伪狂犬病病毒上海株gE-/gI-/GFP+缺失株的生物学特性初步研究

在构建了伪狂犬病病毒上海株的缺失载体pgEI-GFP基础上,将pgEI-GFP转染感染了PRV-SH株的BHK-21细胞,待出现80%以上的细胞病变时收获病毒,并以绿色荧光蛋白为标志,通过蚀斑法得到纯化重组病毒gE-/gI-/GFP+缺失株.研究了该缺失株的的安全性、在细胞上的生长特性以及对断奶仔猪的安全性、免疫原性等生物学特性.试验结果显示,缺失了gE-和gI-后,不影响其在RK细胞上的生长状况和病毒的滴度.该疫苗株对小鼠的半数致死量比亲本毒低且对家兔的致死性的时间延长了,这表明该疫苗的毒力比亲本毒有所下降.该缺失株对断奶仔猪安全,无不良接种反应,接种断奶仔猪能抵御高剂量PRV-SH株强毒的感染,攻毒后试验猪的发热期、散毒天数均低于对照组.该缺失株接种仔猪后在试验期间一直维持较高水平的中和抗体.     

伪狂犬病病毒上海株gE-/gI-/GFP+缺失株的生物学特性初步研究

在构建了伪狂犬病病毒上海株的缺失载体pgEI-GFP基础上,将pgEI-GFP转染感染了PRV-SH株的BHK-21细胞,待出现80%以上的细胞病变时收获病毒,并以绿色荧光蛋白为标志,通过蚀斑法得到纯化重组病毒gE-/gI-/GFP+缺失株.研究了该缺失株的的安全性、在细胞上的生长特性以及对断奶仔猪的安全性、免疫原性等生物学特性.试验结果显示,缺失了gE-和gI-后,不影响其在RK细胞上的生长状况和病毒的滴度.该疫苗株对小鼠的半数致死量比亲本毒低且对家兔的致死性的时间延长了,这表明该疫苗的毒力比亲本毒有所下降.该缺失株对断奶仔猪安全,无不良接种反应,接种断奶仔猪能抵御高剂量PRV-SH株强毒的感染,攻毒后试验猪的发热期、散毒天数均低于对照组.该缺失株接种仔猪后在试验期间一直维持较高水平的中和抗体.     

伪狂犬病病毒上海株（PRV—SH）gE^—／gI^—／GFP^＋缺失株的构建

在构建了含伪狂犬病病毒（Pseudorabies Virus,PRV）上海株gI基因和gE基因克隆鉴定的基础上,采用酶切的方法构建了载体pgEI。然后用限制性内切酶BamHI和BstPI缺失掉gE基因5‘端363bp,同时把绿色荧光蛋白（GFP）基因表达盒插入到缺失部分,并在下游 引入一个多克隆位点,构建了缺失转移载体pgEI-GFP。用DOTAP转染试剂盒将pgEI-GFP转染了感染PRV-SH的BHK-21细胞,待出现80％病变后收获病毒,并以蚀斑法得到纯化的缺失了gE/gI重组病毒株。小鼠试验证实了缺失株的毒力有所下降。     

伪狂犬病病毒TK-/gG-缺失株的构建及其生物学特性研究

将伪狂犬病病毒TK^-／gG^-／LacZ^ 突变株的基因组DNA与含有缺失的gG基因的转移质粒pUSKBB共转染猪肾传代细胞PK-15,待完全病变后收获病毒进行空斑试验,用PCR筛选gG缺失的重组病毒。空斑纯化3次后,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的TK^-／gG^-缺失株。遗传稳定性试验表明该重组病毒能在PK-15细胞上稳定遗传,动物试验表明该缺失株对Balb／c小鼠极为安全且能保护Balb／c小鼠抵抗致死量PRV强毒的攻击。该突变株的获得为我国伪狂犬病的控制和根除奠定了基础。     

