重组细胞高产型H3N2猪流感病毒株的拯救

为拯救出一株能够在动物传代细胞中高水平复制的H3N2亚型猪流感疫苗株,利用反向遗传操作技术,将A/Goose/Dalian/3/01(H9N2)毒株的PB1、PA、NP、M、NS基因和A/PR/8/34毒株的PB2基因作为内部基因与猪流感病毒A/Swine/Henan/S4/01(H3N2)毒株的HA、NA基因进行重组,成功拯救出了具有高度细胞适应性毒株rH3N2株,该毒株接毒MDCK细胞60h后,血凝价可以达到1∶512,表明该毒株具有高度适应细胞繁殖特性,为H3N2亚型猪流感病毒细胞培养型疫苗的研制奠定了基础。     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

H1N1亚型猪流感病毒的拯救

利用8质粒拯救系统成功拯救出了猪流感病毒毒株A/Swine/TianJin/01/2004(H1N1)(A/S/TJ/04)。将猪流感病毒8个基因节段经RT-PCR合成cDNA后, 分别克隆到RNA聚合酶I/II双向表达载体PHW2000中, 构建成8个重组质粒。用8个重组质粒共转染COS-1细胞, 30 h后加入TPCK-胰酶至终浓度0.5 mg/mL。共转染48小时后收获COS-1细胞及其上清, 经尿囊腔接种9日龄SPF鸡胚。收获死亡鸡胚尿囊液并继续用SPF鸡胚传3代, 得到有感染性的病毒。经血凝、血凝抑制验、测序分析、电镜观察等均证实了A/S/TJ/04猪流感病毒的成功拯救。这是目前国内首次报道拯救出H1N1亚型猪流感病毒, 为进一步研究猪流感病毒基因组结构与功能的关系、流感跨种传播的机制以及构建新型猪流感疫苗株奠定了基础。     

重配技术构建高产H1N1型流感疫苗病毒株

目的以经典重配技术制备高产H1N1流感疫苗病毒株。方法以野生型A1/云南昆明/03/2009(H1N1)作为HA及NA基因的供体株,以WHO疫苗株A/Perth/16/2009(H3N2)作为高产基因供体株,共同感染SPF鸡胚,经抗H3及抗N2血清中和筛选法及终末稀释法筛选高产重配H1N1病毒。结果获得一株重配H1N1流感病毒株,病毒血凝滴度为1∶4 096,病毒滴度为7.8 lg EID50/mL,显示为鸡胚高产病毒株;血凝抑制结果为1∶1 024,单向免疫扩散试验结果为阳性,证明抗原性与野生株一致;基因测序结果表明重配株的HA及NA基因序列与野生株序列一致。结论构建了高产重配H1H1流感疫苗病毒株,并应用经典重配技术建立了制备高产流感疫苗病毒株的技术平台。     

2006～2007年流行流感病毒减毒疫苗株的构建

选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为骨架病毒,对其6个内部基因片段进行了全基因合成,同时人工引入5个氨基酸突变(PB1-391E,581G,661T,PB2-265S,NP-34G).HA和NA来源于2006-2007当年流行株A/New Caledonia/20/99(H1N1).8个基因片段通过与改造后的转录载体pAD3000连接,构建8个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS、pMDV-A-HA、pMDV-A-NA.6质粒与当年流行株的表面基因HA和NA进行"6 2"组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝性的冷适应减毒的重组A型人流感病毒.鸡胚尿囊液中重组病毒的血凝效价为l:279-1:210.构建的A/AA/6/60 6个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发奠定了基础.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

一株H1N1亚型猪流感病毒的分离鉴定及全基因序列分析

2006年从辽宁省某猪场采集具有流感症状猪鼻拭子共30份,经9~11日龄SPF鸡胚分离,对分离株进行了血凝试验、血凝抑制试验、RT-PCR亚型鉴定,全基因序列比对和接种试验动物试验。结果表明:该毒株具有凝集0.5%鸡红细胞的活性,且与抗猪流感H1标准血清发生血凝抑制反应,RT-PCR分别扩增得到全基因8个片段,利用DNAStar生物学软件进行序列分析,HA基因与GenBank登录的H1~H16中的H1基因序列同源性最高,NA基因与N1~N9中的N1基因序列同源性最高,故该LN株分离毒为猪流感H1N1亚型病毒。全基因序列中,除了M基因不是猪源的,其它基因片段均与国内H1N1亚型猪流感参照株高度同源,推测LN株可能是由类人和类禽谱系的流感病毒与古典猪谱系的流感病毒重排形成的;用该病毒接种试验动物可成功复制出猪流感典型症状。该病毒的分离鉴定及全基因组序列分析为我国进一步调查猪流感的流行规律提供了基础数据。     

