牛疱疹病毒糖蛋白D的结构特点及其在疫苗研发中的应用

来自牛疱疹病毒1和5型(BoHV-1和BoHV-5)的病毒囊膜糖蛋白D(gD)是病毒的主要组成成分,并且在疱疹病毒的致病机理中起到关键作用。gD对于病毒侵入细胞是必不可少的,是病毒感染细胞期间中和抗体的主要靶标。鉴于其在诱导保护性免疫方面的作用,已使用该糖蛋白作为主要抗原开发出亚单位疫苗、DNA疫苗和载体疫苗候选物,证明gD具有在宿主中诱导较强病毒中和抗体和细胞介导的免疫应答以及对宿主进行免疫保护的能力。论文着重介绍了BoHV-1、BoHV-5和一些人类疱疹病毒gD的结构和功能特征,阐述了gD与宿主免疫系统的反应及gD在新型疫苗设计中的应用,为全面了解gD结构特点及其在疫苗研发中的作用提供借鉴。     

Hydroxyproline-rich glycoprotein accumulation in tobacco leaves protected against Erysiphe cichoracearum by potato virus Y infection

Tobacco cv. Havana 425 acquired resistance to a compatible isolate of Erysiphe cichoracearum after infection by a strain of potato virus Y (PVY^N) that causes veinal necrosis; another common strain (PVY^O) that does not cause necrosis gave less protection. Hydroxyproline-rich glycoproteins (HRGPs), believed to be involved in resistance, were determined by analysing hydroxyproline (Hyp) in purified cell walls. Hyp content increased significantly in PVY^N-protected leaves, compared with untreated controls, 2–4 days after necrotic lesion symptoms developed. No further increase in Hyp was noted in PVY^N-protected leaves after E. cichoracearum challenge. Hyp increases were significantly higher in protected leaves of plants showing symptoms on day 7 than on day 10. Infection with the PVY^O strain caused significant decrease in Hyp content, compared with uninoculated controls. Inoculation of virus-free plants with E. cichoracearum induced moderate and transitory Hyp increases on day 2 or 3, followed by a quick decrease associated with a weak response by the compatible host. It is suggested that HRGP accumulation induced by PVY^N (but not by PVY^O) causes changes in the host cell wall that result in resistance to E. cichoracearum .     

鱿鱼缠卵腺糖蛋白MGS降血脂及非特异性免疫功能研究

为研究鱿鱼缠卵腺糖蛋白MGS的降血脂及免疫活性，本文先根据体外测定胰脂肪酶活力抑制率的结果初步判定MGS的降血脂效果，而后通过建立高血脂模型小鼠进一步研究MGS的降血脂活性，同时采用动物实验研究MGS的免疫活性。结果表明， MGS具有抑制胰脂肪酶活性的作用；能够显著降低小鼠的TC和LDL-C的含量，显著增加HDL-C的含量( P<0．05)，不能显著降低TG的含量，在降低血脂的同时，也可在一定程度上控制高脂小鼠体重的增加；实验组小鼠的廓清指数和吞噬指数与对照组的在统计学上无显著性差异(P>0．05)。     

狂犬病病毒糖蛋白基因中和抗原表位在大肠杆菌中的串联表达

采用RT-PCR方法从狂犬病病毒HEP株中克隆狂犬病糖蛋白膜外区全长基因。利用突变PCR方法,将糖蛋白基因中三段中和抗原位点串联,克隆至pET-28a表达载体。阳性重组质粒转化大肠杆菌BL21(DE3)后,用IPTG诱导表达并确定最佳诱导条件。SDS-PAGE电泳结果显示所表达的蛋白大小与预期相符。经Western-blotting检测,串联表达的狂犬病糖蛋白基因中和抗原位点可与狂犬病标准阳性血清发生特异性反应,表明重组蛋白具有良好的反应原性。     

猪伪狂犬病毒囊膜糖蛋白gB主要抗原表位区基因的克隆及原核表达

参照GenBank发表的猪伪狂犬病毒囊膜糖蛋白gB主要抗原表位的编码区基因序列,设计一对引物,通过PCR扩增后,将约为600bp的目的片段克隆到pGEM-T载体上,酶切后插入原核表达载体pET-32(a)的T7启动子下游,构建的重组质粒pET-gB经IPTG诱导,在大肠杆菌BL21(DE3)中获得了高效表达。SDS-PAGE结果显示,表达产物分子量约为42.4KDa,主要以包涵体形式存在。BandScan分析表明,表达量约占菌体蛋白的60.5%。利用His亲和层析方法得到了纯化的表达产物。Western blotting结果显示,重组蛋白能与阳性血清发生特异性反应,具有较好的抗原反应原性,可以作为检测用抗原。     

Serum Acute Phase Protein Concentrations in Dogs with Autoimmune Hemolytic Anemia

Background: Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported.
Hypotheses: Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and α1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations.
Animals: Twenty-seven dogs with AIHA and 11 control dogs.
Methods: Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365.
Results: Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1–7; P < .002) and normalized with disease stabilization (days 9–365; P > .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions ( P = .153–.940). PCV correlated with APP concentrations over time (CRP r =−.600, AAG r =−.665, albumin r = .533; P < .0001) as did WBC count (CRP r = .253, AAG r = .486, albumin r =−.246; P < .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 μg/mL) compared with dogs that did not (191.2 μg/mL; P = .02).
Conclusions: An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.     

