RT-PCR检测蓝舌病毒技术的建立

蓝舌病毒(Bluetongue Virus,BTV)是呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)的代表种,含10个节段的双链RNA(dsRNA)作基因组.其中,L2节段编码BTV型特异性抗原VP2,L3节段和S7节段编码群特异性抗原多肽VP3和VP7.其中,VP7是BTV粒子的主要结构多肽之一,其编码基因序列保守.VP7具有高度的抗原性,能刺激被感机体产生强的群特异性免疫反应[1].     

在昆虫细胞中高效表达蓝舌病毒VP7蛋白作为组特异性诊断抗原

对蓝舌病毒结构蛋白ＶＰ７作为组特异性诊断抗原进行了研究,将编码ＢＴＶ１３主要组特异性抗原ＶＰ７的Ｓ７ｃＤＮＡ插入杆状病毒表达载体ｐＦａｓｔＢａｃ１,通过同源重组获得了重组杆状病毒ｅｖＢａｃＢＴＶＰ７。用此重组病毒感染昆虫细胞获得ＶＰ７蛋白的高效表达,表达量可占细胞蛋白总量的１２．４％。     

小双节RNA病毒中国株基因组第二节段的序列测定及分析

１９９７年从河北省一起成人腹泻暴发病人粪便中首次分离到小双节ＲＮＡ病毒（ｐｉｃｏｂｉｒｎａｖｉｒｕｓ,ＰＢＶ）中国株。为研究该病毒复制必需的依赖ＲＮＡ的ＲＮＡ多聚酶（ＲｄＲｐ）活性机理,采用改进的单引物扩增法克隆和测序了病毒基因组第二节段。结果表明：第二节段全长１６９６个核苷酸,只有一个ＯＲＦ,占全长的９３．９％,编码５３０个氨基酸残基的蛋白,５’－ＮＴＲ ＡＴ丰富（７５．４％）,可能是本位调控元     

在昆虫细胞中同时表达蓝舌病毒VP3与VP7蛋白可装配成核心样颗粒

将蓝舌病毒（ＢＴＶ）１３型Ｓ７与Ｌ３基因同时插入杆状病毒双表达载体ｐＥａｓｔＢａｃＤｕａｌ,获得重组杆状病毒ｒｖＢａｃＢＴＶＰ３７。该病毒在昆虫细胞中同时高水平表达ＢＴＶ１３ ＶＰ３与ＶＰ７蛋白,可以高效自动装配出２０面体的６０￣７０ｎｍ空心颗粒。分析表明,所获颗粒为空心的ＢＴＶ核心样颗粒（ＣＬＰ）,其成分为ＶＰ３与ＶＰ７,不含ＢＴＶ其它任何蛋白与核酸。这种装配需要ＶＰ３与ＶＰ７的共同参与,二者缺     

草鱼出血病病毒多肽的基因定位

用聚丙烯酰胺凝胶电泳分离纯化的草鱼出血病病毒ＧＣＨＶ８７３的基因组ｄｓ－ＲＮＡ的１１个片段,分别在麦胚无细胞翻译体系中进行翻译。其翻译产物经ＳＤＳ－ＰＡＧＥ系统分析。结果表明,基因组片段１、２、３、４、５、和１０分别编码病毒核心衣壳的结构多肽ＶＰ１、ＶＰ２、ＶＰ３、ＶＰ４、ＶＰ５和ＶＰ１０,片段６和７分别编码病毒外层衣壳的结构多肽ＶＰ６和ＶＰ７。片段８和９分别编码５２ｋＤ和４１ｋＤ的多肽,片段１１编码两种多肽,分子量分别为２９ｋＤ和１９．５ｋＤ,它们与病毒结构多肽无明显对应关系。病毒基因组与多肽大体是一一对应的关系。     

传染性法氏囊病病毒VP3结构蛋白基因在大肠杆菌中的高效表达

传染性法氏囊病病毒（ＩＢＤＶ）是危害养鸡业的重要病原之一,属双股双节段ＲＮＡ病毒科成员,由Ａ和Ｂ两个片段的ｄｓＲＮＡ构成,大小分别约３３００－３４００ｂｐ和２８００－２９００ｂｐ。基因片段Ａ首先编码ＶＰ２－ＶＰ４－ＶＰ３聚合蛋白,然后再断裂成ＶＰ３、...     

