犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

Crystal structure determination of the β-cyclodextrin-p-aminobenzoic acid inclusion complex from powder X-ray diffraction data

The 1:1 inclusion complex of β-cyclodextrin and p-aminobenzoic acid was prepared and characterized by TG-DTA. The crystal structure of the complex was solved directly from powder X-ray diffraction data using the direct space approach and refined using Rietveld refinement techniques. The complex crystallizes in monoclinic P2₁ space group, with unit cell parameters a = 20.7890 ?, b = 10.2084 ?, c = 15.1091 ?, β = 110.825°, V = 2997 ?³. The amino group is located at the wide side of the β-cyclodextrin cavity, forming hydrogen bonds with β-cyclodextrin, and the carboxyl group is located at the narrow side. The crystallographic data obtained from powder diffraction data were compared with the single crystallographic data, and the result shows that solving crystal structure of cyclodextrins inclusion complexes of such complexity is accessible to powder diffractionists to some extent.     

Trehalose synthase converts glycogen to trehalose

Trehalose (alpha,alpha-1,1-glucosyl-glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose <--> trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S-200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [(3)H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca(2+), but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition-state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild-type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10- to 20-fold more glycogen when grown in high concentrations (> or = 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.     

The molecular basis of glycogen storage disease type 1a: structure and function analysis of mutations in glucose-6-phosphatase.

Glycogen storage disease type 1a is caused by a deficiency in glucose-6-phosphatase (G6Pase), a nine-helical endoplasmic reticulum transmembrane protein required for maintenance of glucose homeostasis. To date, 75 G6Pase mutations have been identified, including 48 mutations resulting in single-amino acid substitutions. However, only 19 missense mutations have been functionally characterized. Here, we report the results of structure and function studies of the 48 missense mutations and the DeltaF327 codon deletion mutation, grouped as active site, helical, and nonhelical mutations. The 5 active site mutations and 22 of the 31 helical mutations completely abolished G6Pase activity, but only 5 of the 13 nonhelical mutants were devoid of activity. Whereas the active site and nonhelical mutants supported the synthesis of G6Pase protein in a manner similar to that of the wild-type enzyme, immunoblot analysis showed that the majority (64.5%) of helical mutations destabilized G6Pase. Furthermore, we show that degradation of both wild-type and mutant G6Pase is inhibited by lactacystin, a potent proteasome inhibitor. Taken together, we have generated a data base of residual G6Pase activity retained by G6Pase mutants, established the critical roles of transmembrane helices in the stability and activity of this phosphatase, and shown that G6Pase is a substrate for proteasome-mediated degradation.     

The angiosomes of the head and neck: anatomic study and clinical applications

The angiosome concept was introduced over a decade ago by Taylor and Palmer, whereby the body was considered to be composed anatomically of multiple three-dimensional composite blocks of tissue supplied by particular source arteries. Since then, detailed studies of the forearm and leg have been examined by Taylor and his coworkers. This study focuses on another region--the head and neck. Six fresh head and neck cadaver specimens were examined after infusion with a radio-opaque lead oxide mixture and correlated with over 24 previous body studies. The vascular anatomy of the skin, superficial musculoaponeurotic system (SMAS), muscles, brain, dura, and bone was examined. Each layer was painstakingly removed, photographed, labeled, and mapped to the respective arteries and veins. A radiologic subtraction technique was used to allow successive layers to be compared. This information was then scanned into a computer, analyzed, color coded, and labeled, thereby producing a three-dimensional study of the head and neck region to identify the respective angiosomes. As in previous detailed examinations of the leg and forearm, the angiosomes were found to be connected usually within tissues, such as muscle, skin, specialized organs or glands, rather than between the tissues. The muscles usually had vessels of two or more angiosomes supplying them and fell into three major groups based on the similarity of their arterial supply. In some areas, the midline anastomoses were rich, especially in the integument of the scalp, forehead, and lips. In other regions, the midline vascular connections were poor, especially in the tongue and palate. No fewer than 13 angiosomes of the head and neck, supplied by the branches of the external carotid, internal carotid, and subclavian arteries, have been defined, mapping their three-dimensional territories in the skin, the deep soft tissues, and the bones. Although most angiosomes spanned between skin and bone, three territories, those of the vertebral, lingual, and ascending pharyngeal vessels, were confined to the deep tissues without cutaneous representation. Finally, this study provides additional data for the surgeon to help plan safer incisions and better reconstructive flap procedures. It also gives information that may help explain the etiology and treatment of head and neck arteriovenous vascular malformations.     

