狂犬病毒核蛋白基因的克隆及表达

目的获得狂犬病毒CVS株核蛋白(N)基因并在大肠杆菌中表达,为进一步研发狂犬血清学检测试剂盒提供抗原。方法RT-PCR扩增狂犬病毒CVS株核蛋白测序、并定向克隆入原核表达载体pET-28a,构建原核重组表达质粒pET-28a-N,转化大肠杆菌E.coliBL21,并经IPTG诱导在大肠杆菌中表达CVS株核蛋白。结果与结论扩增的CVS株N基因与发表的CVS株N基因序列完全一致,并在大肠杆菌中得到了高效表达,且表达产物具有免疫反应性,为进一步研发狂犬血清学检测试剂盒提供了抗原。     

杆状病毒表达SARS冠状病毒核蛋白抗原性的研究

目的 对杆状病毒表达SARS冠状病毒NP抗原性进行分析与比较。方法 利用SDS—PAGE、Western—Blot和ELISA证明重组核蛋白(rSN)在昆虫细胞获得高效表达．具有特异免疫反应原性。结果 表达rSN的昆虫细胞裂解、稀释后直接包被ELISA板．来检测SARS-CoV康复病人血清特异抗体，敏感性稍高于Veto细胞培养的全病毒(OD分别为2．8和0．6)。与健康人血清不发生特异反应(OD值为0．01)；表达rSN昆虫细胞用于间接免疫荧光，快速检测血清特异抗体反应。同样表现了良好的敏感性和特异性。结论昆虫细胞表达rSN有望替代SARS-CoV全病毒，作为安全、敏感和特异的诊断抗原。应用于ELISA和IFA血清学检测。     

狂犬病毒核蛋白基因的克隆表达及其免疫原性

目的构建狂犬病毒核蛋白(NP)基因的重组杆状病毒表达载体,在昆虫细胞中表达出具有免疫原性的抗原,用于狂犬病的检测及诊断。方法应用RT-PCR方法扩增ERA株狂犬病毒NP基因后克隆到PCR2.1TA载体,转化OneShortTMTOP10感受态细胞构建TA克隆载体,并与pFastBacTM1转座质粒载体分别用KpnΙ和NotΙ双酶切,用T4连接酶连接,构建pFast ERA-NP重组转座质粒,经双酶切、PCR扩增及插入序列方向鉴定后,转化到DH10BacTME.coli感受态细胞,经三抗培养基筛选和PCR扩增鉴定后获得重组Bacmid质粒,将重组Bacmid质粒转染Sf9昆虫细胞制备重组杆状病毒毒液,并继续扩增重组杆状病毒,以第三代毒液感染Sf9昆虫细胞,96h收获细胞,用直接免疫荧光(DFA)、SDS-PAGE和Western blot对表达产物进行检测和分析。结果构建了含有ERA NP基因的重组杆状病毒,并在昆虫细胞中获得表达。结论本研究成功表达出了具有免疫原性的狂犬病毒核蛋白。     

H5N1亚型禽流感病毒核蛋白NP的原核表达及抗体制备

目的制备H5N1亚型禽流感病毒NP重组蛋白和抗体,为研制含NP组分的人禽流感亚单位疫苗和建立评价方法提供检测抗原和抗体。方法以H5N1亚型禽流感病毒A/chicken/Hubei/489/2004(H5N1)NP基因cDNA克隆质粒pMD-NP为模板,PCR扩增获得NP基因抗原区片段,克隆到原核表达载体pET-28a,构建重组质粒pET-ΔNP,转化大肠杆菌BL21-CondonPlus(DE3)-RIL,IPTG诱导重组蛋白表达。采用Ni亲和层析方法纯化NP重组蛋白,免疫家兔,收集免疫血清,ELISA测定抗体效价;间接免疫荧光检测所制备抗体与真核细胞表达NP蛋白的反应性。结果与结论成功的制备了高效价兔抗NP抗体,抗体效价在105以上,该抗体能与原核和真核系统中表达的NP蛋白特异性反应,为研制以NP蛋白为抗原组分的人禽流感亚单位疫苗和建立评价方法奠定了基础。     

