H9N2型禽流感病毒HA基因的克隆及其DNA疫苗的动物免疫试验

血凝素(HA)是决定禽流感病毒的毒力强弱和免疫原性的主要蛋白质．根据已发表的149亚型AIV的HA基因序列,设计合成了1对H9HA特异引物,以AIV A／Chicken／Henan／1／1999／(H9N2)核酸为模板,通过RT-PCR扩增出1条1．6kb cDNA片段。将HA基因插入pVAXI中,构建了真核表达质粒pVAX-H9,采用活体电击法免疫3周龄SPF鸡10只,剂量为50μg/只,3周后加强免疫一次,5周后以100倍鸡胚感染剂量(EID)的HA基因同源病毒对所有鸡进行攻毒,其间每周检测抗体水平变化,6周后以棉拭子进行泄殖腔病毒分离,结果为攻毒后免疫组鸡HI效价为9log2-10log2,对照组为2log2-4log2;免疫组病毒分离数为0／10。对照组为10／10,表明所构建的HA基因表达质粒可作为基因疫苗诱导鸡产生免疫保护反应。     

鸡新城疫病毒HeB02分离株F基因的克隆及其DNA疫苗的研究

根据GenBank报道的鸡新城疫病毒F基因序列设计了一对引物,以鸡新城疫病毒HeB02分离株基因组为模板,通过RT-PCR扩增出了1·66kb左右的F基因片段,序列分析表明HeB02株F基因与国内标准强毒株F48E9及弱毒疫苗La Sota和Clone30的F基因核苷酸序列的同源性分别为88·1%、84·9%和83·8%。将HeB02株F基因插入真核表达载体pVAX1中,构建了真核表达质粒pSV-F,通过脂质体转染COS-7细胞,SDS-PAGE分析可见表达的特异蛋白条带;Western blot、ELISA和中和试验检测结果表明:真核表达的蛋白与抗新城疫病毒的抗体发生特异性反应,说明F蛋白具有很好的免疫原性。采用活体电击法以真核表达质粒pSV-F免疫3周龄SPF鸡,剂量为50μg/只,3周后加强免疫1次,5周后以100倍鸡胚感染剂量(EID)的F基因同源病毒对所有鸡进行攻毒,攻毒前后每周分别以喉拭子进行病毒分离和HI效价测定。结果显示对照组在攻毒前一直没有检测到抗体效价,攻毒后检测效价为3·0log2±1·40,并且于攻毒后第9天全部死亡;活疫苗组和实验组免疫后第2周检测到抗体效价,第5周最高,HI效价分别为8·3log2±1·30和7·2log2±1·23,攻毒1周后HI效价分别达9·8log2±1·55和8·9log2±1·77,极显著高于对照组(P<0·01)。免疫组未分离到新城疫病毒,对照组全部分离到新城疫病毒。表明所构建的F基因真核表达质粒可作为候选基因疫苗诱导鸡产生免疫保护反应。     

利用Cre-loxP自动敲除H5亚型禽流感病毒血凝素重组鸡痘病毒中的报告基因

研究去除重组鸡痘病毒中的报告基因,构建一株只含目的基因的重组毒。将H5亚型AIV的HA基因作为靶基因,两侧含loxP序列的GFP表达盒插入鸡痘病毒重组臂基因构建了转移质粒载体,将其与脂质体混合转染CEF细胞,获得了表达H5和GFP的鸡痘病毒重组体。通过二次转染,利用Cre酶自动敲除重组病毒中的GFP基因,最终获得了只含H5血凝素基因表达盒的重组鸡痘病毒。免疫荧光和病毒滴度测定结果表明,经过连续传代后重组病毒仍然稳定复制并表达H5血凝素。用10⁵PFU和2×10⁵PFU rFPV H5免疫SPF鸡,28d后,免疫组鸡抗体平均滴度(HI)分别达到4log 2和4.5log 2,结果表明,H5HA基因重组病毒能刺激鸡群产生较高特异抗体。     

