Immunogenic and antigenic properties of recombinant soluble glycoprotein of rabies virus

Rabies virus glycoprotein is a type I transmembrane protein exposed on the surface on the mature virus particle that induces virus neutralizing antibodies. In the present study, 60 amino acid C-terminal hydrophobic anchor (transmembrane) and cytoplasmic domains of glycoprotein were deleted from full-length glycoprotein and fused with polyhistidine tag. The N-terminal viral signal peptide was also replaced with CD33 signal peptide for efficient secretion in mammalian cells. Following transfection of Madin Darby bovine kidney (MDBK) cells with plasmid encoding this soluble form of glycoprotein, polyclonal populations of stably transfected resistant cells were obtained after G418 selection. The protein was expressed as a glycosylated protein and secreted outside the cells utilizing N-terminal CD33 signal peptide. The secreted soluble glycoprotein was purified from cell culture supernatant by Ni--agarose affinity chromatography utilizing C-terminal polyhistidine tag. Like full-length glycoprotein, the expressed recombinant soluble glycoprotein was found to be immunogenic when injected in rabbits. In this study, we have assessed the potential of recombinant soluble glycoprotein as diagnostic antigen in ELISA and found that this recombinant protein can be used as diagnostic antigen in ELISA for detecting anti-glycoprotein antibodies in immunized host.     

狂犬病病毒糖蛋白在Bac-to-bac杆状病毒系统中的表达及应用

为表达狂犬病病毒(RV)糖蛋白(G),本研究通过RT-PCR方法克隆RV Flury LEP病毒株G基因,将其克隆至质粒pFastBacHTα中,重组质粒pFastBac-RV-G转化DH10Bac感受态细胞,经同源重组获得重组杆状病毒穿梭质粒Bacmid-RV-G,将其转染昆虫细胞sf21获得含有G基因的重组杆状病毒.采用SDS-PAGE和western blot对重组蛋白进行鉴定及抗原性分析,以重组G蛋白作为包被抗原的ELISA检测36份犬血清样品.结果表明,在Bac-to-bac杆状病毒系统中表达的RV G蛋白能与his-tag单克隆抗体及RV阳性血清发生特异性反应,其相对分子量为60 ku;与以RV为包被抗原的商品化ELISA试剂盒相比,以重组RV G蛋白为抗原建立的间接ELISA的敏感性、特异性和符合率分别为80%、81.8%和80.6%,表明杆状病毒系统表达的RV G蛋白是检测RV抗体的理想抗原蛋白,作为一种亚单位疫苗具有潜在的应用前景.     

狂犬病病毒ERA株糖蛋白基因的克隆与序列分析

根据GenBank中已发表的狂犬病病毒G基因序列,设计合成了1对特异性引物,对RV ERA株G基因进行了RT-PCR扩增。将PCR产物纯化后与pMD18-T连接得到重组质粒pMD-G,并进行核苷酸序列测定。结果该基因全长1 575bp,编码524个氨基酸。与GenBank中已发表的其他RV标准毒株(CVS,PV,SRV9,SAD-B19)相比,G基因核苷酸的同源性为89.1%～99.1%,推导的氨基酸序列的同源性为89.7%～99.3%。对推导的RV ERA株G蛋白氨基酸序列分析表明,该蛋白含有3个潜在的N-联糖基化位点。用DNAStar软件对RV ERA株与CVS、SRV9、PV、SAD-B19等G蛋白疏水性、Jame-son-Wolf抗原表位和表面极性分析表明,它们之间非常相似。     

表达狂犬病病毒糖蛋白的复制缺陷型重组腺病毒的构建

利用大肠杆菌内质粒间同源重组的方法,将狂犬病病毒G基因插入腺病毒基因组的E1基因区,构建了带有狂犬病病毒G基因的重组腺病毒质粒。重组质粒经Pme酶切,线性化后转染293细胞,成功获得了均一的复制缺陷型重组腺病毒。荧光显微镜下能观察到重组腺病毒GFP报告基因的表达。用PCR方法证实了重组腺病毒基因组中含有G基因,RT-PCR方法可检测到G基因的转录产物mRNA,Westernblotting方法能检测到重组腺病毒在29细胞中表达的G蛋白。此重组腺病毒在293细胞连续传代10次,PCR方法都能扩增出G基因目的条带。试验结果表明,狂犬病病毒G基因已成功重组到腺病毒基因组中,不但能稳定表达,而且能在重组腺病毒基因组中稳定存在。     