表达猪繁殖与呼吸综合征病毒GP5蛋白重组伪狂犬病毒的构建及其生物学特性分析

将猪繁殖与呼吸综合征病毒（Porcine Reproductive and Respiratory Syndrome Virus,PRRSV）CH-1a株GP5基因插入到伪狂犬病病毒（Pseudorabies Virus,PRV）Bartha-K61株TK基因中,获得了一株TK^-／gE^-表型的重组伪狂犬病病毒,命名为rPRV—GP5。经生长动力学、表达动力学和间接免疫荧光证实PRRSV GP5在重组病毒中获得了表达,表达蛋白的抗原性与亲本病毒相似,rPRV-GP5在不同的细胞上毒价和细胞病变与Bartha—K61比较无显著差异。4只PRV抗体阴性的绵羊,每只接种10^6.0。PFU的rPRV-GP5可以完全抵抗10^3LD50伪狂犬病强毒S株的攻击。10头PRV、PRRSV抗体阴性的仔猪滴鼻接种rPRV-GP5 10^7.0 PFU／头并在接种后63d攻击PRRSV CH-1a株10^5.0 TCID50／头,攻毒后3d和5d出现了PRRSV荧光抗体和ELISA抗体,在政毒后14d检测到了PRRSV中和抗体,Bartha—K61活疫苗组和对照组至实验结束时仍未检测到PRRSV中和抗体。这说明rPRV-GP5免疫产生了针对PRRSV的回忆性免疫应答。     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白(GFP)的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota-EGFP,病毒F1代尿囊病毒液按1×104EID50接种9~10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为28、210.3、211.3和211,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×106EID50病毒量接种9~10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota-EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

伪狂犬病病毒鄂A株TK－／gG－／LacZ＋突变株的构建

为了以国内地方分离株鄂A株为亲本构建伪狂犬病病毒双基因缺失株,采用外切酶Ⅲ和绿豆核酸酶酶切,构建了缺失主要毒力基因TK基因部分编码区的重组质粒pSTK1-4,进一步改造成为转移质粒pUCPB4。用HindⅢ将质粒pUCPB4线性化,然后与用EcoRI消化的伪狂犬病病毒鄂A株TK^-/LacZ^ 突变株基因组DNA共转染PK－15细胞,等完全病变后,收毒作空斑试验,PCR筛选TK缺失的重组病毒。重组病毒空斑纯化3次,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的无TK^-/LacZ^ 突变株污染的TK缺失的重组病毒,分别以TK缺失株病毒与鄂A野毒株为模板对TK基因进行PCR扩增,扩增产物经酶切分析和测序后发现：TK缺失重组病毒的TK基因缺失了205个碱基,XhoⅠ和SalⅠ位点消失,SmaⅠ酶切片段发生变化,两种病毒在PK－15细胞上形成空斑的大小和增殖 滴度无明显的差别。进一步提取TK缺失突变株基因组DNA,与含gG-LacZ的转移质粒pUSKZ通过磷酸钙法共转染PK－15细胞,待完全病变后,在X-gal存在下筛选蓝斑,将桃取的蓝斑纯化3次后,对纯化的重组病毒进行TK、LacZ扩增,结果既能扩增出较以鄂A野毒株为模板扩增的要小的TK基因片段,同时又能扩增出LacZ基因,证实所得到的重组病毒为TK^-/gG^-/LacZ^ 突变株。此双缺失突变株的构建成功,为在我国最终根除伪狂犬病提供了有用的工具。     

表达绿色荧光蛋白重组新城疫病毒 LaSota疫苗株的构建

新城疫病毒是理想的新型活病毒疫苗载体,具有巨大的优势和应用前景。采用生产实践中广泛应用、免疫效果良好的NDV LaSota弱毒疫苗株,建立了反向遗传操作系统。在此基础上,进一步构建了表达绿色荧光蛋白（GFP）的重组NDV基因组cDNA克隆,成功救获了重组病毒rLaSota_EGFP,病毒F1代尿囊病毒液按1×10.4EID50接种9～10日龄SPF鸡胚尿囊腔,接种后分别于24h、48h、72h及96h收获尿囊液,检测平均HA滴度分别为2.8、2 10.3、2 11.3和2 11,每mL尿囊液病毒量EID50分别为108.64、109.22、109.21和109.64,重组病毒与亲本株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。各代次重组病毒按1×10.6EID50病毒量接种9～10日龄SPF鸡胚,96h内完全不致死鸡胚。救获重组病毒保持了LaSota弱毒疫苗亲本毒株对鸡胚良好的高滴度生长适应和低致病特性,并且鸡胚连续传9代次仍保持GFP的稳定表达及生物学特性不变。重组病毒rLaSota_EGFP的成功救获为开展新城疫病毒活载体疫苗研制提供了可行的技术平台。     

汉滩病毒诱导小鼠骨髓瘤SP2／0细胞凋亡的初步研究

为研究汉滩病毒对肿瘤细胞的诱导凋亡作用,以一定量病毒悬液感染体外培养的SP2／0细胞,接种后一定时间将细胞消化甩片行Gimsa染色观察凋亡细胞核的变化,制细胞悬液以流式细胞仪测细胞周期,并用免疫组化的方法检测凋亡分子Fas和FasL的表达。结果示经病毒诱导后细胞出现生长特性及形态学变化,Giemsa染色观察到典型凋亡细胞：流式细胞仪显示有凋亡峰出现;免疫组化检测出感染后SP2／0细胞中Fas和FasL表达明显升高。该结果表明汉滩病毒可诱导体外培养SP2／0细胞凋亡,其发生可能与凋亡分子Fas和FasL有关。     

