人类细小病毒B19壳蛋白VP2在昆虫杆状病毒表达系统中的表达

利用BAC-TO-BAC系统获得了人类细小病毒B19壳蛋白VP2的重组昆虫杆状病毒,并在sf9细胞中表达出VP2。用蚀斑法纯化病毒,终末稀释法测定病毒滴度为3.6×108。Western印迹检测证实了表达蛋白的特异性,间接免疫荧光法可观察到细胞胞浆中的表达蛋白颗粒。     

展示生长抑素的猪细小病毒样颗粒构建及其免疫原性

为了获得既可预防猪细小病毒感染又能促进生长的嵌合病毒样颗粒疫苗,以PPV NJ-a株基因组DNA为模板扩增VP2基因片段,在VP2基因N端融合人工合成的4拷贝生长抑素基因,构建杆状病毒转移载体pFast-SS4-VP2。通过转化DH10Bac感受态细胞,pFast-SS4-VP2与穿梭载体Bacmid重组,获得重组Bacmid,命名为rBacmid-SS4-VP2。rBacmid-SS4-VP2转染Sf-9细胞,获得重组病毒rBac-SS4-VP2。SDS-PAGE与Western blotting鉴定可见约68 kDa的rSS4-VP2条带;rBac-SS4-VP2感染细胞IFA检测产生很强的特异性绿色荧光;感染细胞超薄切片电镜观察到大量特征性病毒样颗粒。将重组蛋白分别辅以铝胶、IMS和白油不同佐剂免疫小鼠,通过检测免疫小鼠VP2特异性ELISA抗体、PPV特异性中和抗体、生长抑素的抗体水平及生长激素水平来评价嵌合病毒样颗粒的免疫原性。结果表明,辅以铝胶与IMS佐剂重组蛋白组均产生了与PPV全毒组相似的ELISA抗体与中和抗体反应;重组蛋白免疫组均产生较好的针对生长抑素的抗体反应;免疫小鼠体内生长激素的水平明显升高;其中以铝胶佐剂组产生的各抗体水平最高,白油佐剂组各抗体水平最低。为以后生产安全、有效的颗粒化亚单位疫苗提供了一个新的设计思路,又为应用病毒样颗粒递呈外源肽,从而生产多联亚单位疫苗奠定了基础。     

抗细小病毒B19-VP2单克隆抗体的建立

制备抗细小病毒B19－VP2单克隆抗体,用于检测人血清中的B19抗原,辅助诊断相关疾病;也可用于制备人类细小病毒基因工程疫苗。用纯化的基因工程表达的B19－VP2蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞和小鼠骨髓瘤Sp2/0细胞融合,有限稀释法克隆细胞。ELISA及IF证明抗体特异性。克隆筛选出4株细胞,并初步建立了检测B19－VP2抗原的双抗体夹心酶联免疫吸附试验,为双抗体夹心法检测B19抗原为临床相关疾病诊断提供了检测手段。     

人细小病毒B19基因变异的研究进展

人细小病毒Ｂ１９简称Ｂ１９病毒,是细小病毒中惟一能感染人类的病毒,也是动物病毒中对人类具有致病性的最小单链线状ＤＮＡ病毒,其与儿童及成人的多种疾病密切相关。Ｂ１９病毒的基因变异对其致病、传播等具有重要影响,且与Ｂ１９病毒诊断试剂,疫苗的制备等关系密切。本文就Ｂ１９病毒基因组的变异状况,基因变异研究方法及研究Ｂ１９病毒基因变异的意义作一概述。     

人细小病毒B19与自然流产关系的研究

无菌留取 5 4例自然流产妇女和 43例妊娠无异常孕妇血清 ,用聚合酶链反应 (PolymeraseChainReaction ,PCR)检测的人细小病毒B19(HumanParvovirusB19,B19)DNA ,在自然流产组中人细小病毒B19DNA有 15例阳性 ,阳性率为 2 7.78%。正常对照组中 ,人细小病毒B19DNA有 2例为阳性 ,阳性率为 4.65 % ,用x2 检验 ,x2 =8.86,P <0 .0 1,两组有非常显著性差异。由此总结 ,人细小病毒B19感染可能是导致自然流产的原因之一     