应用pAdEasy-1系统构建表达狂犬病毒核蛋白的重组腺病毒

目的应用pAdEasy-1系统,将分离自我国的狂犬病毒CTN株N基因重组入人5型腺病毒基因组的E1区,成功获得了表达狂犬病毒核蛋白的复制缺陷型重组腺病毒。为使核蛋白得到高效表达,在构建时,将N基因控制在CMV早期启动子和SV40poly(A)加尾信号下,并对其翻译调控区进行了改造,在其第一个密码子前加入了哺乳动物典型的Kozak序列基本功能单位ACG,在N基因终止密码子的3’端另加了一个终止密码子。Western Blotting方法证实该重组腺病毒能正确表达产生核蛋白。重组腺病毒在细胞上遗传稳定性分析表明N基因能稳定存在于腺病毒基因组而不被切除。     

左旋多巴对帕金森病大鼠海马区酪氨酸羟化酶及α-突触核蛋白表达的影响

目的探讨左旋多巴(L-dopa)对帕金森病(PD)大鼠海马区酪氨酸羟化酶(TH)及α-突触核蛋白(α-Syn)表达的影响。方法选取96只健康雄性Wistar大鼠,按随机数字表法分为对照组、PD模型组和L-dopa干预组各32只;各组再随机分为4 d和8 d两个亚组,各16只。对PD模型组和L-dopa干预组大鼠皮下注射鱼藤酮乳液(2 mg/kg)建立大鼠PD模型,对照组皮下注射等剂量的葵花油。造模成功后对L-dopa干预组大鼠灌胃给予L-dopa干预治疗,对照组及PD模型组大鼠灌胃给予生理盐水,各组中两个亚组均分别持续4 d和8 d。采用Y型电迷宫试验检测各组大鼠的学习与记忆能力,海马区TH和α-Syn的阳性细胞计数及表达水平的检测分别采用免疫组化法及Western蛋白印迹法。结果 PD模型组大鼠的学习与记忆能力显著低于对照组,而L-dopa干预组较PD模型组明显提升(P0.05);PD模型组及L-dopa干预组大鼠的TH阳性细胞数及表达水平均显著低于对照组,L-dopa干预组高于PD模型组,且L-dopa干预组的8 d亚组高于4 d亚组(P0.05);PD模型组及L-dopa干预组的α-Syn表达水平均显著高于对照组,L-dopa干预组低于PD模型组,且L-dopa干预组的8 d亚组低于4 d亚组(P0.05)。结论鱼藤酮可诱导大鼠发生PD,使其学习与记忆能力显著下降,L-dopa可通过增强海马区的TH表达水平,抑制α-Syn的表达,从而治疗PD。     

靶向核壳蛋白基因的siRNA对流感病毒的预防和治疗作用

目的探讨靶向核壳蛋白(nucleoprotein, NP)的siRNA对甲型流感病毒(influenza virus A, IAV)的预防及治疗作用。 方法在对IAV测序的基础上设计对NP保守区特异的小干扰RNA(small interfering RNA, siRNA),并观察在犬肾细胞(Madin-Darby canine kidney, MDCK)中先转染了siRNA然后以IAV感染,或先以IAV感染,然后转染siRNA时MDCK细胞中IAV载量的变化。 结果1．转染了siRNA的MDCK细胞再进行IAV感染,不同剂量siRNA组均较转染剂对照组IVA病毒载量显著降低(20 pmol siRNA组,P<0.05;40 pmol siRNA组,P<0.05;80 pmol siRNA组,P<0.01);2.当细胞在转染siRNA之前感染IAV,siRNA组的病毒载量显著低于对照组(P<0.01),也低于转染试剂组(P<0.05);3.免疫细胞化学结果显示：无论MDCK细胞预先转染siRNA还是感染了IVA后再进行siRNA转染,siRNA组的NP蛋白表达均明显低于对照组(P<0.01)。 结论靶向NP的siRNA通过干扰IVA的NP蛋白合成,从而对IVA的生长产生抑制作用;无论MDCK细胞感染IVA前后应用靶向NP的siRNA转染均可以降低IVA的产生,说明靶向NP的siRNA对于IVA感染具有预防及治疗作用。     