Correlation of five radioimmunoassay systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancy period

The measurement of serum or plasma PAG concentrations is currently used as a specific method for pregnancy diagnosis in cattle. In this study, the correlation between five radioimmunoassay systems (RIA-497, RIA-706, RIA-780, RIA-809 and RIA–Pool) developed for measurement of PAG concentrations in ruminant species was investigated in plasma from pregnant Friesian Holstein females. Plasma PAG concentrations (ng/mL) measured by different RIA systems were significantly correlated between them ( 0.81; P < 0.001). PAG concentrations increased significantly from Day 21 (n = 27) to 30 (n = 37) after AI by use of all PAG–RIA systems. From Day 30 to 80 after AI, lower PAG concentrations were observed when using the homologous system RIA-497. The addition of several proteinase inhibitors changed neither the non specific binding nor the B₀ binding to the tracer. Our results suggest that all tested PAG–RIA (RIA-497, RIA-706, RIA-780, RIA-809 and RIA–Pool) are highly correlated and can be useful to follow PAG concentrations in samples collected during the first trimester of gestation.     

不同转染介质对狂犬病病毒糖蛋白基因免疫的效应

将狂犬病病毒糖蛋白 ( RGP) c DNA Bgl 片段 ( 1 .6 7kb)分别克隆进质粒 p GFP-Cl、p SV2 -dhfr和pc DNA3 ,构建了重组质粒 p GFP-C1 -RGP、p SV2 -RGP和 pc DNA3 -RGP,通过脂质体和聚乙烯亚胺 ( PEI)包裹后分别转染 BHK-2 1细胞和乳鼠脑内接种 ,3种重组质粒均能表达出 RGP。表达水平高低依次为 p GFP-C1 -RGP>pc DNA3 -RGP>p SV2 -RGP。 3种重组质粒分别以全裸质粒、质粒 -脂质体、质粒 -PEI形式 ,经骨骼肌免疫小鼠 ,间接 ELISA检测。结果显示 ,免疫鼠血清中特异性抗体水平明显高于对照组 ;80 %免疫小鼠能抵抗狂犬病病毒强毒的攻击。全裸质粒免疫诱生的抗体水平同其剂量相关 ;而以 PEI或脂质体作为转染介质、质粒接种量超过 2 0μg时 ,诱生抗体水平不再随质粒剂量增大而显著变化。质粒免疫后 1 50 d采用 PCR仍然能从注射部位检测到 RGP基因的存在     

Development of a Latex Agglutination Test Using The Major Epitope Domain of Glycoprotein E of Pseudorabies Virus Expressed in <Emphasis Type="Italic">E. coli</Emphasis> to Differentiate Between Immune Responses in Pigs Naturally Infected or Vaccinated with Pseudorabies Virus

A 0.8 kb DNA fragment encoding the major epitope domain of glycoprotein E (gE) of pseudorabies virus (PRV) was inserted downstream of the T7 promoter of an expression vector, pET-28b, to yield the recombinant plasmid pETgE804. After induction by isopropy1-β-D-thiogalactopyranoside (IPTG), a high level expression of fusion protein was obtained. SDS-PAGE and western immunoblotting analysis showed that the fusion protein was 38 kDa and could bind with antisera against PRV. The protein existed mainly in the form of the inclusion body. After being denatured and renatured, the protein was used to prepare the latex antigen. The concentration of antigen, temperature and time for sensitization were optimized. The latex agglutination test (LAT) was able to differentiate sera of PRV-infected pigs from those of gE-deletion vaccine-immunized pigs. The diagnostic specificity and sensitivity of the developed gE latex agglutination test (gE-LAT) were also evaluated by using sets of sera. The diagnostic specificity and diagnostic sensitivity of the gE-LAT were 96.77% and 95.76%, respectively. For comparison between gE-LAT and a commercial blocking enzyme-linked immunosorbent assays (ELISA), 260 serum samples were tested. The coincidence frequency of both assays was 96.94% (252/260). No significant difference was found between the two methods (p>0.05). For comparison between the abilities of gE-LAT and gE-ELISA to detect sera with low titres of gE-specific antibody, 66 sera from 22 pigs were tested. The data indicate that the gE-LAT is of similar sensitivity to gE-ELISA. These results indicate that gE-LAT using recombinant gE might be very useful as a routine screening method for the differential diagnosis of PRV infection.