鸭坦布苏病毒囊膜蛋白抗原表位的串联表达与抗原性分析

在对鸭坦布苏病毒囊膜蛋白抗原表位分析的基础上,将已经筛选出的8条抗原性良好的多肽采用柔性肽拼接,转入T载体中。将酶切回收的串联基因(命名为TEM)克隆于表达载体p ET32a中,获得重组表达质粒p ET32a-TEM,将该质粒转化于感受态细胞BL21(DE3)中,经IPTG诱导后进行超声裂解,对融合蛋白进行可溶性分析、Western blotting分析和免疫原性分析。重组质粒p ET32a-TEM的酶切和测序结果表明,TEM串联基因已成功插入到p ET32a原核表达载体中;表达的p ET32a-TEM融合蛋白分子质量约为38 000,以包涵体形式存在。Western blotting和小鼠免疫试验结果显示,p ET32a-TEM融合蛋白与鸭坦布苏病毒阳性血清能发生特异性反应,具有良好的抗原性。串联多肽的成功表达,将为鸭坦布苏病血清学检测方法建立、新型工程疫苗的研发奠定基础。     

Demonstration of biological activity of a murine leukemia virus of New Zealand black mice

Although New Zealand Black mouse embryo and adult tissues show evidence of murine leukemia viral particles and antigens, efforts to demonstrate biological activity of a murine leukemia virus by standard methods have proved negative. Cocultivation of tissues of these mice with non-virus-yielding hamster cells transformed by Moloney sarcoma virus, however, has resulted in the rescue of a pseudotype sarcoma virus, presumably carrying the New Zealand Black mouse leukemia virus coat. This virus has an unusual host restriction, producing foci of cell alteration only in rat cells.     

白斑综合征病毒囊膜蛋白VP31与该病毒侵染的相关性

白斑综合征病毒(WSSV)是一种已给世界养虾业造成了严重危害的病原体.根据GenBank上公布的WSSV囊膜蛋白基因VP31的序列,设计并合成特异引物,PCR扩增得到VP31基因,大小为783 bp.通过引物两端的BamHⅠ和EcoRⅠ酶切位点连接该基因,然后将其构建到家蚕杆状病毒表达载体pFAST BAC HTB上.重组的家蚕杆状病毒转染5龄家蚕幼体内表达,表达产物经SDS-PAGE检测,显示与预期31 kD大小相吻合的蛋白带.用Ni2 柱纯化该蛋白免疫新西兰大白兔制备抗体,肌肉注射克氏鳌虾进行活体中和病毒试验.中和试验结果显示,囊膜蛋白VP31与白斑综合征病毒的侵染性相关,对病毒的侵染性可能起着重要作用.     

白斑综合征病毒囊膜蛋白VP31与该病毒侵染的相关性

白斑综合征病毒(WSSV)是一种已给世界养虾业造成了严重危害的病原体.根据GenBank上公布的WSSV囊膜蛋白基因VP31的序列,设计并合成特异引物,PCR扩增得到VP31基因,大小为783 bp.通过引物两端的BamHⅠ和EcoRⅠ酶切位点连接该基因,然后将其构建到家蚕杆状病毒表达载体pFAST BAC HTB上.重组的家蚕杆状病毒转染5龄家蚕幼体内表达,表达产物经SDS-PAGE检测,显示与预期31 kD大小相吻合的蛋白带.用Ni2 柱纯化该蛋白免疫新西兰大白兔制备抗体,肌肉注射克氏鳌虾进行活体中和病毒试验.中和试验结果显示,囊膜蛋白VP31与白斑综合征病毒的侵染性相关,对病毒的侵染性可能起着重要作用.     

