犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCVDXMV)纤突蛋白基因进行了克隆和序列测定。该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白。与GenBank中已发表的11个CCV毒株s基因相比,s基因核苷酸序列同源性在40．2％-99．5％之间;推导的氨基酸序列同源性在15．9％-99．O％之间。DXMV株s基因变异区主要集中在该基因前1／2处,其中350．370、439．478、1718．1818三个区域碱基变异较大,而1060．1700区却十分保守。基于s全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和USpatent株亲源关系最近。推导的DXMV株s蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc．1弱毒多一个;其中第566．568位糖基化位点为多数强毒拥有而弱毒没有的。另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究。     

北京地区人呼吸道合胞病毒核蛋白基因的序列分析及原核表达

为了解北京地区流行的人呼吸道合胞病毒(HRSV)N蛋白基因的特征,并用大肠杆菌表达获得N蛋白,从2006年1月至3月收集的首都儿科研究所附属儿童医院急性呼吸道感染患儿标本中分离到7株HRSV毒株(3株A亚型,4株B亚型),分别经RT-PCR扩增得到HRSV N蛋白全基因,克隆至pUCm-T载体中并进行测序和分析;从重组质粒pUCm-N9968中经PCR扩增获得N基因,亚克隆入原核表达载体pET30a( )中,得到重组表达质粒pET30a-N9968,转化大肠杆菌BL21(DE3),IPTG诱导培养;SDS-PAGE和Western blot检测目的蛋白的表达和抗原性。经扩增并测序,7株HRSV N蛋白基因全长均为1 176 bp,编码一个含391个氨基酸的蛋白;北京地区7株RSV分离株的N蛋白基因之间的核苷酸和推导的氨基酸同源性分别在85.4%~99.7%之间和95.4%~100%之间,进一步证明N蛋白基因的高度保守性。经诱导培养产生大量带6个组氨酸标记的重组N蛋白,主要以包涵体形式存在。N蛋白粗提物经Ni2 亲和层析获得较理想纯化。Western blot结果显示,所表达的蛋白能与特异性单克隆抗体和人血清反应。说明本研究构建的重组pET30a-N9968原核表达质粒使N蛋白获得了高效表达,并且具有特异的抗原活性,为今后进一步研究奠定了基础。     

犬冠状病毒南京株M基因的同源重组分析

对来自腹泻犬粪样的犬冠状病毒(CCV)南京株NJ17株及参考株1-71的M基因进行了克隆、测序,并与GenBank中所有已知CCV毒株及同亚群的猪冠状病毒(TGEV)和猫冠状病毒(FCoV)代表株的M基因进行了同源性比较和系统进化分析,同时对M蛋白的结构和功能进行了预测分析.结果表明,CCV1-71与近年在中国分离到的CCV毒株V1、V2及大熊猫源的毒株具有98.9%～99.5%的同源性,说明这些毒株可能是来自同一毒株的准种.NJ17与其他中国分离株及国外分离株的同源性为87.0%～91.9%,显示国内可能存在一个相对独立进化的CCV毒株.序列比较发现,所有CCV毒株在可能的同源重组"热点"区内都有一个CTTTAG序列,与鸡传染性支气管炎病毒同源重组模板交换位点附近的特征序列相似.CCV NJ17株M蛋白在N端50氨基酸序列与FCoV 79-1683同源性高,而在后212氨基酸序列与TGEV同源性高,提示该毒株可能在M基因上曾经发生过不同病毒的同源重组.CCV M蛋白的结构及功能预测表明,所有毒株都具有分泌型信号肽,有4个螺旋跨膜区,N末端和C末端均位于膜内.M蛋白的两末端具有较强的抗原性,M蛋白上存在多种功能性氨基酸修饰位点且相对保守.N末端的氨基酸变异很大,但是功能性修饰位点相对保守,提示N末端的功能可能与构象有关.     

犬冠状病毒S蛋白主要抗原区基因片断的表达及免疫原性

应用Pichia pastoris酵母表达了犬冠状病毒大熊猫野毒株(CCV DXMV)S蛋白主要抗原区基因片断。用特异性引物扩增出CCV DXMV株S1基因片断,并将其克隆到pGEM-T载体中得到pTS1。用KpnI和Notl双酶切pTS1回收目的基因S1定向克隆到pPICZCαA中,构建出重组质粒pPICZCαAS1。将pPICZCαASl用SacI内切酶线性化后,电转化感受态GS115酵母细胞,用PCR法筛选阳性重组子。用1％的甲醇诱导重组酵母菌,取培养物上清进行重组蛋白的检测。结果重组酵母菌培养物上清用SDS-PAGE电泳可检测到相对分子量为106kDa大小的重组蛋白,Westem-blot证实该重组蛋白可以与CCV多克隆抗体发生特异性血清学反应。凝胶薄层扫描分析表明,3株重组酵母菌在1％甲醇诱导144h后,重组蛋白S1表达量约占培养物上清总蛋白量的6．6-8．6％左右。用重组蛋白S1免疫BALB／C小鼠3次后,小鼠血清CCV中和抗体可达1：8-1：16,表明重组S1蛋白具有一定的免疫原性。     