不依赖甲硫氨酸的翻译起始

Ｓａｓａｋｉ等在ＰｒｏｃＮａｔｌＡｃａｄＳｃｉＵＳＡ 2 0 0 0年第 4期上报道了一种不依赖甲硫氨酸的翻译起始方式[1] 。ＰＳＩＶ (Ｐｌａｕｔｉａｓｔａｌｉｉｎｔｅｓｔｉｎｅｖｉｒｕｓ)是一种昆虫ＲＮＡ病毒 ,属蟋蟀麻痹样病毒组 (Ｃｒｉｃｋｅｔｐａｒａｌｙｓｉｓ ｌｉｋｅｖｉｒｕｓｅｓ) ,为正链ＲＮＡ病毒。属于该组的还有ＤＣＶ、ＲｈＰＶ、ＨｉＰＶ等。该组病毒的理化性质与哺乳动物的小ＲＮＡ病毒 (ｐｉｃｏｒｎａｖｉｒｕｓ)相似 ,但基因图 1　 (Ａ)ＰＳＩＶ基因组结构 ;(Ｂ)预测的外壳蛋白编码区上游的茎环结构组织不同。…     

同时表达蓝舌病毒四个主要结构蛋白可装配成病毒样颗粒

为研制蓝舌病毒（ｂｌｕｅｔｏｎｇｕｅ ｖｉｒｕｓ,ＢＴＶ）基因工程疫苗和进一步研究ＢＴＶ结构与功能的关系,对ＢＴＶ病毒样颗粒（ＶＬＰ）的装配进行了研究。同时在昆虫细胞中表达ＢＴＶ主要结构蛋白ＶＰ７、ＶＰ３、ＶＰ２与ＶＰ５,将细胞裂解液超速离心纯化后,发现主要存在两 形态的颗粒：一种与前文报道的病毒核心颗粒（ＣＬＰ）相同,直径约为６０ｎｍ￣７０ｎｍ,蛋白壳厚１０ｎｍ￣１５ｎｍ;另一种大小为７０ｎｍ￣     

水稻草矮病毒核衣壳蛋白基因克隆及在大肠杆菌中的表达

水稻草状矮化病于70年代曾在南亚、东南亚大面积发生,给当地的水稻生产造成严重损失,在我国也有分布[1]。其病原是水稻草矮病毒(Ｒｉｃｅｇｒａｓｓｙｓｔｕｎｔｖｉｒｕｓ,ＲＧＳＶ),为纤细病毒属(Ｔｅｎｕｉｖｉｒｕｓ)的一个成员,病毒粒体丝状,由核衣壳蛋白和基因组ＲＮＡ组成[2]。基因组含六个ｓｓＲＮＡ片段,均为双义编码[3,4],其中ＲＮＡ5的毒义互补链编码核衣壳蛋白(ｎｕｃｌｅｏｃａｐｓｉｄｐｒｏｔｅｉｎ,ＮＣＰ)[3]。本文报道应用ＲＴＰＣＲ技术获得ＲＧＳＶ沙县分离株(ＲＧＳＶＳＸ)ＮＣＰ基因的ｃＤＮＡ克隆,并得到其在大肠杆…     