鹅坦布苏病毒囊膜蛋白结构域Ⅱ在大肠杆菌中的表达及免疫学鉴定

为了实现鹅坦布苏病毒囊膜蛋白(E蛋白)结构域Ⅱ在大肠杆菌中的表达并对其进行免疫学鉴定,根据鹅坦布苏病毒JS804株囊膜蛋白基因序列,利用人工合成法获得鹅坦布苏病毒囊膜蛋白结构域Ⅱ编码基因并引入限制性酶切位点,片段大小为507 bp。将合成的基因片段克隆入pGEX-4t-1原核表达载体中,构建重组表达载体pGEX-EII并转化至BL21(DE3)中。通过优化表达条件获得重组蛋白质并进行免疫学鉴定。结果显示,重组蛋白质分子量约为4.4×104,Western-blot和间接免疫荧光检测结果表明重组蛋白质具有良好的免疫原性。     

传染性支气管炎病毒(IBV)小囊膜蛋白基因的原核表达

以传染性支气管炎病毒(IBV)M41毒株为材料,克隆该毒株的小囊膜蛋白(E)基因,并将其连接到表达载体pGEX-6P-1中,转化到大肠杆菌BL21中。经IPTG诱导后,SDS-PAGE分析目的蛋白与谷胱苷肽硫转移酶(GST,大小约为26ku)融合后大小约为38ku,表明目的蛋白相对分子质量约为12.4。这一结论为E蛋白的进一步研究提供了条件。     

1-alpha-Tocopherylacetate: biological activity

l-alpha-Tocopheryl acetate has 42 percent of the activity of d-alpha-tocopheryl acetate when compared in the rat hemolysis test. Based on this activity a potency ratio of 1.4 : 1.0 for d-alpha-tocopheryl acetate compared to dl-alpha-tocopheryl acetate was established; this confirms the currently accepted biological ratio. Preparations of dl-alpha-tocopheryl acetate from phytol and from isophytol were equally active in the rat hemolysis test.     

猪繁殖与呼吸综合征病毒GP5蛋白中和性单克隆抗体的制备与免疫学特性测定

利用重组真核质粒pcDNA3-GP5免疫BALB／c小鼠,取脾细胞和骨髓瘤细胞SP2／0融合,经间接ELISA筛选和3次有限稀释法克隆,得到3株能稳定分泌抗猪繁殖与呼吸综合征病毒（PRRSV）GP5蛋白的单克隆抗体（McAb）杂交瘤细胞株,分别命名为4C9、5D10、5F12,其中5D10的Ig亚类为IgM。ELISA检测杂交瘤细胞培养上清液抗体的效价为1：64～1：1024,腹水的效价为1：3200～1：10240,IFA和IPMA的检测结果均为阳性。Western-blot检测证明,这3株McAb均针对PRRSV的GP5蛋白。中和试验表明,5D10和5F12具有病毒中和活性,中和效价分别为1：40和1：80。5D10相对亲和力大于5F12,3株细胞连续培养20代后仍能稳定分泌抗体,表明抗GP5蛋白中和性McAb制备成功。     

Structure of genes coding the envelope proteins of the avian influenza a virus and bovine leucosis virus

This paper comparatively analyzes the density of nucleotide sequences potentially predisposed to the formation of noncanonical DNA structures (G4 quadruplexes) on the hemagglutinin (HA) and neuraminidase (NA) genes of the avian influenza A virus (H5N1) and the env and pol genes of the bovine leucosis virus. The higher genetic heterogeneity of the HA gene in comparison with the NA gene coincided with the high frequency of the nucleotide sequences predisposed to the formation of G4 quadruplexes. The same tendency was observed when comparing the distribution of potential G4 quadruplexes in the env and pol genes of the bovine leucosis virus. We discuss a possible relation between the increased density of the noncanonical DNA structures in the genes whose products interact with the receptor system of target cells of a retrovirus infection and their genetic heterogeneity maintained in the host-pathogen system.     

Deuterium: natural variations used as a biological tracer

The suggestion is made that isotope tracing be carried out by monitoring the natural variations in deuterium concentrations. As an example, the natural variations in deuterium concentrations between food and water collected in Illinois and food and water collected in Colorado were used to determine the residence time of water in the blood and urine of rats. We observed not only a 5(1/2)-day turnover time of water in the blood and urine, but also evidence for the influx of water vapor from the atmosphere through the lungs into the blood.     

P1 of strawberry vein banding virus,a multilocalized protein,functions as a movement protein and interacts with the coat protein

Although the complete nucleotide sequence of strawberry vein banding virus (SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV (SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast two-hybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.     

