H5亚型流感病毒HA头部球状结构域在毕赤酵母中的高效表达及其免疫原性分析

为了研究H5亚型流感病毒HA蛋白中头部球状结构的免疫原性及其基因优化对蛋白表达的影响,本研究构建了重组真核表达载体pPICZ?A-H5HA,并将其转化至毕赤酵母,经筛选获得重组菌株。SDS-PAGE和Western blotting结果分析显示,目的蛋白可在酵母中高效分泌表达,发酵液上清中目的蛋白浓度高达0.2 mg/mL,分子量约为37kDa。将酵母发酵上清经浓缩、纯化后,获得重组目的蛋白H5HA。将不同剂量的H5HA与不同佐剂联用后分别以滴鼻和肌肉注射两种方式免疫试验动物,进行免疫效力的评价。试验结果表明,H5HA具有较好的免疫原性,可诱导SPF鸡产生较高水平的IgG (血凝抑制效价达1∶64、病毒中和抗体效价为1∶218),最佳使用剂量为50μg/羽,与白油佐剂联用时效果最佳,且肌肉注射方式的免疫效果优于滴鼻方式。研究结果为H5亚型流感病毒亚单位疫苗的研制提供了理论支撑。     

毕赤酵母表达gp96-scFv抗体及生物活性测定

旨在利用毕赤酵母分泌表达gp96-scFv抗体,纯化后得到能特异性结合gp96抗原的小分子抗体片段(scFv)。根据gp96-scFv抗体基因序列,合成gp96-scFv抗体基因序列,将gp96-scFv抗体序列克隆到毕赤酵母表达质粒pPICZα-A,线性化的重组表达载体电转化到毕赤酵母X33,甲醇诱导目的蛋白表达,通过亲和层析法纯化目的蛋白,并以SDS-PAGE和Western blotting进行鉴定。通过Western blotting、Immunofluorescence、ELISA、FACS方法对gp96-scFv抗体的生物活性进行了检测。结果成功地构建了分泌表达抗gp96蛋白scFv抗体的毕赤酵母菌,每升毕赤酵母菌培养上清经纯化可获约50 mg gp96-scFv抗体,所获抗体其分子量大约为15 kDa,具有与gp96抗原特异性结合的活性。本研究通过毕赤酵母菌成功表达了gp96-scFv抗体,生物活性Western blotting、Immunofluorescence、ELISA、FACS分析表明该抗体能特异性结合gp96。     

GM-CSF作为Ⅱ型登革病毒E蛋白免疫佐剂的免疫效果观察

目的观察粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)作为II型登革病毒E蛋白免疫佐剂的保护效果,评估GM-CSF作为蛋白疫苗免疫佐剂的可行性。方法提取GM-CSF质粒(pCAG-GM)、将表达II型登革病毒E蛋白的重组质粒(E/pGEX-6P-1)进行诱导,表达产物用亲和层析纯化。将BALB/c小鼠随机分为E蛋白组、佐剂组(E蛋白+GM-CSF)和对照组进行免疫,采用酶联免疫吸附试验(ELISA)检测血清抗体终点效价;采用蚀斑减少中和试验(PRNT)检测II型登革病毒中和抗体水平;酶联免疫斑点试验(ELISPOT)检测小鼠免疫后细胞因子的水平;用攻毒试验观察各组小鼠保护率。结果 E蛋白组和佐剂组小鼠血清中抗体终点效价、中和抗体水平均在免疫后有一定的升高,差异无统计学意义(P0.05);佐剂组细胞因子(IFN-γ和IL-10)水平较E蛋白组均有明显上升,差异具有统计学意义(P0.05);但攻毒试验显示,佐剂组小鼠全部死亡,生存率为0,而E蛋白组的小鼠保护率为33%。结论 GM-CSF免疫佐剂可抑制II型登革病毒E蛋白的免疫保护作用,其作为蛋白疫苗免疫佐剂仍需慎重。     

HBsAg/GM-CSF融合蛋白在毕赤酵母中的表达及其免疫原性研究

为探索一种提高乙肝病毒表面抗原免疫原性的新方法,用PCR和基因重组技术构建HBsAg与GM-CSF的融合基因,并在毕赤酵母中分泌表达HBsAg/GM-CSF(S-GM)融合蛋白。表达产物用SDS-PAGE检测,W estern b lot分析,离子交换柱纯化后免疫昆明鼠,ELISA检测免疫小鼠血清中抗HBsAg的抗体水平。结果显示S-GM融合蛋白在毕赤酵母中获得了表达,离子交换柱一步纯化即可得到纯度达90%以上的S-GM。W estern b lot分析S-GM可分别与抗HBsAg及抗GM-CSF的抗体特异结合。ELISA检测发现第一次免疫后4w出现抗HBsAg的抗体,加强免疫后融合蛋白组几乎全部阳转,且抗体水平较HBsAg组(P=0.009<0.05)及HBsAg和GM-CSF的混合物组(P=0.032<0.05)高。HBsAg/GM-CSF融合蛋白能够在毕赤酵母中表达,且可增强HBsAg的免疫原性,为提高乙肝疫苗的免疫效果提供了新的思路与方法。     

