II型鲤疱疹病毒ORF4的多克隆抗体制备及其组织分布

【目的】制备II型鲤疱疹病毒(Cy HV-2)ORF4多克隆抗体,利用免疫学方法研究ORF4编码蛋白在病毒感染过程中的组织表达特征。【方法】利用在线软件SMART和BLASTx分析Cy HV-2 ORF4序列,采用PCR技术从Cy HV-2基因组中扩增得到ORF4基因,克隆至表达载体PGEX-4T-3中,将获得的重组表达质粒转化到大肠杆菌BL21(DE3)中,用IPTG诱导表达,表达产物经过SDS-PAGE和Western blot鉴定,再利用亲和层析法纯化出重组蛋白。用纯化的重组ORF4蛋白免疫新西兰兔,收集兔血,分离兔血清获得抗ORF4蛋白的多克隆抗体,再利用Western blot检测抗体与ORF4蛋白之间的特异性。提取攻毒后的异育银鲫肌肉、脑、鳃、脾脏、肝胰脏、心脏、肾脏等组织DNA,用荧光定量PCR技术监测其病毒在各组织的复制水平。使用RIPA裂解液提取上述各组织总蛋白,再利用Western blot技术检测ORF4在感染Cy HV-2的异育银鲫各组织中的表达情况。【结果】原核表达的重组ORF4蛋白分子量为65 k D,与预期大小一致;获得的ORF4抗血清能特异性识别重组ORF4蛋白。病毒感染的异育银鲫体内ORF4主要表达在肾脏、脾脏和鳃上;荧光定量PCR证明Cy HV-2主要在肾脏、脾脏和鳃上富集。【结论】Cy HV-2的非结构蛋白ORF4可能参与病毒的复制,是病毒复制感染周期的特征性指示蛋白之一,这为深入研究ORF4在Cy HV-2感染过程中的作用机制提供参考。     

基于猪德尔塔冠状病毒重组核衣壳蛋白的ELISA抗体检测方法的建立与评价

【目的】猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)是近年来新发现的一种猪肠道冠状病毒,2014年首次暴发于美国,随后亚洲多个国家也相继报道,对养猪业构成了巨大威胁。以大肠杆菌表达纯化的PDCoV重组核衣壳(N)蛋白为包被抗原,建立检测PDCoV抗体的间接ELISA方法,为PDCoV的血清抗体检测和流行病学调查提供工具。【方法】以PDCoV CHN-HN-2014株的基因组RNA为模板,通过RT-PCR扩增PDCoV核衣壳蛋白(N)基因的全长cDNA,将其插入原核表达载体pET-30a中,构建原核表达质粒p ET30a-N,转化大肠杆菌Rosetta(DE3),经异丙基-β-D-硫代半乳糖苷(Isopropyl-beta-D-thiogalactopyranoside,IPTG)诱导表达,以纯化的重组N蛋白为包被抗原,建立PDCoV N-ELISA抗体检测方法,评估其特异性、敏感性、稳定性,并用于临床血清的检测。【结果】SDS-PAGE电泳检测证实表达的重组N蛋白主要以可溶性形式存在,Western blotting证实表达的重组蛋白具有反应活性。用纯化的重组蛋白建立的N-ELISA具有良好的特异性、敏感性、稳定性。与中和试验同时检测148份免疫猪血清和102份临床血清,两种方法的阳性符合率为88.99%,阴性符合率为92.90%,总符合率为91.20%。用建立的ELISA方法检测267份临床血清,PDCoV抗体阳性血清的比率为66.67%。【结论】建立的猪德尔塔冠状病毒N-ELISA抗体检测方法与中和试验的符合率高,可用于PDCoV血清抗体检测和流行病学调查。     