Gene immunization of mice with plasmid DNA expressing rabies virus glycoprotein

Gene immunization can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses. We constructed plasmid vectors expressing the full-length Vnukovo-32 rabies virus glycoprotein G under the control of CMV IE promoter and enhancer, adenovirus tripartite leader sequences and poly A signal of SV40. The gene vaccines were evaluated for the ability to elicit neutralizing antibodies and to protect BALB/c mice against lethal rabies virus challenge. First, mice were injected intramuscularly (i.m.) into the left hind leg and by the intradermoplantar (i.d.p.) route with equal amounts of plasmid DNA (0.25-0.1 mg). Two weeks later, immunization was boosted with an additional dose of the DNA. The immunized mice were challenged by intracerebral (i.c.) inoculation of CVS-27 (10-50 LD50) rabies virus. All mice produced anti-rabies virus neutralizing antibodies with a titre of > or = 1:45 after immunization with 0.1-0.4 mg of DNA. In challenge experiments, 83 to 91.6% protection was observed. These results confirm that a DNA vaccine could be a simple and effective solution for preventing the spread of rabies.     

云南狂犬病监测及病毒部分N基因序列比较

狂犬病是由狂犬病毒感染引起的一种严重侵害中枢神经系统的致死性人畜共患传染病,呈世界性分布,我国属于疫病高发区.基于病毒N基因结构和变异特征,将狂犬病毒分为2个进化组群、7个基因型[1].国内外已研究建立了一批狂犬病诊断方法[2-4],开展病毒基因结构特征及分子流行病学研究[5-7].至今未见对近年云南狂犬病研究报道.本文在云南省4个地区开展狂犬病病原学监测,并对病毒N基因进行克隆测序,以期认识狂犬病分布状况、病毒特征及与已知毒株的遗传相关性.     

Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein.

Striped skunks (Mephitis mephitis) were vaccinated with a vaccinia virus recombinant expressing the rabies virus glycoprotein. Virus neutralizing antibodies to rabies virus were present at 14 days postvaccination by the following routes: scarification (6/6), intramuscular (4/4) and intestinal (5/8). Six out of seven skunks that ate vaccine filled baits had virus neutralizing antibodies at 28 days. When challenged intramuscularly with street virus, the survival rates were 5/7 for the bait-fed group, 4/8 for the intestinal group, 3/4 for the intramuscular group, 5/6 for the animals that were scarified, and 0/8 for controls. This is the first report of a high rate of immunization of skunks with a rabies vaccine administered orally.     

狂犬病毒分离株糖蛋白基因的克隆与序列分析

为分析狂犬病毒(RV)分离株糖蛋白基因之间的差异,本研究采用RT-PCR的方法扩增由广东某地临床表现正常犬的唾液中分离的RV(GD33)分离株的G基因,将其克隆、测序后与国内外部分毒株相应基因进行核苷酸序列比较,结果表明,该分离株与其他毒株相比核苷酸同源性为94%～97.1%;氨基酸同源性为97%～99.2%。而GD33分离株与HEP-Flury株在跨膜区、膜内区有很高的一致性;另外,GD33分离株与HEP-Flury和FluryLEP株在333位点出现精氨酸被取代的现象,证实GD33分离株这2个疫苗株没有显著差异,对监控我国狂犬病疫情具有一定的意义。     

Pyrosequencing of the rabies virus glycoprotein gene to demonstrate absence of vaccine-associated rabies cases following oral vaccination

Replication competent vaccines have been used successfully for the control of terrestrial rabies, mainly in wildlife; however, these vaccine strains occasionally may induce rabies. In this study, a pyrosequencing protocol for the rapid identification of vaccine-associated rabies viruses was applied to the 2008–2011 Italian epidemic. There was no evidence of vaccine-associated rabies cases following oral vaccination of foxes with the SAG2 and SADB19 vaccine strains.     