人副流感病毒三型感染对豚鼠咳嗽敏感性的影响

目的 研究人副流感病毒3型(PIV3)感染对豚鼠咳嗽敏感性(CRS)的影响.方法 将雄性SPF级豚鼠60只按随机数字表法分成对照组、感染6 d组、感染12 d组、感染28 d组、感染42 d组和哮喘组,每组10只.病毒株为PIV3,经体外细胞培养扩增后,通过滴鼻方法接种于感染组豚鼠呼吸道;对照组接种细胞培养基;哮喘组给予卵清白蛋白致敏及诱发哮喘.辣椒素刺激后用BUXCO肺功能仪测定各组豚鼠CRS,同时测定气道高反应性(AHR),并行BALF细胞学检测及肺组织病理学观察.结果感染后6、12、28和42 d组豚鼠CRS[中位数(四分位数间距)]分别为7.50(5.25)、7.30(7.25)、8.40(9.75)和8.20(5.50)CCnt(咳嗽总次数),均高于对照组的2.50(3.00)CCnt(均P<0.05),感染后42 d组升高最明显;哮喘组CRS为3.90(1.75)CCnt,与对照组比较差异无统计学意义(P=0.18).感染后6 d组豚鼠AHR明显;感染组豚鼠BALF中炎症细胞总数均较对照组明显升高,感染6 d和12 d组淋巴细胞明显升高.病理学检查显示感染组豚鼠急性期气道炎症表现为主,未见明显的肺实质炎症.结论 PIV3感染导致豚鼠CRS在急性和亚急性咳嗽期动态增高,CRS升高可能是豚鼠病毒感染引起咳嗽的关键特征.     

重组人α-突触核蛋白原核表达系统的建立及纯化方法

目的 构建α-突触核蛋白的原核表达系统,通过蛋白纯化的定量测定,确定表达这一蛋白的最合适载体和纯化方法,为进一步研究岶金森病病理机制奠定基础.方法 将含有α-突触核蛋白DNA序列的质粒pET3a-Syn和pCEX-4T-1-Syn分别转入BL21(DE3),IPTG(异丙基-D-硫代半乳糖苷)诱导表达,分别采用HPLC和亲和层析的方法进行纯化.纯化后的蛋白进行SDS-PAGE电泳检测纯度,BCA法进行定量比较.结果 构建了α-突触核蛋白两种载体的原核表达系统.pET3a-Syn表达的蛋白,每升菌液可获得20.5 mg目的蛋白;pGEX-Syn表达的蛋白,每升菌液可获得4.8 mg目的蛋白.结论 比较了两种α-突触核蛋白载体表达情况,确定了能够得到大量、高纯度α-突触核蛋白的纯化方法.     

双歧杆菌完整肽聚糖对汉坦病毒核蛋白诱导细胞免疫应答的影响

目的探讨双歧杆菌完整肽聚糖作为佐剂对汉坦病毒核蛋白(HTNV-NP)诱导细胞免疫应答的影响。方法提取双歧杆菌完整肽聚糖(WPG),并将其与HTNV-NP混合后免疫BALB/c小鼠。于加强免疫后4周,检测小鼠NK细胞和CTL的杀伤活性、淋巴细胞增殖反应,采用ELISA法检测IL-2和IFN-γ分泌水平,同时观察WPG的安全性。结果注射HTNV-NP组和注射HTNV-NP+WPG组小鼠NK细胞活性、CTL细胞毒活性、淋巴细胞增殖反应刺激指数(SI)以及IL-2、IFN-γ的分泌水平分别为(5.16±1.34)%(、28.46±4.16)%、1.7(、89.65±7.63)pg/ml(、245.68±56.74)pg/ml和(9.85±0.87)%(、43.29±5.54)%、3.1、(204.48±36.17)pg/ml、(473.29±74.94)pg/ml,差异有显著性(P<0.01)。其间小鼠未出现明显不良反应。结论WPG可显著增强HTNV-NP的细胞免疫应答,是良好的疫苗佐剂。     