抗禽流感病毒多表位DNA疫苗的构建及其免疫效力研究

多表位DNA疫苗是建立在常规DNA疫苗基础上的一种新型疫苗。它是用表位作免疫原,这样就比较容易在一个表达载体上克隆病原体的多个抗原基因中具有免疫活性的部分。本试验以H5N1亚型禽流感病毒的HA和NP基因及其表位为基础构建了4个重组质粒：1 pIRES/HA（表达全长的HA基因）;2 pIRES/tHA（只表达HA基因的主要抗原表位区）;3 pIRES/tHANpep（融合表达HA基因的抗原表位区和NP基因的3个CTL表位）;4 pIRES/tHANpep-IFN-γ（用鸡的IFN-γ基因取代质粒pIRES/tHANpep中的neo基因）。分别用这4个重组质粒和空载体质粒pIRES1neo肌注免疫30日龄SPF鸡。免疫3次,间隔为2周,每次每只鸡的剂量为200μg。第3次免疫后两周以高致病性禽流感病毒H5N1强毒攻击,免疫及攻毒前后均采血检测HI抗体效价和外周血CD4⁺、CD8⁺T细胞的变化。结果发现,攻毒前各质粒免疫组均检测不到HI抗体,攻毒后1周存活鸡HI抗体效价迅速升高到64～256。流式细胞仪检测显示外周血CD4⁺、CD8⁺T细胞在疫苗免疫后都有不同程度的升高。空载体质粒对照组鸡（10只）在攻毒后3～8 d内全部死亡,其他各重组质粒免疫组鸡都获得了部分保护,保护率分别是：pIRES/HA组为545%（6/11）,pIRES/tHA组为30%（3/10）,pIRES/tHANPep组为36.3%（4/11）, pIRES/tHANPepIFNγ组为50%（5/10）。这些结果表明我们构建的多表位DNA疫苗能够诱导机体产生特异性免疫应答,并在同型禽流感强毒攻击时对鸡只提供了一定的保护。     

利用反向遗传技术产生8基因全禽源流感病毒疫苗候选株

利用反向遗传技术将含有A/Chicken/Shanghai/F/98(H9N2)株禽流感病毒(avian influenza virus,AIV)的6个内部基因与H5N1亚型AIV的2个表面基因HA和NA共转染COS-1细胞,产生了6 2全禽源的重配AIV。将H5N1亚型AIV的HA基因经基因突变致弱,然后将A/Chicken/Shanghai/F/98(H9N2)AIV的6个内部基因的cD-NA和以上致弱的禽源HA基因及NA基因的cDNA分别克隆到转录/表达载体pHW2000中,构建成8个转录/表达质粒。将8个质粒共转染COS-1细胞,24h后收获细胞及上清接种SPF鸡胚,72~90h后鸡胚死亡,收取鸡胚尿囊液进行血凝、血凝抑制试验、序列分析、病毒致病性试验和动物免疫保护试验,最终证实产生了致弱的全禽源AIV疫苗候选株。     

表达禽流感病毒M2基因的重组马立克氏病病毒的构建

将禽流感病毒M2基因克隆于真核表达质粒pIRES-EGFP中,使其位于pCMV启动子的调控下,并与绿色荧光蛋白基因（EGFP）串联后,将上述串联基因插入到含MDV CVI988的非必需区US基因的重组质粒pUS2中,构建带标记的重组质粒,然后将此重组质粒转染感染了MDV CVI988的鸡胚成纤维细胞,利用同源重组的方法,筛选了表达禽流感病毒M2基因的重组病毒MDV1。经PCR、Dot-blotting,Western-blotting等实验的结果表明,禽流感病毒M2基因的确插入到MDV1（CVI988）基因组中并获得表达。重组MDV1免疫1日龄SPF鸡21天后,用ELISA可检测到M2蛋白的特异性抗体。接种了重组病毒rMDV的鸡体内针对H9N2疫苗血凝素的抗体滴度(p<0.05)明显提高,以禽流感病毒AIV A/Chicken/Guangdong/00（H9N2）攻毒后进行病毒重分离试验的结果发现,重组病毒能有效地降低病毒的排出量(p<0.01),说明该重组病毒可以用于防制禽流感的免疫。     