日本脑炎病毒SA14-14-2 E蛋白结构域Ⅲ的抗原性和免疫原性分析

为了表达日本脑炎病毒囊膜蛋白(E蛋白)结构域DⅢ区,了解其作为亚单位疫苗的可能性,本研究根据SA14-14-2病毒株序列(GenBank Accession No.D90195)设计两条引物,以全长JEV感染性克隆pBR-JTF为模板,通过PCR扩增出JEVE蛋白DⅢ的cDNA片段,构建了原核表达载体pET-JEDⅢ,转化大肠杆菌Rosetta(DE3)进行融合表达。融合蛋白为可溶性表达,表达量约占菌体蛋白的75%。用纯化后蛋白免疫新西兰兔和BALB/C鼠,通过ELISA,Western blotting,噬斑减少实验,及乳鼠攻毒实验验证JEDⅢ的抗原性和免疫原性。Western blotting及ELISA结果表明纯化后的表达产物具有良好的抗原性,纯化的JEDⅢ蛋白免疫新西兰兔,可以获得高达1:7×105滴度的抗JEV特异性抗体;JEDⅢ蛋白免疫BALB/C鼠,可以获得1:8.2×104滴度的抗JEV特异性抗体。并且获得1:256滴度的中和抗体,乳鼠攻毒实验能达到75%的保护效果。以上结果说明本研究表达、纯化的重组JEDⅢ蛋白,免疫小鼠以及兔后,能产生抗JEV的特异性抗体,中和性抗体,能够保护部分乳鼠接受毒...     

含分子内佐剂的SARS-CoV-2 RBD域重组蛋白制备及免疫效果评价北大核心CSCD

制备含破伤风毒素肽(tetanus toxin,TT)、促吞噬肽(tuftsin)和新型冠状病毒刺突蛋白(spike,S蛋白)受体结合域(receptor-binding domain,RBD)的融合蛋白,探讨分子内佐剂对RBD蛋白体液免疫和细胞免疫效果的影响。将破伤风毒素肽、促吞噬肽与S蛋白RBD区域通过柔性多肽串联,密码子优化后构建重组载体,原核表达纯化制备重组S-TT-tuftsin蛋白,与铝佐剂混合后免疫BALB/c小鼠,对其体液及细胞免疫效果进行评价。重组S-TT-tuftsin蛋白以包涵体形式表达,离子交换层析纯化后采用梯度透析进行复性,复性蛋白经Dot blotting鉴定,可与新冠亚单位疫苗(安徽智飞公司)免疫后人血清发生反应。小鼠免疫实验结果表明,免疫35 d时抗体水平到达平台期,含分子内佐剂重组蛋白(铝佐剂)免疫小鼠后血清ELISA抗体效价高达1︰66240,显著高于S-RBD蛋白(铝佐剂)免疫小鼠抗体效价(P<0.05)。同时,含分子内佐剂重组蛋白刺激小鼠产生更强的淋巴细胞增殖能力,刺激指数可达4.71±0.15,相较于S-RBD蛋白的刺激指数1.83±0.09具有显著性差异(P<0.0001)。分子内佐剂破伤风毒素肽和促吞噬肽可显著增强新冠S蛋白RBD域的体液免疫和细胞免疫效果,可为新冠亚单位疫苗和其他病毒亚单位疫苗的研制提供理论基础和参考。     