犬冠状病毒南京株M基因的同源重组分析

对来自腹泻犬粪样的犬冠状病毒(CCV)南京株NJ17株及参考株171的M基因进行了克隆、测序,并与GenBank中所有已知CCV毒株及同亚群的猪冠状病毒(TGEV)和猫冠状病毒(FCoV)代表株的M基因进行了同源性比较和系统进化分析,同时对M蛋白的结构和功能进行了预测分析。结果表明,CCV171与近年在中国分离到的CCV毒株V1、V2及大熊猫源的毒株具有98.9%～99.5%的同源性,说明这些毒株可能是来自同一毒株的准种。NJ17与其他中国分离株及国外分离株的同源性为87.0%～91.9%,显示国内可能存在一个相对独立进化的CCV毒株。序列比较发现,所有CCV毒株在可能的同源重组“热点”区内都有一个CTTTAG序列,与鸡传染性支气管炎病毒同源重组模板交换位点附近的特征序列相似。CCVNJ17株M蛋白在N端50氨基酸序列与FCoV791683同源性高,而在后212氨基酸序列与TGEV同源性高,提示该毒株可能在M基因上曾经发生过不同病毒的同源重组。CCVM蛋白的结构及功能预测表明,所有毒株都具有分泌型信号肽,有4个螺旋跨膜区,N末端和C末端均位于膜内。M蛋白的两末端具有较强的抗原性,M蛋白上存在多种功能性氨基酸修饰位点且相对保守。N末端的氨基酸变异很大,但是功能性修饰位点相对保守,提示N末端的功能可能与构象有关。     

流感病毒nsl基因的克隆及其原核表达载体的构建

为了解H5N1亚型流感病毒株的nsl基因特性及其规模制备NSl蛋白,首先将病毒在鸡胚中传代,从收获的尿囊液中提取RNA,采用RT-PCR技术扩增流感病毒全长ns基因。测序显示H5N1亚型流感病毒NSlcDNA全长678bp,编码225个氨基酸。BLAST分析表明,Qa/ST／852,01(H5N1)病毒株nsl基因与近年来从华南地区分离的禽H5N1毒株的nsl基因有很高的同源性。之后采用PCR方法扩增nsl基因的cDNA片段,将其克隆到pGEX-4T-3载体中,与谷胱甘肽巯基转移酶(GST)基因融合,构建重组质粒pGEX-4T-3／NSlcDNA,转化大肠杆菌BL21(DE3)并进行诱导表达。SDS,PAGE和凝胶扫描分析,GS~NSl融合蛋白在大肠杆菌中获得了高效表达,并且以可溶形式存在,重组融合蛋白的表达量占菌体总蛋白的28．5％,表达产物经亲和层析纯化后蛋白质纯度达96％以上。经免疫印记证实重组融合蛋白可以被GST特异性抗体所识别。该表达载体的构建为获得大量NSl蛋白进行功能研究及抗体制备提供了基础。     

小鼠肝炎病毒N基因的原核表达和免疫活性初步分析

目的对小鼠肝炎病毒（mouse hapetitis virus）A59毒株核蛋白（N）基因进行了克隆、表达和重组蛋白的免疫活性分析。方法根据GenBank公布的MHV-A59 N基因序列（AY700211）,设计了一对特异性引物,通过RT-PCR扩增出N基因的主要抗原片段NS,将目的片段纯化后与pGEM-T-easy载体连接得到重组质粒pTN,双酶切回收目的基因片段克隆到原核表达载体pGEX-6p-1中,构建了重组质粒pGEX-NS,转化大肠杆菌BL21,用IPTG进行了诱导表达。对表达裂解物进行SDS-PAGE和Western-blotting验证。结果表达产物相对分子质量约56×103与预期相符;能与MHV阳性血清发生特异性反应,出现单一条带。结论原核表达的NP（S）蛋白有良好的抗原性,为检测小鼠肝炎病毒抗体的ELISA检测方法的研究奠定了基础。     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCV DXMV)纤突蛋白基因进行了克隆和序列测定.该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白.与GenBank中已发表的11个CCV毒株S基因相比,S基因核苷酸序列同源性在40.2%～99.5%之间;推导的氨基酸序列同源性在15.9%～99.0%之间.DXMV株S基因变异区主要集中在该基因前1/2处,其中350-370、439-478、1718-1818三个区域碱基变异较大,而1060-1700区却十分保守.基于S全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和US patent株亲源关系最近.推导的DXMV株S蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc-1弱毒多一个;其中第566-568位糖基化位点为多数强毒拥有而弱毒没有的.另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究.     

