埃博拉病毒NP蛋白的单克隆抗体制备及抗原肽的定位

埃博拉病毒(Ebola virus,EBOV)是一种能导致人类及脊椎动物出血热的致死性病毒,对公共卫生具有较严重的危害。EBOV的NP蛋白在病毒复制中具有重要作用,也是诊断该病重要的靶蛋白。文中原核表达重组扎伊尔型EBOV的NP蛋白,重组蛋白免疫bal/c小鼠,制备了一株小鼠抗EBOV-NP的单克隆抗体。利用Western blotting方法,该抗体能特异识别真核表达和原核表达的重组EBOV-NP,并能同莱斯顿型(RestonEbola virus,REBOV)、科特迪瓦型(Cote-d’Ivoire Ebola virus,CIEBOV)和本迪布焦型(Bundibugyo Ebola virus,BEBOV)埃博拉病毒产生交叉反应,而不与苏丹型(the Sudan Ebola virus,SEBOV)和马堡型(the Marburgvirus,MARV)埃博拉病毒产生反应。利用突变PCR和Western blotting方法,定位了该抗体识别的抗原决定簇序列,该序列(PPLESD)位于EBOV-NP蛋白的C端583-588aa。生物信息学研究表明,该序列在已经公布的ZEBOV、CIEBOV、BEBOV共16个型和REBOV的4个型中高度保守。研究结果为建立以上各型埃博拉病毒的检测方法提供了工具,也为研究埃博拉病毒复制及致病机理提供了基础。     

SYBR Green Ⅰ荧光定量RT-PCR检测埃博拉病毒方法的建立

为了建立一种快速准确的检测埃博拉病毒(EBOV)亚型的方法。本研究根据GenBank中公布的EBOV NP基因序列,通过设计引物和优化反应条件,建立了一种SYBR GreenⅠ荧光定量RT-PCR检测方法检测EBOV。以体外转录的EBOV RNA为模板进行试验,该方法检测的灵敏度可以达到1.0×102个拷贝/μL,检测范围达到9个数量级为102～1010,可检测5种亚型EBOV。建立的方法对马尔堡病毒(MARV)、登革病毒(DENV)、新疆出血热病毒(XHFV)、乙型脑炎病毒(JEV)、流感病毒(H1N1和H3N2)和猪繁殖和呼吸综合征病毒(PRRSV)E基因组RNA无非特异性扩增。本文将荧光定量RT-PCR技术用于埃博拉病毒的定量检测中,并且建立了EBOV SYBRGreenⅠ荧光定量RT-PCR检测方法。     

重组杆状病毒表达尼帕病毒融合蛋白和受体结合蛋白的研究

构建了表达尼帕病毒(Nipah virus,NiV)囊膜功能糖蛋白F和G的重组杆状病毒rBac-NF、rBac-NG。Western-blot证实大小分别为61kD和66kD的重组融合蛋白(rNF)和受体结合蛋白(rNG)分别在rBac-NF、rBac-NG感染的昆虫细胞中获得表达,并且rNF前体F0可在昆虫细胞内进一步有效裂解为F1(~49kD)和F2;采用兔抗NiV病毒高免血清间接免疫荧光检测重组杆状病毒表达F和G蛋白显示出良好的特异免疫反应原性。以rBac-NF、rBac-NG感染的昆虫细胞裂解液稀释后直接包被ELISA板,间接ELISA检测兔抗灭活NiV全病毒高免血清中的F和G蛋白特异性抗体,同样具有良好的敏感性和特异性;以rBac-NF和rBac-NG感染昆虫细胞培养物直接免疫BALB/c小鼠,可诱导显著的NiVF和G蛋白特异体液免疫反应,产生的特异抗体可有效中和NiV囊膜蛋白F和G介导的伪型VSV重组病毒侵入NiV易感宿主细胞的感染性。结果表明,杆状病毒表达重组F和G蛋白抗原具有替代NiV全病毒,作为安全、经济、敏感和特异的诊断抗原的潜力,并为重组病毒亚单位疫苗防制尼帕病毒性脑炎的探索研究奠定了基础。     

对虾白斑综合征病毒ORF220基因在昆虫细胞内的表达及其分布

对虾白斑综合征病毒厦门分离株ORF220编码真核生物GP130受体同源蛋白。将ORF220和绿色荧光蛋白编码基因融合在一起克隆到昆虫杆状病毒表达载体pFastBacI,然后与AcBacmid共同转染DH10B细胞。用PCR鉴定含有ORF220和EGFP基因的重组质粒,提取纯化重组质粒并转染昆虫细胞进行表达。结果发现,DNA转染后3-5d可以在荧光显微镜下观察到绿色荧光,表明融合蛋白在昆虫系统内成功表达。用病毒上清液感染昆虫细胞进行时相观察,结果表明,ORF220蛋白在昆虫细胞的细胞质和细胞核内呈随机分布,没有特异的细胞定位。     

对虾白斑综合征病毒ORF220基因在昆虫细胞内的表达及其分布

对虾白斑综合征病毒厦门分离株ORF220编码真核生物GP130受体同源蛋白.将ORF220和绿色荧光蛋白编码基因融合在一起克隆到昆虫杆状病毒表达载体pFastBacI,然后与AcBacmid共同转染DH10B细胞.用PCR鉴定含有ORF220和EGFP基因的重组质粒,提取纯化重组质粒并转染昆虫细胞进行表达.结果发现,DNA转染后3-5d可以在荧光显微镜下观察到绿色荧光,表明融合蛋白在昆虫系统内成功表达.用病毒上清液感染昆虫细胞进行时相观察,结果表明,ORF220蛋白在昆虫细胞的细胞质和细胞核内呈随机分布,没有特异的细胞定位.     