鸭疫里氏杆菌多种血清型间接ELISA检测方法的建立

【目的】鸭疫里氏杆菌(Riemerella anatipestifer,RA)是一种重要的禽病病原,分为21个血清型。但一直缺乏一种针对多种血清型广泛适用的抗体检测方法。前期的研究表明,外膜蛋白A (Outer membrane protein A,OmpA)广泛存在于多种血清型的RA菌株中,是一种重要的免疫原性蛋白,并且其基因序列在RA血清型之间具有高度的保守性,提示其可以作为RA感染血清抗体检测的靶点分子。以重组蛋白OmpA建立间接酶联免疫吸附试验检测RA的抗体。【方法】通过诱导表达条件的摸索及蛋白纯化,获得适用于ELISA包被的重组OmpA抗原。通过Western-blot证明重组蛋白OmpA是否与RA多种血清型发生免疫学反应。进行方阵试验以确定ELISA抗原的最佳包被浓度、被检测血清的反应浓度。重复性、特异性和敏感性试验检查该方法的实用性。【结果】实验证实加入1%乙醇的诱导培养基有利于重组蛋白的可溶性表达。Western-blot结果表明,重组蛋白OmpA可以与1、2、6、10、11、13、14和17型多种RA主要流行血清型有良好的免疫反应性。经方阵试验确定抗原的最佳包被浓度为8 mg/L,待检血清的最佳稀释度为1:160。所建立检测方法具有良好的重复性、特异性和敏感性。【结论】实验建立的鸭疫里氏杆菌多种血清型间接ELISA检测方法可以用于免疫后抗体消长以及感染性抗体的检测。     

中东呼吸综合征冠状病毒S1蛋白在昆虫细胞中的重组表达及免疫效果评价

目的:利用昆虫杆状病毒表达系统重组表达中东呼吸综合征冠状病毒(MERS-Co V)S1蛋白,并对其免疫效果进行评价。方法:构建含有MERS-Co V S1基因的重组杆状病毒质粒,转染Sf9细胞包装杆状病毒;重组病毒传代3次获得种子病毒,感染Sf9细胞,收获感染上清,通过镍离子亲和层析纯化获得S1重组蛋白;用纯化的S1蛋白免疫BALB/c小鼠,采用ELISA检测免疫小鼠血清抗原特异性的抗体水平;采用假病毒中和试验检测血清中抗体的中和活性。结果:获得了表达MERS-Co V S1蛋白的重组病毒株,在昆虫细胞中表达并纯化了S1重组蛋白;利用重组表达的S1蛋白免疫小鼠3次,血清S1特异性Ig G抗体滴度可达1∶102 400,免疫小鼠血清稀释至1/5120后中和百分比仍达50%以上。结论:利用昆虫细胞重组表达的MERS-Co V S1蛋白具有良好的免疫原性,并能有效诱导产生高滴度中和抗体,为发展MERS-Co V重组蛋白疫苗奠定了基础。     

猪肺炎支原体Mhp367蛋白不同区段与恢复期血清反应能力的比较

【背景】课题组前期研究发现猪肺炎支原体Mhp367蛋白是体液免疫显性蛋白,但该蛋白不同区段与猪肺炎支原体恢复期血清的反应能力尚不明确。【目的】鉴定Mhp367蛋白不同区段与猪肺炎支原体恢复期血清的反应能力。【方法】利用不同的引物组合扩增mhp367基因片段,扩增的片段连接pGEX-6P-1、pGEX-4T-3或pGEX-5X-3载体,转化大肠杆菌DH5α感受态细胞。提取的质粒经Bam H I和Xho I双酶切及测序确定重组质粒是否构建成功。正确的重组质粒转化大肠杆菌BL21(DE3)感受态细胞构建重组菌。重组菌经IPTG诱导和超声破菌后,经与谷胱甘肽beads结合和SDS-PAGE电泳检测目的蛋白表达情况。表达目的蛋白的重组菌破菌后上清包被谷胱甘肽板,ELISA方法鉴定Mhp367蛋白不同区段与猪肺炎支原体恢复期血清的反应能力。【结果】构建了9个能以可溶形式表达目的蛋白的重组菌;9个Mhp367蛋白片段均为体液免疫显性,第394-524位氨基酸区段与猪肺炎支原体恢复期血清反应最强,是一个良好的疫苗候选抗原区段。【结论】本研究为猪肺炎支原体基因工程亚单位疫苗的研发提供了候选抗原靶标。     