禽流感病毒NP基因的酵母双杂交饵载体表达及自激活检测

目的构建H5N1亚型禽流感病毒NP基因的酵母双杂交诱饵载体,验证其在酵母中的表达并检测其自激活作用。方法以PCR法从pGEMT/H5NP扩增NP基因编码区序列,将其定向克隆到PGBKT7载体,经测序鉴定后,PEG/Li-Ac法转化酵母菌株AH109,用表型筛选法及颜色筛选法检测其自激活作用同时Westernblot验证诱饵蛋白的表达。结果获得NP编码区基因,并成功构建酵母双杂交系统中的诱饵载体pGBKT7-NP,对宿主细胞酵母菌株AH109无毒性和自激活作用,并能在酵母细胞中稳定表达。结论诱饵载体pGBKT7-NP可用于GAL4酵母双杂交系统钓取与禽流感病毒核蛋白相互作用的蛋白。     

Discrimination of influenza A subtype by antibodies recognizing host-specific amino acids in the viral nucleoprotein

Please cite this paper as: Miyoshi‐Akiyama et al. (2012) Discrimination of influenza A subtype by antibodies recognizing host‐specific amino acids in the viral nucleoprotein. Influenza and Other Respiratory Viruses 6(6), 434–441. Background Nucleoprotein (NP) of influenza viruses is utilized to differentiate between the A, B, and C viral serotypes. The availability of influenza genome sequence data has allowed us to identify specific amino acids at particular positions in viral proteins, including NP, known as “signature residues,” which can be used to discriminate human influenza A viruses from H5N1 highly pathogenic avian influenza in human cases (HPAI) and pandemic H1N1(2009) (H1N1/2009) viruses. Methods Screening and epitope mapping of monoclonal antibodies (mAb) against NP of influenza A, which reacted differently with NP from human influenza A virus from HPAI and H1N1/2009 A virus. To identify the epitope(s) responsible for the discrimination of viral NP by mAbs, we prepared mutant NP proteins in the 293 cell expression system because some of the mAbs reacted with non‐linear epitopes. Results and Conclusions In the present study, we identified 3 mAbs. The results of epitope mapping showed that the epitopes were located at the signature residues. These results indicated that signature residues of NP could discriminate influenza A viruses from different origin.     

Influenza A virus nucleoprotein selectively decreases neuraminidase gene-segment packaging while enhancing viral fitness and transmissibility