水稻矮缩病毒第10号片段编码区的cDNA克隆及序列分析

采用聚合酶链式反应技术,扩增了水稻矮缩病毒(RDV)基因组第10号片段的编码序列,该片段编码病毒的非结构蛋白。对扩增产物进行了克隆和限制性内切酶分析,并绘制了物理图谱。克隆片段大小为1150 bp,含Sac I、Hind III、Nde I、BamH I、Sai I 等酶切位点,引物设计时还在该片段两侧增加了Bgl II和 EcoR I 切点,以便克隆到植物中间载体质粒。利用上述酶切位点对该片段进行了亚克隆和序列分析,结果表明,本研究克隆的RDV中国流行林基因组第10号片段的编码区与日本流行株的相应区域比较,核酸的同源率为96.03％,编码的氨基酸的同源率为97.17％。     

在CHO细胞中表达具有高效成骨活性的BMP-2/7异源二聚体

PCR扩增BMP-2与BMP-7的编码基因, 利用重叠PCR以柔性肽(Gly4Ser)5编码序列将二者串连并克隆到质粒pIRESneo3上, 转染CHO-K1细胞得到混合稳定克隆。ELISA检测培养液中BMP-2/7异源二聚体蛋白的表达水平为230.75±13.34 ng/mL, 以此为条件培养基处理成骨细胞株MC3T3, 对照组为分别含有CHO-K1细胞及大肠杆菌表达的BMP-2同源二聚体以及PBS的条件培养基。结果发现碱性磷酸酶染色与茜素红染色差异明显, 定量RT-PCR显示分子指标OC、ALP、Runx2与Osx的转录水平明显增高(P<0.05), Luciferase报告基因检测BMP/Smad通路活性较对照组升高明显(P<0.05)。首次设计构建了BMP-2/7异源二聚体蛋白, 其成骨活性显著高于BMP-2同源二聚体。     

转铁蛋白受体在土拉弗朗西斯菌LVS入侵巨噬细胞期间的作用

评估转铁蛋白受体-1在土拉弗朗西斯菌LVS感染鼠巨噬细胞期间的作用.用表达GFP的土拉弗朗西斯菌LVS感染鼠巨噬细胞J774A.1.结合单抗的转铁蛋白受体-1分别用键合了Alexa594的羊抗鼠二抗显色,用小干扰RNA下调转铁蛋白受体-1的表达,进而用土拉弗朗西斯菌LVS分别感染转铁蛋白受体-1表达下调的细胞和转染无关siRNA的细胞,并进行细菌计数.分布在细胞膜上的转铁蛋白受体-1参与巨噬细胞对弗朗西斯菌的摄入.免疫印迹结果表明小于扰RNA对转铁蛋白受体-1的表达下调了大约75%.细菌入侵试验显示,在感染1 h时,转铁蛋白受体-1表达下调的细胞内细菌数量等同于对照(F=1.06.P=0.3265);而在感染24 h时,Tfr1下调样本中的细菌数量明显低于对照样本(F=24.12,P=0.0006).这些发现说明在感染早期转铁蛋白受体-1参与了细菌的摄入,这可能与弗朗西斯菌获取铁以利在胞内生存有关.转铁蛋白受体-1的下调不影响细菌的入侵,但抑制细菌在细胞内的增殖.     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。