脊髓灰质炎2型病毒样颗粒在毕赤酵母内的表达与鉴定

目的构建可稳定表达脊髓灰质炎病毒(poliovirus,PV)类病毒颗粒(virus-like particles,VLPs)的整合型重组毕赤酵母,鉴定PV VLPs在毕赤酵母细胞中的表达及组装情况。方法根据毕赤酵母密码子偏好性优化salk株II型P1和3CD基因并连接到p Pic ZA载体,构建p Pic ZA-P1-3CD表达载体;用Bgl II线性化p Pic ZA-P1-3CD载体,电转至毕赤酵母GS115中。通过Zeocin抗性筛选获得整合型重组毕赤酵母,随后用高浓度Zeocin抗性筛选得到高表达菌株。甲醇诱导后,用Western Blot检测目的蛋白表达;蔗糖密度梯度离心纯化PV VLPs并进行透射电镜观察。结果成功构建p Pic ZA-P1-3CD表达载体,获得PV VLPs重组毕赤酵母。Western Blot在重组毕赤酵母裂解上清中检测到目的蛋白的表达;蔗糖密度梯度离心纯化后,在透射电镜中观察到直径为30 nm左右的VLPs,其形态与天然的PV颗粒相似。结论成功构建PV-2型VLPs的整合型重组酵母系统,并在毕赤酵母中组装形成了VLPs,为酵母表达系统中PV VLPs疫苗的研制奠定了基础。     

呈现新型冠状病毒RBD抗原病毒样颗粒的表达与鉴定*

目的:以乙型肝炎病毒核心抗原HBcAg为载体,构建呈现新冠病毒刺突蛋白受体结合域的病毒样颗粒,并鉴定其免疫原性,为新冠病毒疫苗的开发提供新思路。方法:在乙型肝炎病毒核心蛋白氨基酸编码序列第78和81位插入新冠病毒刺突蛋白受体结合域(RBD),并通过柔性linker(G4S)3进行连接,序列优化后将融合基因克隆到原核表达载体pET-28a(+),转化表达菌Rosetta,在自诱导培养基中诱导表达,菌体破碎后经蔗糖密度梯度离心,透析浓缩的方法纯化病毒样颗粒。SDS-PAGE、Western blot、透射电子显微镜检测和鉴定VLPs。将制备的VLPs与佐剂等比例混合经皮下免疫BALB/c小鼠,ELISA检测小鼠血清中特异性抗体,分析该HBc-RBD VLPs的免疫原性。结果:在自诱导培养基中,大肠埃希菌可表达部分可溶的VLPs,经蔗糖密度梯度离心纯化后在透射电子显微镜下可以观察到病毒样颗粒的存在。动物实验表明HBc-RBD VLPs刺激小鼠产生了特异性抗体。结论:在原核表达系统中成功表达了展示RBD抗原的VLPs,并通过小鼠实验初步验证了免疫原性,为新冠病毒疫苗的研发提供了新方向。     

人SM22α在巴斯德毕赤酵母中的表达、纯化及抗体制备

目的：用酵母表达重组人平滑肌22α（SM22α）蛋白,制备兔抗人SM22α多克隆抗体。方法：利用PCR从pGEM3z—SM22α质粒扩增得到人SM22α基因编码区,重组至pPIC9构建酵母表达载体,转染巴斯德毕赤酵母,进行分泌型表达。表达产物经分步盐析和CM-纤维素柱层析纯化后,免疫家兔,制备兔抗人sM22α多克隆抗体。结果：所构建的pPIC9-SM22α酵母表达载体转染酵母感受态细胞后,外源性SM22α可整合至酵母染色体中,经甲醇诱导84h,可实现SM22α的高效表达与分泌。用70％硫酸铵处理上清液,收集沉淀进行离子交换层析纯化后,经变性的聚丙烯酰胺凝胶电泳（SDS-PAGE）可见一分子量为22kD的单一区带。用该纯化产物免疫家兔制备的兔抗人SM22α抗血清可用于检测人或大鼠血管壁中SM22α的表达水平。结论：重组人SM22α可在巴斯德毕赤酵母中进行高效表达与分泌,纯化蛋白可用于抗体制备,为研究SM22α功能提供了检测工具。     

牛乳源金黄色葡萄球菌EsxA蛋白的表达及免疫原性分析

为分析牛乳源金黄色葡萄球菌(Staphylococcus aureus)EsxA蛋白的免疫原性,构建EsxA-p ET-28a重组表达质粒,重组质粒经诱导表达后进行SDS-PAGE和Western blotting鉴定。用纯化后重组EsxA蛋白免疫小鼠,用间接ELISA检测免疫小鼠血清中的IgG、IgG1和IgG2a水平;免疫小鼠经S.aureus菌株攻击后,检测小鼠肝、脾、肾组织荷菌数和免疫保护率,观察S.aureus菌株攻击后小鼠肝、脾、肾病理组织学变化。结果表明,成功诱导表达了EsxA重组蛋白,该重组蛋白免疫小鼠后血清抗体效价可达1∶900,与对照相比,重组蛋白免疫后可减少小鼠肝、脾、肾组织的荷菌数,减轻这些脏器的病理损伤,对免疫小鼠保护率达75%。上述结果表明,该重组Esx A蛋白具有良好的免疫原性。     