兔出血症病毒结构多肽分析

粗提病毒经Ｓｅｐｈａｒｏｓｅ４Ｂ层析后,获得纯化的兔出血症病毒（ＲＨＤＶ）。提纯病毒经过ＳＤＳ－ＰＡＧＥ经考马斯亮蓝染色显示Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ７条多肽,凝胶扫描显示Ａ为ＲＨＤＶ主要结构多肽。用多抗和单抗作免疫转印分析,证实Ａ、Ｂ、Ｃ、Ｄ、Ｅ、和Ｇ为结构多肽,此６条结构多肽间的抗原关系十分密切。     

心脑血管疾病大额住院消费统计分析

通过对医院2004-2006年心脑血管疾病大额住院消费(消费大于50000元,以下简称大额消费)病例发病率高的前五种疾病的构成情况、药费、材料费消耗情况进行分析,认为加强大额病例中发病率高的病种的重点管理,是降低医疗费用的有效途径。建议制定常见病大额病种预定额付费方案和审查报销制度;采用适宜技术;控制药费,防止过度医疗,有效地遏制医疗费用的过快增长。     

Evidence that the C-terminus of OprM is involved in the assembly of the VceAB-OprM efflux pump

Although the architecture of tripartite multiple drug resistance (MDR) efflux pumps of Gram-negative bacteria has been well characterized, the means by which the components recognize each other and assemble into a functional pump remains obscure. In this study we present evidence that the C-terminal domain of the Pseudomonas aeruginosa OprM and the α-helical hairpin domain of Vibrio cholerae VceA play an important role in the recognition/specificity/recruitment step in the assembly of a functional, VceAB-OprM chimeric efflux pump. To our knowledge, this is the first evidence directly linking the C-terminal domain of an outer membrane efflux protein to its recruitment during the assembly of a tripartite efflux pump.     

两个猪苓菌株生长速度及酯酶同工酶比较研究

对不同地理分布的猪苓纯培养菌株进行了种性和酯酶同工酶的比较研究,结果表明,鸡爪苓（Z）纯培养菌株和猪屎苓（ZJ）纯培养菌株的种性有很大不同,两个纯培养菌株的酯酶同工酶酶带类型差异较大,亲缘关系较远。     

Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by 60–85%) and the γ-subunit of nerve growth factor (by 55–93%). Tetrapeptide (VI) proved to be an effective inhibitor of tissue activator of plasminogen and the γ-subunit of nerve growth factor (by 96 and 93%, respectively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the activity of streptokinase [(III), IV), and (VI)], papain [(I), (II), IV), and (VI)], subtilisin [(V) and (VI)], α-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except for (I) and (III)] when applied on fibrin plates at a concentration of 1 × 10^?2 M, while, at the concentration of 1 × 10^?3 M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.     

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed P/e₂ = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H₂O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II.

2. The initial phase of H⁺ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H⁺ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.

3. Dibromothymoquinone reduction, observed as O₂ evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O₂ evolution agreeing well with the steady-state rate observed under continuous illumination.

4. Such measurements of the initial rates of O₂ evolution and H⁺ uptake yield an H⁺/e⁻ ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H⁺/e⁻ ratio of 1.7 at pH 7.6.)

5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H⁺ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, P_i and hexokinase, when added individually, have no effect on proton uptake in this system.

6. The involvement of a proton uptake reaction with an H⁺/e⁻ ratio of 0.5 in the Photosystem II partial reaction H₂O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e₂) and proton uptake (H⁺/e⁻) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.     

林带阻力系数与透风系数关系的理论分析

根据冲量定理分析了林带对气流的阻力,首次得到了林带阻力系数的估算模式（Ｃｄ＝（１．８＋０．２α）（１－α）ｓｉｎ２ω,并利用有关文献发表的资料进行了验证．文章还对来流平行林带时林带对气流的阻力进行了讨论,指出来流平行林带时林带对气流的阻力仅为来流垂直林带时林带对气流阻力的０．７—１．１％,可以不予考虑．     