H5N1亚型禽流感病毒拯救体系的建立

选择鸡胚高产的鸭源H5N1亚型禽流感病毒A/Duck/Shandong/093/2004株作为骨架病毒,在完成了全基因组序列测定基础上,设计合成的11对引物对病毒的8个基因分11段进行扩增。通过与转录载体PHW2000连接,构建A/SD/04的8个基因的拯救载体,经测序获得序列准确的拯救质粒:2412、42、243、244、245、246、247和248。A/SD/04的8质粒与PR8(H1N1)进行不同组合的拯救,获得8个均含A/SD/04 HA基因的H5重组流感病毒。鸡胚尿囊液中重组病毒的血凝效价在28~210,EID50在10-8.5~10-9之间,MDT在34~46h之间,均与野生A/SD/04(wt A/SD/04)相似。重组病毒对6周龄的SPF鸡的静脉接种指数(IVPI)与wt A/SD/04却有明显的差异,说明不同组合的内部基因影响病毒对鸡的致病力,但不影响病毒的鸡胚致死能力、对鸡胚的感染能力和病毒在鸡胚中的繁殖能力。构建的A/SD/04的8个质粒拯救系统,为H5N1的基因功能研究和新型疫苗开发奠定基础。     

神经氨酸酶茎部氨基酸缺失对H5N1亚型禽流感病毒生物学特性的影响

近年来H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)茎部15~20个氨基酸的自发缺失时有报道,突变对于AIV生物学特性的影响还没有得到系统研究。应用反向遗传操作技术,拯救获得5株具有不同NA茎部长度的H5N1/PR8重组AIV。重组病毒的内部基因和血凝素(HA)基因来源相同,NA基因来源不同,并在NA茎部进行20个氨基酸的删除或添加突变。通过研究其生物学特性发现,5株重组病毒在SPF鸡胚中繁殖良好,其EID50、MDT和平均病毒滴度相似;NA茎部长短影响病毒的解凝能力,长茎病毒红细胞解脱能力比短茎病毒强;NA茎部15或20个氨基酸删除突变提高了重组病毒在MDCK细胞上的繁殖能力,短茎病毒释放出的病毒粒子数量是长茎病毒的10~100倍,释放时间提前6~10h,短茎病毒在MDCK细胞上形成的空斑也明显比长茎病毒的空斑大。实验结果揭示了AIV NA茎部氨基酸缺失突变的生物学意义,NA茎部15或20个氨基酸删除突变增强了AIV的细胞适应性,可能与现阶段H5N1亚型AIV宿主范围进一步扩大有关。利用反向遗传技术成功拯救了5株H5N1/PR8重组流感病毒,为流感病毒基因功能研究和重组疫苗研究建立了技术平台。通过对AIV NA茎部氨基酸的删除突变提高了病毒在MDCK细胞上的繁殖产量,为流感病毒细胞苗的生产提供了新的思路。     

应用焦磷酸测序技术快速检测猪流感病毒金刚烷胺耐药性的分子标签

【目的】本研究旨在通过焦磷酸测序技术对我国分离的H1N1、H3N2、H9N2等3种基因型的10株猪流感病毒分离株进行金刚烷胺耐药性鉴定。【方法】流感病毒M2蛋白5个关键位点氨基酸残基(第26、27、30、31和34位)中的任何一个发生突变会导致抗流感病毒药物中金刚烷胺抗药性的产生。本研究利用焦磷酸测序技术对2004-2008年国内分离的10株猪流感病毒M基因金刚烷胺耐药性分子决定区进行了鉴定,并进行抗药性分析。【结果】基于M2蛋白基因保守区序列建立的焦磷酸测序技术能用于国内猪流感病毒的快速检测,且具有较好的特异性和重复性。抗药性分析表明10株猪流感病毒国内分离株中5株H1N1分离株全部耐药,主要存在M2蛋白的V27T、V27I或S31N位点的突变,而4株H3N2和1株H9N2猪流感病毒分离株在M2蛋白5个关键位点上均未出现变异,表明其对金刚烷胺敏感。【结论】基于M基因的焦磷酸测序技术可以用于我国猪流感病毒金刚烷胺耐药性快速鉴定。     

Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

RNA病毒的反向遗传学

反向遗传操作作为一种新兴技术在RNA病毒的研究中发挥着重要作用。本文介绍了RNA病毒反向遗传学的研究方法以及RNA病毒反向遗传技术的最新研究进展。     

Studies on non-disjunction of the major autosomes in Drosophila melanogaster. I. Methodology and rate of induction by x-rays for the compound second chromosome

The induction of non-disjunction by X-irradiation of the second chromosome in stage-7 oocytes of Drosophila melanogaster has been studied by employing isochromosome stocks. This makes the quantitative recovery possible of progeny resulting from disomic and nullosomic eggs. Determination of egg hatchability has been used to correct for varying degrees of segregation in males carrying different isochromosomes. Even at exposures as low as 250 R the frequency of non-disjunction is significantly higher than in the controls. No evidence has been obtained for the existence of a threshold. In the stage-7 oocytes, the induction of non-disjunction increased linearly with radiation exposure over a range of 250–3000 R and thus seems to reflect a single-hit event. These findings could be of significance for the evaluation of genetic radiation hazards in man. In slightly younger oocyte stages the induction of disomic eggs followed dose-square kinetics. The frequency of nullosomic eggs rises exponentially with radiation exposure, presumably as a consequence of increasing chromosome loss resulting from unrestituted breaks in each of the two maternal isochromosomes. Furthermore, it was observed that the late stage-7 oocytes were more sensitivie to the induction of non-disjunction than earlier stages.     

Influence of Water on the Primary Photosynthetic Activity of Rhodospirillum Rubrum in Reverse Micelles

The effect of water on the primary photosynthetic activity of purple bacterium Rhodospirillum rubrum was studied in Hexadecane-Tween-Spane (HTS)- and phospholipid (PLC)-reverse micelles. Reverse micelles offer the possibility of modulating the amount of water to which enzymes and multienzymatic complexes are exposed. Fast bacteriochlorophyll (BChl) fluorescence induction kinetics and reaction centre absorption changes at 820 nm were used as an assay for the functional transfer of bacterial cells into HTS-reverse micelles and bacterial photosynthetic complexes (BPC) into PLC-reverse micelles. Both the bacterial cells and BPC showed an increase in the rate of primary photosynthetic activity by increasing the concentration of water in the reverse micelles. The bacterial cells could be kept viable for many hours in HTS-reverse micelles in presence of 6% (v/v) water. NMR studies indicated that the photosynthetic activity was affected by the availability of water in reverse micelles. The bacterial cells in HTS or BPC in PLC reverse micelles could be used to further understand the influence of water on the organisation and function of photosynthetic complexes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of trypsin microenvironment on the rate constants of elementary stages of the hydrolysis reaction of N α-Benzoyl-L-arginine ethyl ester

The hydrolysis reaction of N ^α-benzoyl-L-arginine ethyl ester catalyzed by trypsin from pig pancreas was comparatively studied in an aqueous buffer solution and in the system of reversed micelles of Aerosol OT in octane (pH 8.5) to determine the mechanisms of influence of the enzyme microenvironment on the rate constants of the elementary stages of the enzymatic reaction. The temperature dependences of the catalytic constant k _cat and the rate constant of the second order k _cat/K _m (s, catalysis efficiency) allowed the determination of the rate constants and the activation energy of elementary stages of the enzymatic reaction. It was revealed that a decrease in the efficiency of catalytic action of trypsin in reverse micelles in comparison with an aqueous solution is first of all determined by a decrease in the rate constant of formation of the enzyme-substrate complex k ₁. Possible mechanisms of the effect of the microenvironment on the elementary stages of catalytic action of the enzyme are discussed.