锦鲤疱疹病毒囊膜蛋白ORF132的克隆及分析

提取感染锦鲤疱疹病毒(KHV)的锦鲤(Cyprinus carpio koi)肾脏组织DNA,通过PCR扩增了KHV ORF132基因。该基因全长513 bp,所编码的蛋白包含170个氨基酸,分子量18.5 ku,等电点8.11,有信号肽以及2个潜在的O糖基化位点。应用DNA Star程序,通过综合分析二级结构柔性区、蛋白的亲水性、表面可能性、抗原性指数,预测KHV ORF132蛋白主要B细胞表位区段位于Leu40～Ala45、Asn53～Pro66、Arg97～Thr118、Ala125～Pro136、Glu140～Arg145、Asn160～Arg170。     

beta-Arrestin: a protein that regulates beta-adrenergic receptor function

Homologous or agonist-specific desensitization of beta-adrenergic receptors is thought to be mediated by a specific kinase, the beta-adrenergic receptor kinase (beta ARK). However, recent data suggest that a cofactor is required for this kinase to inhibit receptor function. The complementary DNA for such a cofactor was cloned and found to encode a 418-amino acid protein homologous to the retinal protein arrestin. The protein, termed beta-arrestin, was expressed and partially purified. It inhibited the signaling function of beta ARK-phosphorylated beta-adrenergic receptors by more than 75 percent, but not that of rhodopsin. It is proposed that beta-arrestin in concert with beta ARK effects homologous desensitization of beta-adrenergic receptors.     

Expression of interleukin-10 activity by Epstein-Barr virus protein BCRF1

Cytokine synthesis inhibitory factor (CSIF; interleukin-10), a product of mouse TH2 T cell clones that inhibits synthesis of cytokines by mouse TH1 T cell clones, exhibits extensive sequence similarity to an uncharacterized open reading frame in the Epstein-Barr virus BCRF1. Recombinant BCRF1 protein mimics the activity of interleukin-10, suggesting that BCRF1 may have a role in the interaction of the virus with the host's immune system.     

Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene

Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41.     

梭鱼白细胞介素-22原核表达及活性蛋白的制备

根据梭鱼IL-22cDNA序列设计特异性引物,从梭鱼脾脏中扩增IL-22用于表达片段,连接至载体pUC57。分别以BamHⅠ和KpnⅠ对含有目的基因的质粒和pQE-30进行酶切,连接并转化到大肠杆菌M15中,以IPTG进行诱导表达。表达产物通过Ni-NTA亲和层析进行纯化。经SDS-PAGE对表达产物进行鉴定。SDS-PAGE显示该IL-22表达质粒经IPTG诱导后,IL-22重组蛋白主要在菌体培养上清中表达。通过优化蛋白表达及纯化方法,成功获得了梭鱼IL-22活性蛋白。     

中药调控MAPK信号通路在疾病治疗中的研究进展

目的分离丽江猪丝裂原活化蛋白激酶10 (mitogen-activated protein kinase,MAPK10) 基因序列,并分析其组织mRNA和蛋白表达情况。方法采用PCR技术扩增丽江猪MAPK10基因,并利用生物信息学软件对获得的序列进行分析,实时荧光定量PCR检测丽江猪脑、肝脏、心脏、脾脏、肺脏、肾脏、胰脏、背脂和背最长肌等9种组织中MAPK10基因的相对表达量;以大白猪为对照,实时荧光定量PCR 和Western-blotting检测丽江猪与大白猪背脂中mRNA和蛋白表达差异。结果MAPK10基因序列总长1281 bp,编码426个氨基酸;MAPK10蛋白属亲水蛋白,无跨膜结构和信号肽,有44个磷酸化位点和2个糖基化位点;二级结构以α-螺旋和无规则卷曲为主;丽江猪MAPK10基因序列与人、牛、山羊、绵羊、鼠、狗、猴和鸡的同源性分别为95.3%、93.5%、93.2%、93.0%、92.6%、91.2%、90.5%和85.6%;MAPK10基因在丽江猪9个组织中差异表达,以脑中最高;丽江猪背脂MAPK10基因的mRNA表达量极显著低于大白猪 (P<0.01),蛋白表达量也有低于大白猪的趋势 (P>0.05)。结论MAPK10基因可作为研究丽江猪脂肪沉积性状的候选基因,结果可为研究猪脂肪沉积提供参考。