狂犬病病毒糖蛋白膜外区基因在大肠埃希氏菌中的高效表达

为获得狂犬病病毒（RV）糖蛋白抗原，采用RT-PCR方法从狂犬病病毒ERA株中扩增了编码RV糖蛋白的全长基因，将其克隆于pMD18-T载体，再经PCR扩增出糖蛋白全长基因和膜外区基因，将其分别亚克隆至原核表达载体pET-28a（＋）、pET-32a（＋）和pGEX-4T-1中，经PCR和双酶切鉴定以及序列分析，表明已成功构建了重组质粒。将重组质粒转化到大肠埃希氏菌BL21（DE3）中进行表达，结果显示，克隆到pET-32a（＋）的糖蛋白膜外区基因表达量最高，目的蛋白表达量占菌体总蛋白的45．4％。经Western-blotting检测，不同载体表达的糖蛋白膜外区产物均可与兔抗RV多抗发生特异性反应，表明，重组蛋白具有良好的反应原性。     

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。     

Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.     

Genetic and phylogenetic analysis of glycoprotein of rabies virus isolated from several species in Brazil

Genetic and phylogenetic analyses of the region containing the glycoprotein (G) gene, which is related to pathogenicity and antigenicity, and the G-L intergenic region were carried out in 14 Brazilian rabies virus isolates. The isolates were classified as dog-related rabies virus (DRRV) or vampire bat-related rabies virus (VRRV), by nucleoprotein (N) analysis. The nucleotide and amino acid (AA) homologies of the area containing the G protein gene and G-L intergenic region were generally lower than those of the ectodomain. In both regions, nucleotide and deduced AA homologies were lower among VRRVs than among DRRVs. There were AA differences between DRRV and VRRV at 3 antigenic sites and epitopes (IIa, WB+ and III), suggesting that DRRV and VRRV can be distinguished by differences of antigenicity. In a comparison of phylogenetic trees between the ectodomain and the area containing the G protein gene and G-L intergenic region, the branching patterns of the chiropteran and carnivoran rabies virus groups differed, whereas there were clear similarities in patterns within the DRRV and VRRV groups. Additionally, the VRRV isolates were more closely related to chiropteran strains isolated from Latin America than to Brazilian DRRV. These results indicate that Brazilian rabies virus isolates can be classified as DRRV or VRRV by analysis of the G gene and the G-L intergenic region, as well as by N gene analysis.     

狂犬病病毒糖蛋白基因正向重组伪狂犬病病毒（疫苗株TK^-／gI^-）的构建

构建以伪狂犬病病毒(PRV)为载体表达狂犬病病毒糖蛋白的重组活载体疫苗。采用AseⅠ/MluⅠ切下EG-FP-rgp的表达盒,将其克隆入p8-AA载体的酶切位点处,经酶切鉴定为正向连接,阳性重组子命名为p8-EGFP/rgp。将该质粒与PRV TK-/gI-基因组在质脂体介导下共转染PK-15细胞,获得PRV-EGFP/rgp重组病毒;通过噬斑克隆对其进行筛选、纯化,并通过RT-PCR、Southern-blot、Western-blot和间接免疫荧光对其进行鉴定,并对重组病毒的滴度、外源基因的遗传稳定性及其与亲本病毒株生长特性的关系进行了测定。结果表明:重组病毒的滴度(TCID50)为108.125/mL;外源基因在细胞内得到了有效的表达,并具有良好的免疫反应性和遗传稳定性;PRV-EGFP/rgp与亲本病毒PRV TK-/gI-生长趋势差异不显著。通过构建表达狂犬病病毒糖蛋白正向重组PRV的活载体疫苗的研制,为狂犬病疫苗的研制提供了新的思路和方法。     

Effect of inhibitors of cytoplasmic structures and functions on rabies virus infection in vitro

The effect in vitro of some cytoplasmic structure and function inhibitors on the different stages of rabies virus infection was investigated. Treatment of fibroblasts (CER) and human neuroblastoma cells (IMR-32) with substances acting on low pH intracellular compartments (methylamine and monensin) prevented rabies virus genome delivery in the cytosol. An early inhibition of viral infection was also obtained in the presence of B and D cytochalasins and trifluoperazine which interact with microfilament structures. Treatment with colchicine and vinblastine did not affect rabies multiplication, suggesting that microtubules are not involved in this process. However, the multiplication of prebound virions did not take place in the presence of inhibitors of oxidative phosphorylation (sodium azide and CCCP) and of glycolysis (2-deoxy-D-glucose) indicating that rabies virus replication is largely energy-dependent in both host cells examined.     