表达ApxIA的血清7型胸膜肺炎放线杆菌弱毒菌株的构建及特性分析

猪传染性胸膜肺炎是由胸膜肺炎放线杆菌引起的一种高度接触传染疾病,严重阻碍着全球养猪业的发展,疫苗接种是控制该病的有效措施。为提高胸膜肺炎放线杆菌弱毒疫苗的免疫效力,以及探索胸膜肺炎放线杆菌弱毒疫苗作为呼吸系统病原疫苗载体的可行性,通过穿梭质粒pJFF224-XN将完整的apxIA基因导入apxIIC基因缺失突变株HB04C-中,构建了含有apxIA和apxIIA基因的弱毒疫苗菌株HB04C2(apxIIC-/apxIIA+/apxIA+)。通过对HB04C2的生物学特性分析发现,穿梭质粒可稳定传代,并表达ApxIA,其生长特性未受穿梭质粒的影响。将HB04C2以气管接种方式免疫仔猪,可产生针对ApxIA和ApxIIA的抗体。二免后2周以高致病性的血清1型胸膜肺炎放线杆菌攻毒,该弱毒疫苗可提供良好的免疫保护效果。     

Analysis of the two active sites of the hyaluronan synthase and the chondroitin synthase of Pasteurella multocida

Type A Pasteurella multocida produces a hyaluronan (HA) capsule to enhance infection. The 972-residue HA synthase, pmHAS, polymerizes the linear HA polysaccharide composed of alternating beta3N-acetylglucosamine (GlcNAc)-beta4glucuronic acid (GlcUA). We demonstrated previously that pmHAS possesses two independent glycosyltransferase sites. Here we further define the sites and putative motifs. Deletion of residues 1-117 does not affect HA polymerizing activity. The carboxyl-terminal boundary of the GlcUA-transferase resides within residues 686-703. Both transferase sites contain a DXD motif essential for HA synthase activity. D247N or D249N mutants possessed only GlcUA-transferase activity, whereas D527N or D529N mutants possessed only GlcNAc-transferase activity, further confirming our assignment of the two active sites within the synthase polypeptide. A potential role of the DXD motif in substrate binding was supported by experiments utilizing high UDP-sugar concentrations that partially rescued the activity of certain mutants. The WGGED sequence motif is involved in GlcNAc-transferase activity because mutants with substitutions at E369 or D370 possessed only GlcUA-transferase activity. Type F P. multocida synthesizes an unsulfated chondroitin (beta3GalNAc-beta4GlcUA) capsule. A chimeric enzyme consisting of residues 1-427 of pmHAS and residues 421-704 of pmCS, the homologous chondroitin synthase, was an active HA synthase. The converse chimeric enzyme consisting of residues 1-420 of pmCS and residues 428-703 of pmHAS was a functional chondroitin synthase. Analyses of a panel of pmHAS/pmCS chimeric enzymes identified a 44-residue region, corresponding to pmHAS residues 225-265, involved in UDP-hexosamine selectivity. Overall, these findings further support the model of two independent transferase sites within a single polypeptide.     

Quantification of glycosaminoglycans by reversed-phase HPLC separation of fluorescent isoindole derivatives

Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.     

Topology and mutational analysis of the single Emb arabinofuranosyltransferase of Corynebacterium glutamicum as a model of Emb proteins of Mycobacterium tuberculosis

The cell wall mycolyl-arabinogalactan (AG)--peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis, and is the target of several antitubercular drugs. For instance, ethambutol (EMB) targets AG biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB, as well as the single Emb from Corynebacterium glutamicum. Here, we present for the first time an experimental analysis of the membrane topology of Emb. The domain organization clearly positions highly conserved loop regions, like the recognized glycosyltransferase C motif and the hydrophilic C-terminus towards the periplasmic side of the cell. Moreover, the assignment and orientation of hydrophobic segments identified a loop region, which might dip into the membrane and could possibly line a transportation channel for the emerging substrate. Site-directed mutations introduced into plasmid-encoded Cg-emb were analyzed in a C. glutamicumDeltaemb strain for their AG glycosyl composition and linkage analysis. Mutations analyzed did not perturb galactan synthesis; however, D297A produced a dramatically reduced arabinan content and prevented growth, indicating an inactive Emb. A second D298A mutation also drastically reduced arabinan content; however, growth of the corresponding mutant was not altered, indicating a certain tolerance of this mutation in terms of Emb function. A W659L-P667A-Q674E triple mutation in the chain length regulation motif (Pro-motif) resulted in a reduced arabinose deposition in AG but retained all arabinofuranosyl linkages. Taken together, the data clearly define important residues of Emb involved in arabinan domain formation and, for the first time, shed new light on the topology of this important enzyme.     

