agent network model

The synchronization of time-delayed multi-agent networks with connected and directed topology is studied. Based on the correlative work about the agent synchronization, a modified model is presented, in which each communication receiver is distributed a delay τ. In addition, a proportional term k is introduced to modulate the delay range and to guarantee the synchronization of each agent. Two new parameters mentioned above are only correlative to the network topology, and a theorem about their connections is derived by both frequency domain method and geometric method. Finally, the theoretical result is illustrated by numerical simulations. Foundation item: the National Natural Science Foundation of China (No. 70571017) and the Research Foundation from Provincial Education Department of Zhejiang of China (No. 20070928)     

癌基因MDM2与异构酶PIN1在瘤细胞中的表达

目的　研究MDM 2癌基因产物与脯氨酰异构酶PIN1之间的相互关系。方法　在U2OS骨髓瘤细胞中分别用MDM2对PIN1的单抗通过免疫沉淀法分离出MDM2或PIN1的结合蛋白 ,再用Westernblot对蛋白进行分离。结果　U2OS骨髓瘤细胞的蛋白天然提取物中MDM 2与P1 8的共沉淀物移行位置与用PIN1单抗检测的免疫沉淀物的移行位置相同。同时PIN1与P90的共沉淀物的移行位置与用MDM2单抗检测的免疫沉淀物的移行位置相同。结论　U2OS骨髓瘤细胞中MDM2与PIN1形成稳定的复合物 ,这可能是MDM2在调控G2 /M过渡点的重要机制之一。     

重组人单链白细胞介素12融合基因(rhscIL-12)的构建和真核表达

克隆人 IL - 1 2 ( h IL - 1 2 ) p40和 p35亚单位 c DNA,构建人单链 IL- 1 2 ( rhsc IL- 1 2 )融合基因 ,并在哺乳动物细胞中进行表达。方法　从经 PDBu刺激的 EBV转化的人 B淋巴母细胞株 NC37中提取 m RNA,经 RT- PCR分别获得 h IL- 1 2 p40和 p35亚单位编码序列的 c DNA,运用重组 PCR技术将两段基因通过一疏水性多肽接头 ( Gly4 Ser) 3 DNA序列进行体外基因重组 ,构建 rhsc IL- 1 2融合基因 ,将其插入 pc DNA3.1 ( + )真核表达载体 ,经脂质体转染 COS7细胞进行表达 ,Western blot进行分析。结果　所克隆的 h IL- 1 2 p40、p35c DNA序列和构建的 rhsc IL - 1 2融合基因 DNA序列均经测序得以证实 ,融合基因可在 COS7中表达其产物 rhsc IL- 1 2融合蛋白 ,其分子量为 70 KD,可与鼠抗人 IL - 1 2 p40 /p70单克隆抗体特异性结合。结论　本研究结果为进一步探讨 rhsc IL- 1 2融合蛋白的生物学活性和特性奠定了基础 ,也为 rhsc IL- 1 2融合基因在原核细胞中的表达提供了可能性     

Surface reconstruction of scanned human body using radial basis functions and adaptive partition of unity

It is important to reconstruct a continuous surface representation of the point cloud scanned from a human body. In this paper a new implicit surface method is proposed to reconstruct the human body surface from the points based on the combination of radial basis functions (RBFs) and adaptive partition of unity (PoU). The whole 3D domain of the scanned human body is firstly subdivided into a set of overlapping subdomains based on the improved octrees. The smooth local surfaces are then computed in the subdomains based on RBFs. And finally the global human body surface is reconstructed by blending the local surfaces with the adaptive PoU functions. This method is robust for the surface reconstruction of the scanned human body even with large or non-uniform point cloud which has a sharp density variation. Foundation item: the National Natural Science Foundation of China (No. 50575139) and the Shanghai Special Fund of Informatization (No. 088)     

