简化cDNA末端快速扩增技术测定基因Ⅲ、Ⅵb和Ⅶ d型新城疫病毒基因组末端序列及分析

[目的]简化cDNA末端快速扩增技术(Rapid amplification of cDNA ends,fLAcE)流程,测定基因Ⅲ、VIb)和VIId型新城疫病毒(Newcastle disease virus,NDV)基因组两侧末端序列,并对NDV的leader和trailer进行分析.[方法]利用T4 RNA连接酶将特定寡聚核苷酸片段的连接于病毒基因组RNA和eDNA,再利用RT-PCR或PCR方法对病毒基因组的末端进行快速的扩增.[结果]建立一套操作简单、低成本、可重复性高的RACE方法,测定了三种基因型5株NDV 3'末端leader和5'末端trailer序列比对分析.[结论]本实验测定的鹅源VII型毒株JS/7/05/Ch基因组的15,184 nt由一个T变为了C,5'端trailer与3'端leader的连续互补序列由8 nt变为12 nt,而其它4株基因Ⅲ型和VI型NDV均未发现该突变.通过RNA的二级结构分析,NDV基因组和反向基因组RNA的3'末端形成一个发卡结构.JS/7/05/Ch等3株NDV U→C(T→C)的突变位于发卡环上,不影响二级结构的形成,发卡环的RNA序列突变为3'-UCUC-5',与基因组3'端发卡环的3'-UCUUA-5'相似,推测可能影响了基因组RNA的复制速度.     

cDNA末端快速扩增技术的研究进展

cDNA末端快速扩增技术是一种基于多聚酶链式反应的技术 ,它的发展大大便利了应用其它方法获得的部分cDNA序列后克隆全长cDNA 5’和 3’末端的工作。不仅RACE方法能在短时间内得到完整的cDNA末端序列 ,而且一些截短的cDNA末端常常也能在RACE的过程中被扩增 ,而这些截短的产物破坏了全长cDNA克隆的获取。许多研究者对RACE的流程提出了改进方案 ,从而提高了该技术的效力。本文介绍了许多已发表的RACE技术关键步骤的改良 ,包括一些具体有效的操作流程 ,如RNA连接酶介导的RACE/连接锚定PCR等 ,还有其有效性的例证。     

水稻黄矮弹状病毒的leader区和trailer区的序列分析及其体内转录物的鉴定

克隆及测定了水稻黄矮病毒基因组RNA的 3′端leader区和 5′端trailer区的序列 .结果表明 ,RYSV的 3′leader长 2 0 3个核苷酸 ,5′trailer长 191个核苷酸 .两者末端的 9个核苷酸完全互补 ,可形成弹状病毒基因组RNA的典型的锅柄结构 .与其他弹状病毒的leader和trailer相比较 ,RYSV的leader和trailer较长 ,除leader的 3′末端和trailer的 5′末端序列具有一定的保守性外 ,其余的核苷酸序列无明显的同源性 .用 3′RACE方法在感染RYSV的水稻中检测出leader的转录物 ,并证明该leaderRNA具有polyA尾巴 .这是继苦苣菜黄脉病毒的leaderRNA后第 2例弹状病毒leaderRNA带polyA尾巴的报道 .用类似的方法没有检测到带polyA尾巴的trailer的正链转录物 .     

鹅源新城疫病毒ZJ1株全基因组的序列测定

依据GenBank上公布的新城疫病毒(NDV)的基因序列,自行设计了9对引物,运用反转录一聚合酶链反应(RT—PCR)获取覆盖NDV鹅源毒株ZJ1株全基因组的部分重叠片段。病毒RNA的5’及3’端的序列由cT)NA末端快速扩增技术(RACE)获取。整个病毒基因组序列由15192个碱基组成,比GenBank上公布的其它4个NDV毒株Beaudettec、B1、Lasota及Clone-30的全基因组序列长6个核苷酸。这6个核苷酸位于核衣壳蛋白(NP)基因内,相对于NDV毒株Lasota株全基因序列的1647nt～1648nt位。其它10个NDV毒株中的9个毒株也被验证具有此特征,且它们分属于3个不同的基因型。     

cDNA末端快速扩增技术新进展

全长cDNA的获得是基因克隆的重要内容,也是目前人类基因组研究中后 个重要方面,cDNA末端快速扩增（RACE）技术是以PCR为基础,从部分已知的cDNA序列扩增出其他未知部分的5′端和3′端的cDNA序列的一种方法,本文从RACE技术的基本原理方法出发,详细阐述了其存在的缺陷,并对RACE最新研究进展,如Marathon-PCR,Smart-PACE以及“电子”cDNA克隆等作一综述。     