Increased expression of protein C-mannosylation in the aortic vessels of diabetic Zucker rats

C-Mannosylation is a novel type of glycosylation in proteins. There are several examples of proteins in which the specific motif Trp-X-X-Trp is mannosylated at the first Trp to produce C-mannosylated Trp (CMW). Although C-mannosylation modifies Trp-X-X-Trp, predicted to be a functional motif of various integral proteins such as cytokine receptors, the physiological or pathological relevance of C-mannosylation in the cell is still not known. In this study, to characterize C-mannosylation in biological samples, we generated specific polyclonal antibodies against CMW by using a chemically synthesized CMW as an antigen. Using the antibody, we investigated the effect of hyperglycemic conditions on protein C-mannosylation in cultured cells and diabetic Zucker fatty rats. We found that protein C-mannosylation was increased in macrophage-like RAW264.7 cells under hyperglycemic conditions compared to low-glucose conditions. Furthermore, C-mannosylation was increased in the aortic vessel wall of Zucker fatty rats. Thrombospondin-1 was identified as a protein modified with C-mannosylation, and its expression was also increased in the aortic tissues of Zucker fatty rats. These results indicate that C-mannosylation is increased in specific tissues or cell types under hyperglycemic conditions, suggesting a pathological role for the increased C-mannosylation in the development of diabetic complications.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

Pathogenesis of primary defects in mitochondrial ATP synthesis

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

Alteration of neural tissue structure by expression of polysialic acid induced by viral delivery of PST polysialyltransferase

The expression of polysialic acid (PSA) on neural cell adhesion molecule (NCAM) is known to attenuate cell-cell interactions. During neural development the widespread expression of PSA-NCAM creates permissive conditions for the migration of neuronal and glial precursors and the guidance and targeting of axons. NCAM polysialylation can occur via either of two specific sialyltransferases, ST8SiaII (STX) and ST8SiaIV (PST), and the purpose of this study was to determine if retroviral delivery of either PST or STX could induce PSA expression in vivo and thereby alter tissue plasticity. Retroviruses expressing GFP-PST or GFP-STX were injected into embryonic retina, and development was evaluated by examining neuroepithelial structure, the expression of markers for specific cell types, cellular proliferation, and apoptosis. Chick retina was chosen because it down-regulates PSA early in its development and has a highly stereotyped program of morphogenesis. Retroviral expression of PST induced PSA expression in retina and resulted in severe but localized alterations in retinal morphogenesis, including an early disruption of radial glial cell morphology, highly disorganized retinal layers, and invasion of pigmented cells into the neural retina. In contrast, retroviral delivery of STX did not induce PSA expression or affect morphogenesis. These findings demonstrate that expression of PSA is sufficient to promote morphological alterations in a relatively nonplastic neural tissue.     

Effects of the sugar headgroup of a glycoglycerolipid on the phase behavior of phospholipid model membranes in the dry state

Glycolipids are important components of almost all biological membranes. They possess unique properties that have only been incompletely characterized so far. The plant glycolipid digalactosyldiacylglycerol (DGDG) strongly influences the physical behavior of phospholipid model membranes in both the dry and hydrated state. It was, however, unclear whether the strong effect of DGDG on the gel to liquid-crystalline phase transition temperature (Tm) in dry phosphatidylcholine (PC) bilayers is mainly due to the high degree of unsaturation of the DGDG fatty acyl chains or to interactions between the DGDG and PC headgroups. Also, no information on the relative effectiveness of membrane bound and free sugars on membrane phase behavior was available. We have used Fourier-transform infrared spectroscopy (FTIR) to investigate the phase properties and H-bonding patterns in dry membranes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) containing one saturated and one monounsaturated (16:0/18:1) fatty acid and different fractions of DGDG or 1,2-dilinolenoyl-sn-glycero-3-phosphatidylcholine (DLPC) (18:3/18:3). This was compared to the effects of galactose (Gal) and digalactose (diGal). All additives depressed Tm of the dry membranes, but DGDG was much more effective than DLPC or Gal. diGal had a similar effect as DGDG, pointing to the sugar headgroup as the component with the strongest influence on membrane phase behavior. A combination of DLPC and diGal, which should theoretically be equivalent to DGDG, was much more effective than the galactolipid. H-bonding interactions with the P = O group of PC were also stronger for free diGal than for DGDG, indicating that the free sugar may be structurally more flexible to adopt an optimal conformation for interactions with the PC headgroup.