犬瘟热病毒南京株H蛋白基因的克隆与表达

根据发表的犬瘟热病毒(CDV)参考株Ondetstepoort的序列设计一对引物,以犬瘟热病毒南京株(CDVNJ．15)感染的Vero细胞总RNA为模板,RT-PCR扩增出1．9kb的全长H蛋白基因,双酶切该全长基因,得到1058bp片段,以正确的阅读框架定向克隆于pET-28b( )中,然后将重组质粒转化宿主菌RosettaTM,在37℃1．0mmol／LIPTG诱导下获得良好表达。经SDS—PAGE鉴定,表达的融合蛋白质约38kDa,与预期值一致。免疫转印试验显示,该重组蛋白可被CDVOnderstepoort兔抗血清识别,表明该重组蛋白具备部分抗原性。     

犬瘟热病毒小熊猫株反向遗传系统的构建及其鉴定

以驯化致弱的犬瘟热病毒小熊猫株(Canine distemper virus,CDV)为模板,构建犬痘热病毒感染性cDNA克隆.对其全基因组序列测定后,用RT-PCR的方法获得组成全长基因的7个片段,通过酶切、拼接将7段CDVcDNA序列插入到真核表达载体pCI的MCS上,构建犬瘟热病毒小熊猫株的全长cDNA质粒(pCI-CDV-LP),同时分别克隆CDV小熊猫株N、P、L蛋白ORF构建三个辅助质粒.酶切鉴定和序列测定表明,pCI载体中插入的核酶及CDV cDNA序列正确无误,使用转染试剂Lipofectamine TM 2000将全长质粒和三个辅助质粒共转染中国仓鼠肾细胞(BSR),经RT-PCR、间接免疫荧光和病毒感染VERO-SLAM细胞试验鉴定,成功拯救出CDV小熊猫株,显示CDV小熊猫株反向遗传系统构建完成,为犬瘟热病毒致病机理及免疫研究奠定基础.     

犬瘟热病毒核蛋白基因重组腺病毒的构建、鉴定与表达

核蛋白基因(N)位于犬瘟热病毒基因组的108-1679位置处,是保守性较强的免疫原性蛋白,因此选择N基因作为目的基因,利用酶切、连接等方法构建了含犬瘟热病毒核蛋白基因的穿梭质粒pVAXAE3LPN.以含CAV-2 SY株全基因组的pPoly2-CAV-2为载体,构建了重组质粒pCAV-2-CDVLPN,利用脂质体介导法转染MDCK细胞,转染三次后,细胞出现了典型的腺病毒样病变.电镜负染、切片观察,酶切、PCR.扩增及测序鉴定的结果表明表达犬瘟热核蛋白基因的重组犬2型腺病毒构建成功,表达的核蛋白分子量为58kDa.     

犬瘟热病毒核衣壳蛋白基因片段的克隆和表达

目的　构建pMal N重组表达载体 ,转化E .coliDH5α ,诱导表达犬瘟热病毒 (CDV)重组核衣壳 (N)蛋白。方法　采用RT PCR技术 ,从CDVRNA中扩增编码N蛋白的基因片段 ,通过连接反应 ,构建重组克隆载体和重组表达载体 ,转化感受态。E .coliDH5α细胞。通过IPTG诱导表达CDV重组N蛋白。结果　扩增出约 1 7kbCDV全长的主要结构蛋白N蛋白基因 ,通过PCR获得 5 36bpN蛋白基因片段。将N蛋白基因片段克隆入原核表达载体pMal C2 ,表达产物麦芽糖结合蛋白 (MAP)与N蛋白的融合蛋白的相对分子质量约 6 0× 10 3,与预期大小一致。结论　构建的pMal N重组载体所表达的CDVN蛋白为进一步研究CDV的特异、敏感的抗体检测方法打下基础     

犬瘟热病毒核蛋白基因重组腺病毒的构建、鉴定与表达

核蛋白基因(N)位于犬瘟热病毒基因组的108—1679位置处,是保守性较强的免疫原性蛋白,因此选择N基因作为目的基因,利用酶切、连接等方法构建了含犬瘟热病毒核蛋白基因的穿梭质粒pVAX?E3LPN。以含CAV-2SY株全基因组的pPoly2-CAV-2为载体,构建了重组质粒pCAV-2-CDVLPN,利用脂质体介导法转染MDCK细胞,转染三次后,细胞出现了典型的腺病毒样病变。电镜负染、切片观察,酶切、PCR扩增及测序鉴定的结果表明表达犬瘟热核蛋白基因的重组犬2型腺病毒构建成功,表达的核蛋白分子量为58kDa。     