细小病毒B19诊断芯片的初步研究

初步探讨并制备细小病毒B19诊断芯片,进行实验室验证．用基因芯片点样仪将细小病毒B19诊断探针固定在特殊处理的玻片上,以细小病毒B19质粒重复检测．运用限制性显示(RD)技术,用Cy5标记的通用引物进行荧光标记,通过与基因芯片杂交,严谨洗涤,将非特异性的标记片段洗脱后,经扫描仪扫描,计算机解读．杂交结果显示,Cy5标记的探针均出现杂交信号,而阴性对照和空白对照的杂交信号均很弱：芯片检测具有高特异性、敏感性和可重复性．初步建立了较可靠的制备与检测细小病毒B19诊断芯片的方法,经验证诊断准确率高,假阳性率低．     

细小病毒B19壳抗原VP2在大肠杆菌中的表达及血清学检测

为了进行B19感染临床的血清学诊断,利用原核表达载体PQE31克隆和表达B19壳蛋白VP2,酶切鉴定PCR产物及PQE31-VP2克隆的正确性,Western-blot证明表达蛋白的特异性,并对其表达条件和纯化条件进行了优选。在D600为0.7,诱导时间为5h时表达量最高。Ni2+亲和色谱,用0.5mol/L咪唑洗脱液洗脱,获得纯化蛋白。利用纯化蛋白检测100份人群血清,免疫斑点法结果为阳性94例,阴性6例;ELISA结果为阳性84例,阴性16例,两种方法结果一致(0.25>P>0.1)。     

新型鸭细小病毒样颗粒的制备及鉴定

【背景】鸭短喙侏儒综合征(beak atrophy and dwarfism syndrome, BADS)是由新型鸭细小病毒(novel duck Parvovirus, NDPV)感染导致雏鸭生长发育迟缓、上下喙萎缩的疾病。BADS的暴发给我国养鸭业造成了巨大的经济损失。【目的】利用大肠杆菌表达系统制备NDPV病毒样颗粒(virus-like particles, VLPs),为研制NDPV相关疫苗奠定基础。【方法】对NDPV VP2序列全长进行密码子优化、合成,连接至pColdTF表达载体,获得pColdTF-NDPV-VP2重组质粒,酶切、测序鉴定正确后将重组质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)对蛋白表达进行可溶性分析;使用凝血酶(thrombin)切除trigger factor (TF)标签,利用镍柱(Ni-NTA)亲和层析方法纯化重组蛋白;利用Western blotting对纯化后的VP2蛋白进行反应原性分析;利用透射电镜、动态光散射观察重组蛋白形态以及能否形成VLPs。【结果】构建了pColdTF-NDPV-VP2重组质粒,在大肠杆菌中主要以可溶性形式表达,融合蛋白TF-VP2大小约为115 kDa,去除TF标签经镍柱纯化后得到67 kDa的VP2蛋白;Western blotting试验表明VP2蛋白能与NDPV鸭阳性血清发生特异性结合;通过透射电镜可以观察到形状规则、直径约为20−25 nm的病毒样颗粒。【结论】利用大肠杆菌表达系统制备了NDPV VLPs,为下一步研发BADS相关亚单位疫苗及生物相关制品提供了基础。     

细小病毒B19感染实验室诊断技术的研究进展

人类细小病毒感染,临床可有多种表现,尤其可导致胎儿早产、流产、死胎、贫血、发育迟缓及形成积水型胎儿。本文拟从电镜检查、ＥＬＩＳＡ、固相放射免疫技术（ＲＩＡ）、分子杂交及多聚酶链反应（ＰＣＲ）等几个方面对该病毒感染实验室诊断的国外最新研究进展作一介绍。     

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.     