狂犬病毒糖蛋白单、双拷贝基因重组腺病毒的构建

将携带狂犬病毒糖蛋白单、双拷贝基因的穿梭栽体pShuttle-G和pShuttle-dG分别经PmeⅠ线性化后电转化含E1和E3区缺失的人5型腺病毒骨架质粒pAdEasy-1的感受态菌株BJ5183-AD-1进行细菌内同源重组,经抗性筛选,PcR、PacⅠ酶切及测序鉴定.构建了狂犬病毒糖蛋白单、双拷贝基因重组腺病毒质粒pAdHu5-G和pAdHu5-dG,线性化后分别转染Ad-293细胞进行病毒包装.结果显示,在第4天发生细胞病变效应,且荧光显微镜下观察到绿色荧光蛋白的表达;western-blot证实2株重组腺病毒表达的糖蛋白能被特异性单抗识别;遗传稳定性分析显示,病毒传至10代时单G和双G基因均稳定遗传,没有发生缺失和突变.为比较分析2株重组腺病毒糖蛋白的表达时效及免疫效果奠定了基础.     

云南牟定狂犬病病毒株N基因和G基因序列分析

自英国引进3株荧光素标记的针对狂犬病毒核蛋白的单克隆抗体,建立狂犬病直接免疫荧光诊断技术。根据已知狂犬病毒核蛋白和糖蛋白基因序列,参照已发表文献,设计合成4对PCR引物和2对克隆引物,以弱毒疫苗毒株为阳性对照,建立狂犬病RT-PCR及套式PCR诊断技术。自牟定采集发病犬脑组织样品15份,免疫荧光及PCR诊断结果均为阳性。对病毒N基因和G基因全序列经RT-PCR扩增,克隆至pMD18-T载体进行测序（登录号分别为：EU095330、EU253477）,并与已知代表毒株对应序列进行比对及系统发育分析,结果表明：云南牟定狂犬病毒属于基因Ⅰ型和血清Ⅰ型毒株,与近年从广西、浙江、江苏、湖北等省分离的毒株遗传关系密切,在系统发育树中形成同一小分支并均属于亚组群Ⅱ毒株。     

两株狂犬病病毒强毒株糖蛋白基因的克隆及潜在抗原表位分析

通过RT-PCR分别获得了狂犬病病毒强毒CVS株、DRV82株糖蛋白基因,进行克隆及测序,并推导出氨基酸序列,与犬用疫苗弱毒株ERA、SRV9、犬源性街毒株CGX及人用疫苗株PG的糖蛋白序列进行比较。结果表明,以上狂犬病病毒毒株间的核苷酸同源性为83.1%～99.2%,氨基酸序列同源性为87.0%～98.5%。经Jameson-Wolf抗原表位优势图分析,CVS株与其他各株相比发现在304位、372位抗原表位优势升高;而DRV82株与其它各株差异不明显。抗原优势变化可能导致狂犬病病毒糖蛋白出现新的潜在抗原位点,为下一步构建不同毒株的狂犬病病毒糖蛋白重组疫苗奠定了基础。     

Induction of protective immunity by topic application of a recombinant adenovirus expressing rabies virus glycoprotein

The objective of this study was to determine if a replication defective recombinant adenovirus expressing rabies virus glycoprotein (Adrab.gp) given through a non-invasive vaccination route (by topical application) onto the skin (NIVS) could elicit an immune response and/or protection against rabies. Groups of mice were immunized by NIVS with various doses of Adrab.gp. For comparison, groups of mice were immunized intramuscularly, subcutaneously, or intradermally with Adrab.gp. Mice received two booster immunizations at 1 and 2 months after the first immunization. Virus neutralizing antibody (VNA) titers were measured at day 21 after the first and second immunizations and at day 14 after the third immunization. Fifty percent of the mice immunized by NIVS with 2 x 10(7) and 2 x 10(8)pfu Adrab.gp vaccine developed VNA, whereas none of the control mice or the mice immunized by NIVS with the lowest dose (2 x 10(6)pfu) of Adrab.gp virus developed VNA. However, this low dose induced high titers of VNA in mice immunized by parenteral routes. Two weeks after the last immunization, all the mice were challenged with a lethal dose of rabies virus. More than 70% of the animals immunized by NIVS with > or = 2 x 10(7)pfu Adrab.gp virus survived the challenge, whereas all the mice in the negative control group and the group immunized by NIVS with the lowest dose of Adrab.gp succumbed to rabies. Taken together, the results suggest that NIVS with Adrab.gp can induce VNA production and protection against lethal challenge with rabies virus in mice.