Serum renalase inversely related to heart rate variability in dialysis patients

Heart rate variability （HRV） is a noninvasive technique for autonomic nerve activity assessment and an indirect index of adrenergic cardiovascular drive. Renalase is the only known enzyme degrading circulatory catecholamines, participating in regulations of the cardiovascular and sympathetic nervous systems. This study aims at the relation between serum renalase and HRV indexes in dialysis patients. There were 14 hemodialysis （HD） cases, 16 continuous ambulatory peritoneal dialysis （PD） cases, and 16 healthy volunteers enrolled in this study. All the subjects underwent 24 h Holter testing by using a dynamic electocardiogram monitor system to determine HRV parameters, and serum renalase levels were measured. The standard deviation of normal to normal R-R intervals （SDNN, a measure of beat-to-beat variability）, root mean square successive difference （RMSSD）, and the proportion derived by dividing NN50 （the number of interval differences of successive normal to normal intervals greater than 50 ms） by the total number of NN intervals （pNN50） were determined to assess HRV. Both HD and PD patients presented increased maximum heart rate （Max-HR）, minimum heart rate （Min-HR） and average heart rate （Ave-HR）, and presented decreased SDNN, RMSSD and pNN50 relative to healthy controls. Serum levels of renalase were highly negatively correlated with Ave-HR （correlation coefficient r = -0.68, probability P 〈 0.01）, while positively correlated with SDNN （r = 0.64, P 〈 0.01） in both the HD and PD groups. Serum level of renalase was positively correlated with 24 h urine volume （r = 0.73, P 〈 0.01） in PD patients. Renalase might be reciprocally related to HRV and inversely related to Ave-HR in dialysis patients, which will help to probe into renalase＇s physiology and may pave the way to explore renalase replacement therapy in patients with end stage renal disease （ESRD）.     

General admissibility for linear estimators of multivariate random regression coefficients and parameters with respect to a restricted parameter set

This paper considers the linear model effected by random disturbance, Y = XB + ɛ, where $ \left[ \begin{gathered} B \hfill \\ \varepsilon \hfill \\ \end{gathered} \right] \sim \left( {\left[ \begin{gathered} A\Theta \hfill \\ 0 \hfill \\ \end{gathered} \right],V \otimes \Sigma } \right) $ \left[ \begin{gathered} B \hfill \\ \varepsilon \hfill \\ \end{gathered} \right] \sim \left( {\left[ \begin{gathered} A\Theta \hfill \\ 0 \hfill \\ \end{gathered} \right],V \otimes \Sigma } \right) , and Θ ^T A ^T X ^T NXAΘ ⩽ Σ. It gives a definition for general admissible estimator of a linear function SΘ + GB of random regression coefficients and parameters. The necessary and sufficient conditions for LY and LY + C to be general admissible estimators of SΘ + GB in the class of both homogenous and non-homogenous linear estimators are obtained. The conclusion is not dependent of whether or not SΘ +GB is estimable.     

小鼠抗人T淋巴细胞表面抗原噬菌体抗体库的构建及筛选

目的　构建并筛选小鼠抗人静息 /活化T淋巴细胞表面抗原的噬菌体展示抗体库。方法　以人静息 /活化T淋巴细胞免疫Balb/c小鼠 ,取脾细胞 ,RT -PCR扩增VH 和VK 基因 ,拼接为ScFv ,插入噬菌粒载体转化大肠杆菌 ,构建了噬菌体展示抗体库 ,以人T淋巴细胞为靶抗原 ,对该库进行了 3轮淘洗 ,ELISA方法鉴定噬菌体抗体。结果　成功构建了最大实际库容量为 1 .75× 1 0 6的小鼠抗人静息T淋巴细胞表面抗原的噬菌体展示ScFv抗体库和小鼠抗人活化T淋巴细胞表面抗原的噬菌体展示ScFv抗体库。结论　所构建的抗体库可进一步用于对T淋巴细胞表面蛋白抗原的差异显示分析及构建基因工程抗体。     

Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coil BL21, the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG), and it was analyzed with sodium dodecyl sulfate-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyelonai antibody, with which we studied the function of NSSATP5 by determining the different liver tissues with the streptavidin-perosidase (SP) immunohistochemistry method. Results Recombinant NS5ATP5 (molecular weight: 65 kD) and polyclonal antibody were successfully prepared. NS5ATP5 expression in the liver of patients with chronic HCV infection was much higher than that of a normal person, and it was detected mainly in the cytoplasm. Conclusion The findings of the expression difference between HCV patients and normal people led to a novel diagnostic marker to detect HCV infection.     

The settlement characteristic of underground urban complex in Shanghai

The effects of excavation unloading, construction reloading and underground water on basal heave of excavation projects were presented and analyzed based on the measurement results of an underground urban complex which was located in Shanghai. The effects on water pressure and building settlements were analyzed as well. The numerical analyses by finite element method (FEM) were conducted. It showed that the soil under the excavation base continued to heave during the following certain construction stage. It also found that the bearing capacity of uplift piles which supported the buildings affected the structure quality significantly. The conclusions can be applied in future projects. Foundation item: the National Natural Science Foundation of China (No. 50679041)