用小鼠模型分析可溶性表达结核分枝杆菌Ag85A的免疫原性

【目的】可溶性表达结核分枝杆菌Ag85A蛋白,并评价其免疫原性。【方法】利用冷休克表达质粒和含有伴侣质粒的大肠杆菌对Ag85A蛋白进行可溶性原核表达,并进行纯化与鉴定,通过C57BL/6小鼠模型对Ag85A蛋白的免疫原性,包括诱导机体特异性体液免疫应答和细胞免疫应答水平进行分析。【结果】重组菌诱导后裂解上清中检测到可溶性Ag85A蛋白的表达,经过亲和层析纯化收获了纯度在90%以上的Ag85A蛋白,Western blot鉴定显示其具有较好的免疫反应性。Ag85A蛋白免疫小鼠后,血清中可以检测到高水平的Ig G抗体效价,其中Ig G2b水平要高于Ig G1。通过特异性多肽、蛋白刺激脾脏和腹股沟淋巴结细胞可分泌高水平的IFN-γ、TNF-α等Th1型细胞因子。【结论】实现了Ag85A蛋白的可溶性表达,免疫特性评价显示Ag85A蛋白可诱导机体产生强烈的特异性体液免疫应答及Th1型的细胞免疫应答,从而为其进一步免疫学功能的研究奠定了重要基础。     

草地贪夜蛾组织蛋白酶L的基因克隆、原核表达及多克隆抗体制备

【目的】克隆草地贪夜蛾Spodoptera frugiperda的组织蛋白酶L(cathepsin L,CatL)基因,分析该基因的序列特征并制备该酶多克隆抗体,为探析其生理功能奠定基础。【方法】根据甜菜夜蛾Spodoptera exigua的组织蛋白酶L基因开放阅读框(ORF)序列两端直接设计引物克隆草地贪夜蛾组织蛋白酶L基因。利用生物信息学软件分析该基因的序列特征,利用ClustalX2软件进行同源比对和进化分析,利用同源建模预测该酶三维结构。通过原核表达重组蛋白,多次免疫新西兰大白兔制备该酶多克隆抗体。【结果】克隆获得了草地贪夜蛾的组织蛋白酶L基因SfCatL(GenBank登录号:HQ110065),ORF序列长1 035 bp,编码344个氨基酸,预测N-末端含有长度为16个氨基酸残基的信号肽序列,去除信号肽序列后,预测成熟蛋白分子量为36.8 kD,等电点为6.69。SfCatL氨基酸序列与其他13个物种的组织蛋白酶L氨基酸序列比较有53.7%~96.8%的一致性,与甜菜夜蛾组织蛋白酶L氨基酸序列一致性最高,达96.8%。同源建模预测表明,SfCatL折叠成紧密而稳定的元宝状结构,含3个对结构有稳定作用的二硫键,亲水性氨基酸主要包被在蛋白的表面。原核表达、纯化SfCatL蛋白制备抗血清,其效价超过1∶40 000,Western blot鉴定结果表明抗血清与草地贪夜蛾Sf9细胞SfCatL蛋白能够特异性结合。【结论】获得了草地贪夜蛾SfCatL完整ORF序列,分析了其特征,经原核表达、纯化获得高纯度的融合蛋白,成功获得多克隆抗体。本研究为进一步研究该基因的功能并开发组织蛋白酶抑制剂类杀虫剂提供理论依据。     

含禽流感病毒M2e 氨基端抗原表位的重组传染性法氏囊病毒VP2 蛋白的免疫原性鉴定

【目的】构建传染性法氏囊病毒VP2蛋白展示禽流感M2e抗原表位的重组蛋白,研发预防H5或H9亚型禽流感和传染性法氏囊的基因工程疫苗。【方法】根据现有禽流感疫苗株M2e的氨基端12个氨基酸多肽序列(nM2e)序列,结合GenBank中H5和H9亚型禽流感病毒nM2e的比对结果,确定nM2e序列。用融合PCR分别将1拷贝H5或H9的nM2e序列插入IBD B87株VP2基因的PBC区,获得VP2BCnM2e重组基因。将重组基因克隆至杆状病毒表达系统,转染Sf9细胞进行表达。经间接免疫荧光和Western blotting检测Sf9细胞表达重组基因后,扩繁重组病毒,制备疫苗,间隔4周对非免鸡作2次重复免疫,用间接ELISA和鸡胚成纤维细胞中的病毒血清中和试验检测血清中VP2和nM2e的抗体效价。【结果】成功构建含H5或H9 nM2e的VP2BCnM2e重组基因,该重组基因在Sf9细胞中得到表达。经免疫鸡,两重组蛋白均能激发针对VP2和nM2e的抗体,VP2BCnM2eH5组抗体效价高于VP2BCnM2eH9组。【结论】两重组蛋白均具有免疫原性,VP2BCnM2eH5免疫原性更佳。     