人细小病毒B19 VP1u多克隆抗体的制备及其保守区外N端氨基酸对sPLA2活性影响的初步研究

【目的】制备人细小病毒B19-VP1u的多克隆抗体,探究VP1u多克隆抗体及其保守区外N端氨基酸对病毒磷脂酶A2活性的影响。【方法】首先通过分子克隆方法构建相应原核表达载体;利用原核表达系统纯化含MBP标签的VP1u全长及N端系列截短突变融合蛋白;接着免疫新西兰大白兔制备全长VP1u蛋白的多克隆抗体;最后利用磷脂酶A2活性检测试剂盒检测了纯化蛋白的磷脂酶A2活性。【结果】Western blot及免疫荧光实验证实制备的多克隆抗体具有较高的特异性;磷脂酶A2活性检测发现全长VP1u-MBP融合蛋白具有一定的活性,该活性可以被VP1u的抗体抑制;N端保守区外截短系列蛋白的酶活检测发现,N端截掉12个氨基酸时酶活降低53%,截掉67个氨基酸时酶活性几乎完全丧失。【结论】首次发现VP1u保守区外N端氨基酸,尤其是第12个氨基酸前的区域以及第22-67个氨基酸之间的区域,对sPLA2活性的保持具有重要意义,推测该区域可能对维持正常的蛋白构象起重要的作用;而其特异性多克隆抗体的制备也为进一步研究B19病毒VP1u在病毒复制周期的作用奠定基础。     

细小病毒B19 Oligo探针设计

利用BLAST软件对细小病毒B19的序列进行序列比对,获得特异序列;利用生物学软件Oligo6.40设计特异性高、Tm值接近、长度均一的Oligo探针。结果获得了13条70bp的Oligo探针,用于芯片打印及细小病毒B19的检测。表明利用BLAST系统和生物学软件Oligo6.40设计细小病毒B19诊断芯片的探针是一种简便而有效的方法。     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

Clinical features and laboratory findings of human parvovirus B19 in human immunodeficiency virus-infected patients

Immunocompromised patients may develop severe chronic anaemia when infected by human parvovirus B19 (B19V). However, this is not the case in human immunodeficiency virus (HIV)-infected patients with good adherence to highly active antiretroviral treatment (HAART). In this study, we investigated the clinical evolution of five HIV-infected patients receiving HAART who had B19V infections confirmed by serum polymerase chain reaction. Four of the patients were infected with genotype 1a strains and the remaining patient was infected with a genotype 3b strain. Anaemia was detected in three of the patients, but all patients recovered without requiring immunoglobulin and/or blood transfusions. In all cases, the attending physicians did not suspect the B19V infections. There was no apparent relationship between the infecting genotype and the clinical course. In the HAART era, B19V infections in HIV-positive patients may be limited, subtle or unapparent.     

Enhanced kinetic extraction of parvovirus B19 structural proteins

Recombinant structural proteins (VP1 and VP2) of the human parvovirus B19 have been expressed simultaneously using the baculovirus expression system to form virus-like particles (VLPs) that have potential use as vaccines. In this study, we report optimization of extraction conditions to recover these VLPs from cell paste. Under hypotonic conditions with neutral pH these VLPs were poorly extracted (up to 3% extraction). Addition of reducing agents, detergents, salts, and sonication did not improve the extractability. While screening for conditions to improve the extractability of the VLPs, we discovered that a combination of higher pH and elevated processing temperature significantly increased the extraction. Whereas increasing pH alone increased extractability from 3% to 6% (pH increased from 8.0 to 9.5), the effect of elevated temperature was much more substantial. At 50 degrees C, we observed the extraction to be more than fivefold higher than that at room temperature (up to 25% extracted at pH 9.0). The kinetics of extraction at elevated temperatures showed a rapid initial rate of extraction (on the order of minutes) followed by a plateau. In addition, we compared the extraction of VP1 expressed alone. VP1 expressed alone is incapable of forming VLPs. We observed that non-VLP VP1 was easily extractable (up to 60% extracted) under conditions in which the VP1 + VP2 VLPs were not extractable. From these studies we conclude that parvovirus B19 structural proteins expressed to form VLPs have a hindered extractability as compared with non-VLP protein. This hindrance to extraction can be significantly reduced by processing at elevated temperatures and an increased pH, possibly due to the enhanced rates of solubilization and diffusion.     

Production of porcine parvovirus virus-like particles using silkworm larvae

Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.