犬冠状病毒S蛋白主要抗原区基因片断的表达及免疫原性

应用Pichiapastoris酵母表达了犬冠状病毒大熊猫野毒株(CCV DXMV)S蛋白主要抗原区基因片断.用特异性引物扩增出CCVDXMV株S1基因片断,并将其克隆到pGEM-T载体中得到pTS1.用KpnI和NotI双酶切pTS1回收目的基因S1定向克隆到pPICZαA中,构建出重组质粒pPICZ αAS1.将pPICZαAS1用SacI内切酶线性化后,电转化感受态GS115酵母细胞,用PCR法筛选阳性重组子.用1%的甲醇诱导重组酵母菌,取培养物上清进行重组蛋白的检测.结果重组酵母菌培养物上清用SDS-PAGE电泳可检测到相对分子量为106kDa大小的重组蛋白,Western-blot证实该重组蛋白可以与CCV多克隆抗体发生特异性血清学反应.凝胶薄层扫描分析表明,3株重组酵母菌在1%甲醇诱导144h后,重组蛋白S1表达量约占培养物上清总蛋白量的6.6-8.6%左右.用重组蛋白S1免疫BALB/C小鼠3次后,小鼠血清CCV中和抗体可达18-116,表明重组S1蛋白具有一定的免疫原性.     

鸡传染性支气管炎病毒核蛋白基因在大肠杆菌中的表达及抗原性初步研究

为了研究重组鸡传染性支气管炎病毒核蛋白能否作为群特异性诊断抗原加以应用,将鸡传染性支气管炎病毒国内分离株IBV—LX4核蛋白基因亚克隆于大肠杆菌原核表达载体pPROEX^TM HT中。构建拟表达重组鸡传染性支气管炎病毒核蛋白的重组质粒pPROEX^TM HT—N。经核苷酸序列测定后,阳性质粒转化大肠杆菌DH5α,并进行诱导表达。表达产物经SDS-PAGE、Western blot鉴定,表明核蛋白在大肠杆菌中获得了表达,表达产物是分子量分别为约56kD和45kD的蛋白,其中分子量为56kD的蛋白表达量约为菌体总蛋白的13％。包涵体被6mol盐酸胍裂解后。通过镍离子亲和树脂进行了纯化,并用纯化的分子量为56kD的重组蛋白免疫新西兰白兔,所获得的兔抗鸡传染性支气管炎病毒核蛋白多克隆抗体,分别与不同致病型的鸡传染性支气管炎病毒进行琼脂扩散反应,结果表明。该多克隆抗体可与各不同致病型毒株发生反应。这初步表明重组鸡传染性支气管炎病毒核蛋白。可作为群特异性诊断抗原用于该病毒的诊断中。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Summary Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l⁻¹ glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l⁻¹) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.     

Specificity of action of piperidylcholine derivatives as substrates of cholinesterases of various origin

The review deals with study of enzymologic properties of a novel highly specific acetylcholinesterase substrate, N-(β-acetoxyethyl) piperidinium iodomethylate (“piperidylcholine”), and its 30 derivatives that were tested as effectors of cholinesterases of mammals and various species of Pacific squids. It was proven for the first time that responsible for specificity of action was structure of cyclic ammonium grouping of the alcohol part of molecule of the ester substrate. Analysis of specificity is performed based on enzymatic hydrolysis parameters—activity of catalytic center of cholinesterases and bimolecular constant of the reaction rate that are determined at optimal and low substrate concentrations. Among the specially synthesized group of thioester compounds there is revealed one more highly specific acetylcholinesterase substrate—N-(β-acetoxyethyl) piperidinium.     

Interconnection of parameters of regulation system of lipid peroxidation and of morphophysiological parameters of mouse liver

A complex analysis of seasonal fluctuations of the mean group parameters of the system of regulation of lipid peroxidation has been performed in liver of Balb/c mice. Association of lipid characteristics and morphophysiological parameters is studied in the Balb/c mouse liver. An inter-connection is revealed between the liver index and the amount of lysoforms of phospholipids, the scale and character of the interconnection differing essentially depending on proportion of phos-phatidylcholine in mouse liver phospholipids.