西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli [pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。     

斜纹夜蛾核型多角体病毒ⅡORF146基因的克隆、表达与启动子活性分析

【目的】研究斜纹夜蛾核型多角体病毒II ORF146基因的结构与功能。【方法】根据SpltMNPV IIORF146基因序列设计引物,经PCR扩增克隆ORF146基因。在生物信息学分析基础上进行启动子活性分析和转录时相分析。构建ORF146片段的原核表达载体,表达并纯化融合蛋白后制备多克隆抗体。【结果】核苷酸序列分析表明,读码框含1383 bp,编码460个氨基酸的蛋白质,推定分子量为50.4 kDa。启动子活性分析和转录时相分析都表明该基因是个早、晚期都表达的基因,在病毒感染8 h和18 h有两个转录峰,24 h以后转录水平略有下降,但趋于稳定。pET-28a-ORF13原核表达的融合蛋白经纯化后制备的多克隆抗体特异性高,效价可达1∶3200以上。【结论】SpltMNPV II ORF146基因是一个早期和晚期都表达的病毒组成型结构蛋白基因。推测ORF146基因可能与SpltMNPV II病毒感染宿主细胞后病毒DNA复制有关。制备的多克隆抗体可用于深入研究该蛋白的生物学特性与功能。     

Ectogenesis and the case against the right to the death of the foetus

Ectogenesis, or the use of an artificial womb to allow a foetus to develop, will likely become a reality within a few decades, and could significantly affect the abortion debate. We first examine the implications for Judith Jarvis Thomson’s violinist analogy, which argues for a woman’s right to withdraw life support from the foetus and so terminate her pregnancy, even if the foetus is granted full moral status. We show that on Thomson’s reasoning, there is no right to the death of the foetus, and abortion is not permissible if ectogenesis is available, provided it is safe and inexpensive. This raises the question of whether there are persuasive reasons for the right to the death of the foetus that could be exercised in the context of ectogenesis. Eric Mathison and Jeremy Davis have examined several arguments for this right, doubting that it exists, while Joona Räsänen has recently criticized their reasoning. We respond to Räsänen’s analysis, concluding that his arguments are unsuccessful, and that there is no right to the death of the foetus in these circumstances.     

On the diversity of the Cladocera in the tropics

The mythical concept of an impoverished tropical cladoceran fauna is refuted. On a planetary scale, around half of the cladoceran species presently known occur exclusively in the tropics-subtropics, often with considerable restriction to particular geographical subzones. On a regional (political) scale, the situation is often unclear because of the continued fragmentary nature of studies, and because political units are not a good basis for biogeographical comparisons. At the finest level of resolution (lake-perlake comparisons), there appears to be an upper limit of c. 50 cladoceran species per individual lake. No significant difference between lakes in the temperate zone and in the tropics could be established here. Daphnia is largely absent from the tropics, but is replaced by more Sidids, Moinids, and Bosminids, such that the average cladoceran community in the limnetic zone of a tropical lake is not characterized by less species but rather by lower population densities. This, in turn, is considered a consequence of higher prevalent predation levels in the tropics.     

Tubular networks in the terminal endings of the visual receptor cells in the human,the monkey,the cat and the dog

Summary A tubular network was found in the terminal endings of the visual receptor cells in the human, the monkey (Macaca mulatta), the cat and the dog. These tubules are arranged in close groups in the vicinity of the synaptic lamellae and the invaginated dendrites. According to the form, diameter, density of the tubules and to the consistence of the network formed by them one can distinguish at these places an initial type (type I), a transitory (type II) and a vesicular one (type III). In the the type III branching, bizarre forms are frequent. The diameter of all the tubules reaches 500–600 Å, their density and walls being the same as in the synaptic vesicles.Similar networks also occur in the axons of the visual receptor cells of the monkey.