汉滩病毒M、S基因不同拼接方式在昆虫细胞中融合表达效果比较

将汉滩病毒囊膜糖蛋白G1与核蛋白(NP)部分片段以不同方式拼 接,构建G1S0.7或S0.7G1嵌合基因,分别插入杆状病毒表达载体pFBD,转化DH10Bac致敏菌, 获得含有嵌合基因的重组穿梭质粒Bacmid,用其转染Sf9细胞,快速筛选出含有G1S0.7或S0.7 G1嵌合 基因的重组杆状病毒,在昆虫细胞中表达外源融合蛋白.利用间接免疫荧光、ELISA和免疫 印迹对表达产物进行检测.结果表明,含G1S0.7嵌合基因之重组杆状病毒可在昆虫细胞中表 达出融合蛋白,该蛋白可被抗汉滩病毒核蛋白及糖蛋白G1特异性单抗所识别,其分子量约97 kD;含S0.7G1嵌合基因之重组杆状病毒在昆虫细胞中表达的融合蛋白,只能被抗汉滩病毒核 蛋白特异性单抗所识别,其分子量约43kD.上述结果提示,G1S0.7嵌合基因可能在昆虫细胞 中表达出完整的具有生物学活性的融合蛋白,S0.7G1嵌和基因的昆虫细胞表达产物不完整 ,且生物学活性不如G1S0.7嵌合基因的表达产物.     

禽呼肠孤病毒σC基因在杆状病毒表达系统中的表达及其活性鉴定

本研究利用Bac-To-Bac杆状病毒表达系统构建重组禽呼肠孤病毒(Avian reovirus,ARV)σC基因的杆状病毒,感染sf9细胞获得表达重组蛋白。首先,将ARVσC基因克隆至pFastBacHTA载体,构建重组供体载体pFσC,将其转化大肠杆菌DHl0Bac感受态细胞,使σC基因整合到Bacmid穿梭载体中,获得重组穿梭载体BacmidσC。通过脂质体介导将其转染到sf9昆虫细胞中,获得重组杆状病毒rBacσC。通过Western Blot、间接免疫荧光试验(IFA)进行检测,结果显示:σC蛋白在重组杆状病毒rBacσC感染的sf9昆虫细胞中获得正确表达,分子质量约为37kD,表达的σC蛋白具有良好的反应活性。     

汉滩病毒M、S基因不同拼接方式在昆虫细胞中融合表达效果比较

将汉滩病毒囊膜糖蛋白G1与核蛋白 (NP)部分片段以不同方式拼接 ,构建G1S0 .7或S0 .7G1嵌合基因 ,分别插入杆状病毒表达载体 pFBD ,转化DH10Bac致敏菌 ,获得含有嵌合基因的重组穿梭质粒Bacmid ,用其转染Sf9细胞 ,快速筛选出含有G1S0 .7或S0 .7G1嵌合基因的重组杆状病毒 ,在昆虫细胞中表达外源融合蛋白。利用间接免疫荧光、ELISA和免疫印迹对表达产物进行检测。结果表明 ,含G1S0 .7嵌合基因之重组杆状病毒可在昆虫细胞中表达出融合蛋白 ,该蛋白可被抗汉滩病毒核蛋白及糖蛋白G1特异性单抗所识别 ,其分子量约 97kD ;含S0 .7G1嵌合基因之重组杆状病毒在昆虫细胞中表达的融合蛋白 ,只能被抗汉滩病毒核蛋白特异性单抗所识别 ,其分子量约 4 3kD。上述结果提示 ,G1S0 .7嵌合基因可能在昆虫细胞中表达出完整的具有生物学活性的融合蛋白 ,S0 .7G1嵌和基因的昆虫细胞表达产物不完整 ,且生物学活性不如G1S0 .7嵌合基因的表达产物     

16型人乳头瘤病毒衣壳蛋白在真核细胞中的表达

为了构建HPV16型晚期蛋白重组杆状病毒,并使其在昆虫细胞中获得高效表达.首先构建2株重组杆状病毒转移质粒,分别携带人乳头瘤病毒晚期基因L1及L1和L2,再用线性化的杆状病毒DNA与该重组杆状病毒转移质粒共转染sf9昆虫细胞进行同源重组,获得2株重组杆状病毒.经鉴定该重组病毒中有目的基因存在且可表达所编码的L1或L2晚期蛋白.结果表明HPV16型晚期蛋白在昆虫细胞中获得成功表达,为HPV16型预防性基因工程亚单位疫苗的研制和诊断试剂的研究开发奠定了基础.     