2种嵩草属植物形态特征对生境干旱化的响应

该研究于西藏自治区东南部的东达山高山草甸沿生境干旱化梯度设置10个样地,采用线性回归方法分析优势种高山嵩草(Kobresia pygmaea)和矮生嵩草(K.humilis)叶片数量与其它构件数值(分株数量、茎基直径、根系数量和根系长度)之间的关系,并采用线性回归斜率测度分株功效、叶片萌生能力、根系分生功效和根系伸长功效,以探讨嵩草的分株能力、茎基生长和根系生长对生境干旱化过程的响应机制。结果显示:(1)2种嵩草植物的叶片数量与4种构件数值均为显著线性正相关关系。(2)随着生境干旱化程度增加,高山嵩草分株数量增加,矮生嵩草分株受干旱抑制程度高,分株数量呈下降趋势;2种嵩草分株功效下降,即单株叶片数量因干旱化程度增加而减少。(3)2种嵩草的茎基直径、叶片萌生能力随着生境干旱化程度增加而下降;高山嵩草叶片萌生能力的变化与生境干旱化梯度一致,具有连续性;矮生嵩草叶片萌生能力对生境干旱化的适应性弱,干旱到一定程度发生骤降。(4)随着生境干旱化,高山嵩草的根系数量和长度均增加,矮生嵩草根系长度增加,但数量却无规律变化;2种嵩草的根系分生功效和根系伸长功效均下降。研究表明:嵩草属植物分布的最适宜生境为表面稍有积水的沼生生境。为适应生境干旱化,高山嵩草降低叶片萌生率以减少蒸腾作用,增加分株数量以增强对空间的占有能力,并增加根系数量和长度来提高对土壤水分的吸收能力,因此对干旱有较强适应性且分布范围广;矮生嵩草只通过降低叶片萌生率,增加根系长度响应干旱化生境,其分布范围较窄。     

The detection of salivary enzymes of phytophagous Hemiptera: a compilation of methods

The role of Hemipteran saliva and salivary enzymes is central to an understanding of the etiology of damage that these insects cause to plants. The dilute nature of the salivary secretions and the minute quantities in which they are often obtainable have made analysis and detection of salivary components very difficult. Such investigations in this laboratory have led us to formalise the techniques involved and we believe that the compilation of these methods presented herein may be useful to other research workers in this area. Methods are described for acid and alkaline phosphatase, esterase, /S-glucosidase, carbohydrases, invertase, amylase, proteinase, pectinase, cellulase, catalase, peroxidase, catechol oxidase, superoxide dismutase and ascorbic oxidase.     

民勤绿洲边缘地下水位变化对植物种群生态位的影响

在民勤绿洲边缘,利用空间区域不同地下水位(湖区8～12m,泉山区15～17m,坝区20～23m)和时间序列(1984～1992年)民勤沙井子地区地下水位下降(7.45～11.65m)梯度,研究地下水位下降对荒漠植物种群生态位的影响。结果表明:空间区域地下水位下降,植物种群生态位宽度均减小,种群退化;时间序列地下水位下降,白刺种群在扩展,其它植物种群在退化。白刺种群生态位宽度在民勤绿洲边缘荒漠植物群落中最大,是该区的建群种。由于白刺种群在地下水位7.45～11.65m范围内扩展,民勤绿洲生态环境治理中地下水位达到该范围是一个主要目标。     

中医证候结构表征研究及其前景展望

当前中医理论基础研究,有证候判定规范、证候疗效评价、证候物质基础、方剂配伍规律等的4个主要领域,但其共同的关键科学问题是中医证候结构表征,主要目的都是为了提高辨证论治的准确性和临床疗效。针对中医证候的结构表征研究,以脾气虚证候的发生规律为切入点,通过理论推证结果与临床数据分析结论相互印证,来揭示证候的结构特征,建立起了中医证候的拓扑结构数学模型,挖掘出了中医理论体系的数学科学基础,在证候基础研究上体现出中医理论的自身规律与临床诊疗特色,具有广阔的应用前景。