Evidence for plant viruses in the region of Argentina Islands, Antarctica

This work focused on the assessment of plant virus occurrence among primitive and higher plants in the Antarctic region. Sampling occurred during two seasons (2004/5 and 2005/6) at the Ukrainian Antarctic Station 'Academician Vernadskiy' positioned on Argentina Islands. Collected plant samples of four moss genera (Polytrichum, Plagiatecium, Sanionia and Barbilophozia) and one higher monocot plant species, Deschampsia antarctica, were further subjected to enzyme-linked immunosorbent assay to test for the presence of common plant viruses. Surprisingly, samples of Barbilophozia and Polytrichum mosses were found to contain antigens of viruses from the genus Tobamovirus, Tobacco mosaic virus and Cucumber green mottle mosaic virus, which normally parasitize angiosperms. By contrast, samples of the monocot Deschampsia antarctica were positive for viruses typically infecting dicots: Cucumber green mottle mosaic virus, Cucumber mosaic virus and Tomato spotted wilt virus. Serological data for Deschampsia antarctica were supported in part by transmission electron microscopy observations and bioassay results. The results demonstrate comparatively high diversity of plant viruses detected in Antarctica; the results also raise questions of virus specificity and host susceptibility, as the detected viruses normally infect dicotyledonous plants. However, the means of plant virus emergence in the region remain elusive and are discussed.     

Early onset of virus infection and up-regulation of cytokines in mice treated with cadmium and manganese

A substantial database indicates that a large number of environmental pollutants, chemicals and therapeutic agents to which organisms are exposed cause immunotoxicity. The suppression of immune functions may cause increased susceptibility of the host to a variety of microbial pathogens potentially resulting in a life-threatening state. Evaluation of the immunotoxic potential of chemical xenobiotics is of great concern and, therefore, we have investigated the impact of exposure of inorganic metals, specifically cadmium (Cd) and manganese (Mn) on Encephalomyocarditis virus (EMCV), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEEV) infection. Pretreatment with a single, oral dose of Cd or Mn increased the susceptibility of mice to a sub-lethal infection of these viruses as observed by increased severity of symptoms and mortality compared to untreated controls. An early onset of virus infection was found in brains of Cd and Mn treated animals. Histopathological observations of the brain indicate evidence of inflammation and greater tissue pathology in Cd-or Mn-exposed mice compared to control animals. Meningitis and vascular congestion was seen in virus infected mice in all the metal treated groups, and further, the perivascular inflammation appeared earlier in treated mice compared to control. Encephalitis was maximum in Cd pretreated mice. Widespread environmental contamination of metals and the potential for their exposure and subsequent infection of humans or animals is indicative that further studies of these and all other metals are important to understand the effect of environmental pollution on human health.     

RNA viruses in the sea

Viruses are ubiquitous in the sea and appear to outnumber all other forms of marine life by at least an order of magnitude. Through selective infection, viruses influence nutrient cycling, community structure, and evolution in the ocean. Over the past 20 years we have learned a great deal about the diversity and ecology of the viruses that constitute the marine virioplankton, but until recently the emphasis has been on DNA viruses. Along with expanding knowledge about RNA viruses that infect important marine animals, recent isolations of RNA viruses that infect single-celled eukaryotes and molecular analyses of the RNA virioplankton have revealed that marine RNA viruses are novel, widespread, and genetically diverse. Discoveries in marine RNA virology are broadening our understanding of the biology, ecology, and evolution of viruses, and the epidemiology of viral diseases, but there is still much that we need to learn about the ecology and diversity of RNA viruses before we can fully appreciate their contributions to the dynamics of marine ecosystems. As a step toward making sense of how RNA viruses contribute to the extraordinary viral diversity in the sea, we summarize in this review what is currently known about RNA viruses that infect marine organisms.     

干扰素在皮肤科的临床应用

本文主要介绍国内外干扰素外用制剂在治疗由单纯疱疹病毒、人乳头瘤病毒、水痘－带状疱疹病毒以及皮肤癌等皮肤性疾病的临床应用情况,研究结果显示干扰素在治疗这些皮肤病方面具有很好的疗效。     

Letter: Evidence for non-repetitive subunits in the genome of Rous sarcoma virus

The complexity of Rous sarcoma virus RNA has been determined using molecular hybridization. Relative to poliovirus RNA, the complexity of Rous sarcoma virus is 9·3 × 10⁶ daltons, a value close to its physically-determined molecular weight of about 10⁷. Our interpretation is that the 35 S RNA subunits of the 70 S virus genome are non-repetitive, that is, each possesses a unique nucleotide sequence, although a limited amount of redundancy cannot be excluded.     