貉源阿留申病毒全基因组测序及末端序列分子特征分析

貉源阿留申病毒(Raccoon dog and arctic fox amdoparvovirus,RFAV)是自然感染貉和蓝狐的新种阿留申病毒(Amdoparvovirus),为测序RFAV全基因组序列,预测分析RFAV末端发夹结构序列分子特征。本研究采用分段克隆成功获得3株长4832nt、4827nt、4830nt的RFAV全基因组序列,分别命名为RFAV-Y9J、RFAV-RD15、RFAV-HS-R,利用在线软件预测RFAV末端序列二级结构,并与水貂阿留申病毒(AMDV)末端序列进行同源性比对。结果显示阿留申病毒种间、种内3’末端基因组序列保守性强,均存在116nt的Y型发夹结构;RFAV-Y9J与RFAV-RD15毒株5′末端分别存在310nt、305nt的U型发夹结构,RFAV和AMDV种内5′末端基因组序列保守性强,而种间5′末端基因组序列有较大变异。本研究首次完整测序了RFAV的3′和5′末端序列,为其他种阿留申病毒的末端序列扩增提供一种有效方法,为构建RFAV的全基因组序列感染性克隆奠定了基础。     

蒙古冰草磷脂酶D基因cDNA 3'端的克隆及分析

目的：采用3’RACE方法对蒙古冰草磷脂酶D（PLD）基因3’端全序列进行了快速扩增和序列分析,为得到全序列及研究该基因的功能奠定基础。方法：实验以蒙古冰草幼叶为材料,利用RT-PCR技术得到PLD基因的部分cDNA序列,根据此序列设计一条特异性上游引物,反转录引物中的部分序列即M13PrimerM4作为下游引物,按3’RACE试剂盒（TaKaRa）操作流程进行PLD基因3’末端的快速扩增,成功获得993bp的3’RACE反应产物,采用DNAStar软件对扩增的PLD基因的3’RACE产物和中间片段进行拼接,最终获得2113bp的PLD基因3’端cDNA序列。结果：通过BLAST技术,发现该片段与许多植物磷脂酶D基因的同源性为47.0%～80.0%。结论：通过同源性比较,成功克隆了PLD基因cDNA3’端序列。     

一种新的cDNA末端快速扩增获取全长cDNA的方法

为克隆精子发生相关基因的全长cDNA,根据mRNA差异显示获得的ESTs设计引物。利用一种新的cDNA末端快速扩增方法（SMART RACE）扩增该EST的5′末端,并进行克隆测序,与cDNA差异显示获得ESTs拼接后,获得了三个新的全长cDNA。结果表明：SMAR RACE是一种简便、有效的克隆cDNA5′末端未知序列的技术。     

黄瓜花叶病毒山东株(CMV\|SD)RNA2全长cDNA克隆的构建及全序列分析

分别利用5’RACE和3’RACE确定了CMV\|SD RNA2的5’和 3’末端序列,在此基础上,利用RTPCR得到了RNA2的5’端一半的cDNA克隆pC25和3’端一 半的cDNA克隆pC23,并通过拼接构建了RNA2全长cDNA克隆pC2F。通过对pC25和pC23进行序列 测定,得到了RNA2的全序列。序列分析结果表明CMVSD RNA2由3048nt组成,其中存在2个 部分重叠的阅读框ORF1(79～2652nt)和ORF2(2414～2746nt),分别编码858aa的2a蛋白和111 aa的2b蛋白,并在2a蛋白的序列中发现了动植物病毒复制酶所特有的两个保守序列。该株系 RNA2核苷酸序列与分属CMV I亚组的Fny株系和II亚组的Q株系RNA2的核苷酸序列同源性分别 为917%和756%;2a蛋白的氨基酸序列同源性分别为938%和677%,2b蛋白的氨基酸序 列同源性分别为830%和513%。同源性比较的结果表明SD株系属于CMV I亚组。     

两株基因III型强毒新城疫的全基因组测序及其与I系苗的亲缘性分析

[目的]研究基因Ⅲ型新城疫(Newcastle disease,ND)强毒分离株Js/7/05/Ch和JS/9/05/Go与中等毒力疫苗株Mukteswar的亲缘性关系,分析三株新城疫病毒(Newcastle disease virus,NDV)的全基因差异.[方法]采用RT-PCR方法获得两株NDV分离株的全基因组核苷酸序列,与GenBank中公布的Mukteswar序列比对分析.[结果]强毒分离株与中等毒力疫苗株Mukteswar全基因组核苷酸同源性均为99.7%;病毒6个阅读框核苷酸序列与Mukteswar同源性为99.6%～99.9%;预测的8种病毒编码蛋白同源性在98.8%～99.8%.然而,测定MDT,ICPI和IVPI发现JS/7/05/Ch和JS/9/05/Go毒力明显强于Mukteswar,其中JS/7/05/Ch株的IVPI达到了2.18.[结论]综合3株NDV的遗传分析结果和已有的流行病学资料可以推断分离株JS/7/05/Ch和JS/9/05/Go是由疫苗株Mukteswar自然进化而来的返强毒株.因此,必须停止使用中等毒力疫苗以免造成更大的危害.     

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