The influenza A virus (IAV) genome is divided into eight distinct RNA segments believed to be copackaged into virions with nearly perfect efficiency. Here, we describe a mutation in IAV nucleoprotein (NP) that enhances replication and transmission in guinea pigs while selectively reducing neuraminidase (NA) gene segment packaging into virions. We show that incomplete IAV particles lacking gene segments contribute to the propagation of the viral population through multiplicity reactivation under conditions of widespread coinfection, which we demonstrate commonly occurs in the upper respiratory tract of guinea pigs. NP also dramatically altered the functional balance of the viral glycoproteins on particles by selectively decreasing NA expression. Our findings reveal novel functions for NP in selective control of IAV gene packaging and balancing glycoprotein expression and suggest a role for incomplete gene packaging during host adaptation and transmission.Seasonal influenza A virus (IAV) remains a major public health threat, causing tens of thousands of deaths each year in the United States alone (1). Morbidity and mortality rates can increase dramatically when a zoonotic strain adapts to circulate in humans, triggering a pandemic. The continuing toll exerted by IAV stems from its remarkable adaptability, which enables it to move between widely divergent host species and also evade herd immunity within each species. Defining the specific mechanisms that mediate IAV adaptation is essential to improving anti-IAV vaccines, therapeutics, and pandemic surveillance.The IAV genome consists of eight negative-sense RNA segments, each of which is required for productive infection. Genome segmentation complements the high mutation rate of IAV by facilitating reassortment, which can maximize positive intergenic epistasis (2–5) and allow selective elimination of segments with deleterious mutations (6, 7). Although reassortment is the most obvious and best-characterized benefit of segmentation, there are likely additional evolutionary advantages.Genome segmentation imposes the substantial constraint of maintaining a gene-packaging mechanism to produce fully infectious virions (8). For IAV, it is widely believed that a single copy of each segment is packaged into progeny virions with nearly perfect efficiency, resulting in an equimolar ratio of the segments at the population level (9–12). Confounding this model, we recently demonstrated that most IAV virions fail to express one or more gene products (13). This finding raises the possibility that in some circumstances incomplete influenza gene packaging is evolutionarily neutral and possibly even advantageous (14).The viral glycoproteins HA and neuraminidase (NA) are encoded by separate genome segments. HA attaches IAV to terminal sialic acid residues on the host cell surface, enabling viral entry. By hydrolyzing sialic acids, NA detaches budding virions and neutralizes HA-inhibiting glycoproteins and is required for the spread of IAV within and between hosts (15–18). Because the specificity of HA and NA for different sialic acid linkages and contexts can vary substantially, functional alignment between the yin–yang activities of HA and NA represents a major determinant of host adaptation, transmissibility, and immune escape (19–23). Independently controlling levels of HA and NA expression therefore may be critical for fine-tuning their functional balance.We previously described a single amino acid substitution (F346S) in the nucleoprotein (NP) of mouse-adapted A/Puerto Rico/8/34 (PR8), selected during serial passage in guinea pigs, which enhances replication in the guinea pig respiratory tract and enables contact transmission (4). Here, we report that this adaptive mutation selectively decreases both the expression and packaging of the NA gene segment, thus revealing a surprising role for NP in the regulation of glycoprotein function and demonstrating that decreased gene packaging can be associated with increased in vivo fitness and transmissibility.     

The evaluation of three diagnostic tests for the detection of equine influenza nucleoprotein in nasal swabs

Background

Equine influenza (EI) is a highly contagious respiratory disease of horses.

Objectives

The aim of this study was to evaluate two rapid antigen detection kits (Directigen or DFA, and Espline) and a commercial ELISA for the detection of EI nucleoprotein in nasal swabs.

Method

Nasal swab samples from naturally and experimentally infected horses were used to compare the sensitivity and specificity of these assays to virus isolation (VI) and real-time RT-PCR.

Results

If real-time RT-PCR was considered as the gold standard, the sensitivity of the other tests in field samples was 68% (DFA), 35% (ELISA), 29% (Espline), and 9% (VI). These tests had 100% specificity when compared to real-time RT-PCR. A receiver operating characteristic (ROC) curve indicated that decreasing the cutoff of the ELISA would increase sensitivity with some loss of specificity. In samples from experimentally infected horses, the sensitivity of the tests compared with real-time RT-PCR was 69% (VI), 27% (DFA), 6% (Espline), and 2% (ELISA). The specificity was 100% for Espline and ELISA and 95% for VI and DFA.

Conclusions

This study illustrated that DFA is the most sensitive antigen detection test evaluated for the diagnosis of EI and that it can detect virus in some subclinical infected and vaccinated horses. The results suggest that DFA is a useful adjunct to laboratory tests and may be effective as a screening test in a quarantine station or similar facility where horses are monitored daily.     