流感病毒ns1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ns1基因特性及其规模制备NS1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NS1 cDNA全长678bp,编码225个氨基酸.BLAST分析表明,Qa/ST/852/01(H5N1)病毒株ns1基因与近年来从华南地区分离的禽H5N1毒株的ns1基因有很高的同源性.之后采用PCR方法扩增ns1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/NS1 cDNA,转化大肠杆菌BL21(DE3)并进行诱导表达.SDS-PAGE和凝胶扫描分析,GST-NS1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NS1蛋白进行功能研究及抗体制备提供了基础.     

甲3型流感病毒M2基因在痘苗病毒天坛株中的表达

为获得表达甲3型流感病毒(H3N2)M2蛋白的重组天坛株痘苗病毒RVJ1175M2,使用PCR方法扩增流感病毒全长M2基因,将其克隆到天坛株痘苗病毒同源重组质粒pJSC1175中,获得重组质粒pJSC1175M2,通过与痘苗病毒载体同源重组,构建了含流感病毒M2基因的重组痘苗病毒株RVJ1175M2。PCR检测结果证明,流感病毒(H3N2)M2蛋白基因准确插入到天坛株痘苗病毒TK区;Western blot、免疫荧光和流式细胞计数表明重组病毒RVJ1175M2可以有效地表达M2蛋白,表达的M2蛋白有两条带,分别为15kD和13kD,与相关文献报道一致;M2蛋白可有效分布在感染细胞的细胞膜上。这些结果表明重组痘苗病毒株RVJ1175M2可以有效地表达流感病毒M2蛋白,为使用表达M2蛋白的不同类型疫苗进行广谱流感疫苗效果的比较研究奠定了基础。     

犬瘟热病毒抗原表位T’TB和犬细小病毒VP2蛋白的共表达及免疫小鼠特异性抗体的测定

应用PCR方法扩增犬细小病毒VP2基因,将其克隆至Bac-to-Bac杆状病毒表达系统中的转移载体pFastBacHTc上,命名为pFastBacHTc-VP2,将人工合成的犬瘟热病毒抗原表位基因T'TB克隆至VP2基因的上游,命名为 pFastBacHTc-T'TB-VP2.进而转化含穿梭载体Bacmid的感受态细胞DH10Bac中,获得携带犬瘟热病毒T'TB细胞表位和犬细小病毒VP2基因的重组转染质粒Bacmid-BacHT-T'TB-VP2,将其转染昆虫细胞Sf-9后获得融合重组T'TB-VP2蛋白,大小约为70 ku.经Western blot分析,结果显示:表达的蛋白具有良好的免疫原性.表达的重组蛋白在无佐剂参与的情况下,按确定的免疫程序免疫6～8周龄的BALB/c小鼠,检测小鼠的体液免疫学指标.结果表明:表达蛋白能诱导小鼠产生抗CDV和CPV的特异性中和抗体.本实验为重组犬瘟热与犬细小病毒新型亚单位疫苗的研制奠定了重要的物质基础.     

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。     

流感病毒ns1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ns1基因特性及其规模制备NS1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NS1cDNA全长678bp,编码225个氨基酸。BLAST分析表明,Qa/ST/852/01(H5N1)病毒株ns1基因与近年来从华南地区分离的禽H5N1毒株的ns1基因有很高的同源性。之后采用PCR方法扩增ns1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/NS1cDNA,转化大肠杆菌BL21(DE3)并进行诱导表达。SDS-PAGE和凝胶扫描分析,GST-NS1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NS1蛋白进行功能研究及抗体制备提供了基础。     

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.     

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。     

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.     

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.     

医疗机构合理用药评价模型的构建研究

目的 针对医疗机构的合理用药水平进行评价研究。方法 根据医疗机构合理用药的具体要求,构建医疗机构合理用药评价指标体系,采用基于模糊群决策的方法和多指标评价分析法构建医疗机构合理用药评价模型。结果 构建了基于模糊群决策的医疗机构合理用药评价模型,并通过实例分析证明了评价模型的可行性。结论 建立的基于模糊群决策的医疗机构合理用药评价模型能够对医疗机构的合理用药水平进行科学评价,为提高医疗机构合理用药水平奠定基础。     

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.