寨卡病毒及其疫苗研究

寨卡病毒与黄热病毒、登革热病毒、日本脑炎病毒、西尼罗病毒等都属于蚊媒传播的黄病毒属病毒。寨卡病毒分离于1947年,但由于分布区域有限,所致寨卡热症状较轻,很长一段时间并没有引起太多的关注。最近一些年,特别是2015年后,巴西的寨卡疫情暴发及其与新生儿小头畸形的关联,引起了全球越来越多的关注。疫苗是应对寨卡疫情的重要手段,目前全球有30余个机构在进行寨卡病毒疫苗的研发。本文综述了寨卡病毒的生物学、流行病学、临床特征以及当前不同类型寨卡病毒疫苗研发现状,同时对其他几种黄病毒属病毒批准和临床阶段疫苗情况进行了概述,以为相关研究人员提供参考。     

The incidence of viruses in wild Brassica nigra in Dorset (UK)

Using enzyme‐linked immunosorbent assays, the frequency of occurrence of six viruses was determined in Brassica nigra collected from five coastal sites in Dorset, spanning approximately 24 km. During 1998–2000, the viruses detected were: Turnip mosaic virus (genus Potyvirus) (TuMV), Turnip yellow mosaic virus (genus Tymovirus) (TYMV), Turnip crinkle virus (genus Carmovirus) (TCV), Turnip rosette virus (genus Sobemovirus) (TRoV), Beet western yellows virus (genus Polerovirus) (BWYV) and Cauliflower mosaic virus (genus Caulimovirus) (CaMV). Multiple infections were detected in some individuals (48/447). TuMV was detected infrequently over the three‐year period (5/597). A representative isolate of each virus was tested for its effects on glasshouse‐grown individuals from different half‐sib families of B. nigra from four of the sites. Whether inoculated manually or via aphids (Myzus persicae), TuMV caused a rapid (within 10 days) lethal systemic necrosis in the B. nigra seedlings except when they were near flowering at the time of inoculation. Each of the other viruses invaded systemically but were not lethal. Indeed, BWYV systemically invaded 13/19 glasshouse‐grown B. nigra seedlings but did not produce any visible symptoms. Otherwise, the isolates tested differed in their pathogenicity and in the symptoms they produced in infected B. nigra. With TYMV or TCV viral antigen concentration was closely linked to pathogenicity; for TRoV or CaMV, there was little or no difference in virus concentration between plants with and without symptoms. Substantial and reproducible differences were observed in sensitivity/susceptibility among B. nigra genotypes from different sites in Dorset challenged with the same virus isolate.     

重组乙型肝炎疫苗(CHO细胞)生产工艺病毒灭活验证

目的:验证重组乙型肝炎疫苗(CHO细胞)生产工艺灭活病毒能力,评价疫苗安全性。方法:将乙型肝炎疫苗原液按1∶9比例加入指示病毒(麻疹病毒、VSV病毒),混匀后加入甲醛溶液,使甲醛终浓度达到1/2000和1/4000,放置于37℃,分别于0h、24h、48h、72h取样,用亚硫酸氢钠中和,贮存于-70℃待检或立即检测病毒滴度,对病毒检测阴性的样品盲传3代,进一步检测。结果:疫苗原液加入1/2000和1/4000的甲醛溶液,37℃作用24h、48h、72h,麻疹病毒滴度下降4.0个Log值,VSV病毒滴度下降5.0个Log值,且细胞盲传三代,均未出现细胞病变。结论:乙型肝炎疫苗原液在1/2000和1/4000甲醛浓度37℃72h作用下,均能有效灭活麻疹病毒和VSV病毒。     

Determination of the absolute hand of the helix in the nucleocapsid of haemagglutinating virus (Japan)

The basic hand of the helix of the nucleocapsid of haemagglutinating virus (Japan) was determined to be left-handed from observation of serrated-smooth asymmetry in tilted specimens examined in the electron microscope according to Finch's (1972) technique. Absolute determination of the helical hand was made by comparison with a tilted model of the virus nucleocapsid and by comparing this with the helical sense of the tobacco mosaic virus particle determined by the same method.The left-handed sense of the helix in the nucleocapsid was recognized by examination of cases showing opened-out turns of the helices. Under these conditions the far side of the particle was found to be contrasted predominantly. These conditions are discussed.