一种A型流感病毒NP抗原快速检测试剂的建立

目的建立一种适合现场检测需要的A型流感快速诊断试剂。方法以自制的抗A型流感(FluA)病毒核蛋白(NP)单抗为原料,建立快速检测A型流感病毒的双抗体夹心酶免疫渗滤试验,并对其灵敏度和特异性进行了初步评价。结果该试剂对不同地区流行的各种亚型的A型流感病毒株均有较高的反应性,而对非FluA病毒株无交叉反应。比较该试剂与BD公司的两种流感快速诊断试剂,发现该试剂对随机选取的3株FluA病毒的检测分析灵敏度高出Directigen EZ Flu A试剂5~125倍,对2株FluA病毒的分析灵敏度高出Directigen Flu A试剂约20倍。另外,用该试剂对57份含漱液标本和170份动物拭子标本进行检测,结果显示:本试剂的灵敏度(>85%)和特异性(>95%)均优于当前主流的商品化A型流感快速诊断试剂。结论利用抗FluANP单抗为原料建立了A型流感快速诊断试剂,该试剂的应用无需任何专用仪器,操作简便快速,可满足现场检测需要。     

博尔纳病病毒核蛋白转染人少突胶质细胞的蛋白组学研究

目的 研究博尔纳病病毒(Borna disease virus)核蛋白(Nucleoprotein,p40)质粒转染人少突胶质细胞(Oligodendrocyte,OL)后蛋白表达变化。方法 构建BDVp40质粒转染OL细胞的细胞模型和空载体转染细胞模型,分别提取各组细胞蛋白进行双向凝胶电泳和图像分析,用HPLC-Chip/MS纳流液质联用技术进行差异蛋白分析,NCBI数据库搜索鉴定差异蛋白。结果 BDVp40转染后出现蛋白表达差异,与对照组相比有8个蛋白表达上调,4个蛋白表达下调,1个蛋白只在转染后细胞表达而未转染OL细胞组不表达,其中4个蛋白与神经系统疾病相关。结论 BDVp40转染OL细胞后导致相关蛋白表达改变,其中一部分蛋白与神经系统疾病相关。     

禽流感病毒特异性NP单克隆抗体的鉴定及禽流感特异性检测方法的建立

目的筛选鉴定禽流感病毒特异性单克隆抗体(单抗)并建立一种适合禽源流感病毒特异性检测的抗原检测方法。方法利用分子进化树分析方法对甲型流感病毒核蛋白(NP)基因进行分类,从禽流感病毒分支和人流感病毒分支上各挑选一个代表性流感病毒株的NP基因进行重组抗原的表达及其单抗的筛选制备,最后应用免疫渗滤技术(A-Dot-ELISA)建立抗原快速检测方法。结果根据分子进化树分析结果确定禽流感H5N1病毒株HK/212/2003和人流感H1N1病毒株CA/04/2009的NP基因进行原核表达,获得高纯度的禽流感病毒NP重组抗原rNP-HK/212和人流感病毒NP重组抗原rNP-CA/04;利用上述两种NP抗原制备出抗rNP-HK/212单抗53株,通过差异筛选获得只对禽流感病毒NP蛋白rNP-HK/212有特异性反应而不与人流感病毒NP反应的3株单抗(1F2,5D2及25A2);免疫荧光方法证实1F2等3株单抗只特异识别禽流感病毒;利用单抗1F2成功建立一种适于禽流感病毒特异性检测的抗原快速检测方法 AIV-Dot-ELISA,该方法对病毒滴度为0.025~0.1HA titer的19个不同亚型的禽流感病毒均能检出,对病毒滴度为5HA titer的8个人流感病毒和2个乙型流感病毒的检测均为阴性。结论本研究成功制备出禽流感病毒NP蛋白特异性单抗,并建立了一种仅特异识别禽流感病毒的抗原快速检测方法 AIV-Dot-ELISA,为快速鉴别禽类流感病毒和人类流感病毒感染提供了一种有用的分析检测工具。