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Zusammenfassung In den Endigungen der Photorezeptorzellen von Mensch, Affe (Macaca mulatta), Katze und Hund kommen aus Tubuli bestehende Komplexe vor. Organellenartig in geschlossenen Gruppen angeordnet, liegen sie in Nähe der synaptischen Lamellen und der invaginierten Dendriten. An diesen Stellen kann man nach Form, Durchmesser, Dichte und Konsistenz der von den Tubuli gebildeten Komplexe drei Typen unterscheiden: 1. einen initialen (Typus I), 2. einen Übergangstypus (Typus II) und 3. einen vesiculären Typus (Typus III). In letzterem kommen häufig verzweigte, bizarre Formen vor. Der Durchmesser sämtlicher Tubuli erreicht 500–600 Å. Ihre Dichte und ihre Wand gleicht denen der synaptischen Vesikel.Ähnliche Komplexe fanden wir auch in den Axonen der Photorezeptorzellen vom Affen.

     

Assessing the Physiological State of the Fucoids from the Barents Sea Littoral by the End of the Polar Night

Methods of amperometry and potentiometric titration were used to follow dark respiration (DR) and apparent photosynthesis (AP) in the fucoids Ascophyllum nodosum (L.) Le Jol, Fucus vesiculosus L., and F. serratus L. from the Barents Sea littoral by the end of the 40-day-long polar night. The macroalgae were shown to manifest species-specific low rates of photosynthesis and respiration. However, in spite of their low photosynthetic status due to the effects of subzero temperature and prolonged low or zero illumination, the macroalgae have been able to restore DR and AP to the initial level already by the day 9; the ability to restore AP depended on the level of illumination. The study of the changes in the carbonate–bicarbonate system in the light and darkness demonstrated that the macroalgae grown in darkness, in contrast to those grown in twilight, could absorb bicarbonate in darkness; however, they lost this capacity after two-day-long illumination at an irradiance of 7 mol/(m² s). Bicarbonate uptake in darkness and the capacity to restore the systems of photosynthesis and respiration in fucoid cells are discussed in the context of algal energy metabolism under the polar night conditions.     

Arranging the elements of the potassium channel: the T1 domain occludes the cytoplasmic face of the channel

The voltage-gated potassium channel is currently one of the few membrane proteins where functional roles have been mapped onto specific segments of sequence. Although high-resolution structures of the transmembrane portions of three bacterial potassium channels, the tetramerization domain and the cytoplasmic ball are available, their relative spatial arrangement in mammalian channels remains a matter of ongoing debate. Cryo-electron microscopic images of the six transmembrane voltage-gated Kv channel have been reconstructed at up to 18 Å resolution, revealing that the T1 domain tetramerizes and is suspended below the transmembrane segments. However, the resolution of these images is insufficient to reveal the location of the third piece of the puzzle, the inactivating ball domain. We have used the aberrant interactions observed in a series of chimæric channels to establish that an assembled T1 domain restricts access to the cytoplasmic face of the channel, suggesting that the N-terminal ball and chain may be confined in the space between the T1 domain and the transmembrane portion of the channel.     

Feeding the vertebrate retina from the Cambrian to the Tertiary

The retina of the vertebrate eye is metabolically active and requires nutritive support. During the last 540 million years it has evolved into forms as complicated and nutritionally demanding as those found in avian or primate eyes. Diffusion from the choroid is generally able to supply the metabolic needs of thin retinae. However, when the thickness exceeds the limits of diffusion, structures are needed to supplement the vascular supply from the choroid. These supplemental nutritive devices include the choroidal gland, the falciform process and preretinal vascular plexus of fish, the conus papillaris of lizards, the pecten oculi of birds, the intraretinal vessels of mammals and a few novel systems that remain difficult to classify. These vascular systems are among the most variable features of the vertebrate eye. Here, we review classical and recent findings regarding such retinal nutrition systems, propose a three category classification for them based on histologic origins and speculate on the evolutionary forces which drove their development.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418