不同脊髓灰质炎病毒株3CD蛋白酶在昆虫细胞中的表达及其对P1前体蛋白的剪切

目的获得在昆虫细胞中有效表达脊髓灰质炎病毒P1基因和3CD基因的重组杆状病毒,为制备脊髓灰质炎病毒样颗粒疫苗提供了科学依据。方法将I型脊髓灰质炎病毒(Mahoney株)的P1基因和3CD基因构建到供体质粒中,通过flash BAC ULTRATM系统制备重组杆状病毒;将Mahoney株的P1基因与Sabin株Ⅲ型的3CD基因组合,用同样方法构建重组杆状病毒。通过接种昆虫细胞草地贪夜蛾细胞(sf-9细胞)对两种病毒进行扩增,再接种昆虫细胞粉纹夜蛾细胞(High five细胞)扩大培养,并通过定量PCR对P1和3CD基因的表达进行验证,利用Western blot检测P1蛋白的表达及被3CD蛋白酶剪切的情况。结果获得了两株稳定表达脊髓灰质炎病毒P1和3CD基因的重组杆状病毒。其中,重组杆状病毒(Bac U-Mahoney-P1-3CD)在感染细胞后,3CD蛋白酶表达量较低,不能有效剪切P1前体蛋白;而重组杆状病毒(Bac U-Mahoney-P1-Sabin PV3 3CD)感染细胞后,3CD蛋白酶的表达量和对P1前体蛋白的剪切效力都有明显提高(P<0.05)。结论将Mahoney株P1基因和Sabin株Ⅲ型的3CD基因的组合构建重组杆状病毒,可有效地在昆虫细胞中表达P1和3CD基因,并且Sabin株Ⅲ型的3CD蛋白酶可有效地剪切Mahoney株的P1前体蛋白。     

壳聚糖酶的克隆表达与壳寡糖的制备分析

目的:克隆壳聚糖酶基因于大肠杆菌中实现高表达,制备壳寡糖。方法:以枯草芽孢杆菌总DNA为模板扩增壳聚糖酶基因(CSN),克隆至载体pET23a(+)上,转化菌株BL21(DE3)。重组子经0.5 mmol/L IPTG诱导后,SDS-PAGE和质谱检测与鉴定重组酶。酶纯化后水解壳聚糖,薄层色谱分析其水解产物。结果:质谱证明壳聚糖酶(31.5kDa)成功表达,表达量占菌体总蛋白的45%左右。纯化后重组酶浓度为900 mg/L,纯度95%、回收率85%,酶活力为10 000 U/mg。壳聚糖降解产物为壳二糖至壳四糖。结论:原核表达载体pET23a(+)-CSN构建正确,壳聚糖酶表达量与活性高,适用于水解壳聚糖制备壳寡糖。     

国内外蝗害治理技术现状与展望

本文首先概述了国内外蝗虫发生与为害的态势,总结了现阶段我国蝗虫发生与为害的主要特点:即农田飞蝗暴发频繁而且严重,草原土蝗的发生时常造成严重的经济损失,而且侵入城市干扰市民生活,我国与周边国家之间蝗虫过境迁移频繁,使用化学农药污染环境和农产品;分析了国内外蝗虫防治对策与技术的发展现状,重点介绍了应急防治和可持续治理对策、...     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission

The molecular characterisation of species and genotypes of Cryptosporidium and Giardia is essential for accurately identifying organisms and assessing zoonotic transmission. Results of recent molecular epidemiological studies strongly suggest that zoonotic transmission plays an important role in cryptosporidiosis epidemiology. In such cases the most prevalent zoonotic species is Cryptosporidium parvum. Genotyping and subtyping data suggest that zoonotic transmission is not as prevalent in the epidemiology of giardiasis. Molecular characterisation of Cryptosporidium and Giardia is a relatively recent application that is evolving as new genes are found that increase the accuracy of identification while discovering a greater diversity of species and yet unnamed taxa within these two important genera. As molecular data accumulate, our understanding of the role of zoonotic transmission in epidemiology and clinical manifestations is becoming clearer.     

白术同源四倍体的诱导和鉴定及其与二倍体过氧化物酶的比较

以白术（Atractylodes macrooephala Koidz.）二倍体组培苗为材料,对其四倍体诱导方法进行研究,共获得45个白术同源四倍体株系,为优良株系的选育提供了材料。此外,还分析比较了其中8个白术四倍体株系与二倍体的过氧化物酶同工酶（POD）的酶谱差异,发现四倍体各株系过氧化物酶同工酶谱比二倍体的均多了Rf0.310的谱带,且总过氧化物酶比活力也发生了很大改变,对探讨白术四倍体优良株系的生理生化机理具有一定的参考价值。     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000