共表达H5N1、H7N1亚型AIV HA与鸡IL-18多价重组鸡痘病毒免疫保护性研究

用本实验室构建的重组鸡痘病毒rFPV-H5HA-IL18、rFPV-H5HA-H7HA-IL18和rFPV-H5HA,经翼蹼免疫1日龄SPF鸡和7日龄商品Leghorn蛋鸡,同时以H5亚型AIV全病毒灭活疫苗作为对照.免疫后测定HI抗体效价、淋巴细胞转化指标、重组疫苗对增重的影响、免疫后的攻毒保护效力、免疫后的抑制排毒情况.免疫后不同时间分别测定特异性抗体和淋巴细胞刺激指数.试验结果表明,3株重组鸡痘病毒株均能诱导鸡体产生血凝抑制抗体(HI);共表达鸡IL-18的rFPV-H5HA-IL18和rFPV H5HA-H7HA-IL18诱导商品蛋鸡的细胞免疫水平明显高于非共表达鸡IL-18的rFPV-H5HA.重组鸡痘疫苗免疫SPF和商品蛋鸡后第21d进行攻毒实验,rFPV-H5HA-IL18和rFPV-H5HA-H7HA-IL18免疫攻毒保护率达10/10,rFPV-H5HA免疫攻毒保护率达9/10,与常规疫苗相当.免疫的商品蛋鸡于攻毒后7d采集泄殖腔棉试子样品,检测排毒情况.结果表明,rFPV-H5HA-IL18、rFPV-H5HA-H7HA-IL18免疫组在攻毒后第7d无排毒,其抑制免疫鸡排毒效果优于常规疫苗和单独表达HA的rFPV-H5HA重组鸡痘病毒.rFPV-H5HA-IL18和rFPV-H5HA-H7HA-IL18免疫组鸡,在14日龄时的体重明显高于rFPV-H5HA免疫组和常规疫苗对照免疫组,表明共表达的鸡IL-18能降低鸡痘病毒载体对雏鸡增重的影响.     

共表达H5N1、H7N1亚型AIV HA与鸡IL-18多价重组鸡痘病毒免疫保护性研究

用本实验室构建的重组鸡痘病毒rFPV-H5HA-IL18、rFPV-H5HA-H7HA-IL18和rFPV-H5HA,经翼蹼免疫1日龄SPF鸡和7日龄商品Leghorn蛋鸡,同时以H5亚型AIV全病毒灭活疫苗作为对照.免疫后测定HI抗体效价、淋巴细胞转化指标、重组疫苗对增重的影响、免疫后的攻毒保护效力、免疫后的抑制排毒情况.免疫后不同时间分别测定特异性抗体和淋巴细胞刺激指数.试验结果表明,3株重组鸡痘病毒株均能诱导鸡体产生血凝抑制抗体（HI）;共表达鸡IL-18的rFPV-H5HA-IL18和rFPV H5HA-H7HA-IL18诱导商品蛋鸡的细胞免疫水平明显高于非共表达鸡IL-18的rFPV-H5HA.重组鸡痘疫苗免疫SPF和商品蛋鸡后第21d进行攻毒实验,rFPV-H5HA-IL18和rFPV-H5HA-H7HA-IL18免疫攻毒保护率达10/10,rFPV-H5HA免疫攻毒保护率达9/10,与常规疫苗相当.免疫的商品蛋鸡于攻毒后7d采集泄殖腔棉试子样品,检测排毒情况.结果表明,rFPV-H5HA-IL18、rFPV-H5HA-H7HA-IL18免疫组在攻毒后第7d无排毒,其抑制免疫鸡排毒效果优于常规疫苗和单独表达HA的rFPV-H5HA重组鸡痘病毒.rFPV-H5HA-IL18和rFPV-H5HA-H7HA-IL18免疫组鸡,在14日龄时的体重明显高于rFPV-H5HA免疫组和常规疫苗对照免疫组,表明共表达的鸡IL-18能降低鸡痘病毒载体对雏鸡增重的影响.     

用电穿孔方法研究 H5N1 禽流感病毒DNA 疫苗对动物的免疫保护作用

为了研究 H5N1 DNA 疫苗对小鼠和鸡的保护效率,用 H5N1 禽流感病毒 HA DNA 疫苗免疫 BALB/c 小鼠和 SPF 鸡 . 小鼠和鸡分别经电穿孔和肌肉注射免疫两次,间隔为 3 周 . 二次免疫后,用致死量的同源病毒进行攻毒实验 . 空白对照组在攻毒后全部死亡,而经电穿孔免疫的小鼠和鸡均获得了完全的保护,并能有效地抑制病毒在小鼠肺脏和鸡泄殖腔的繁殖 . 同时,电穿孔免疫的小鼠和鸡均产生了高水平的特异性抗体 . 经电穿孔免疫的小鼠攻毒后 CTL 反应明显加强 . 这些结果表明, HA DNA 疫苗能有效地保护小鼠和鸡对禽流感病毒的感染,同时也表明电穿孔免疫是 DNA 疫苗免疫的有效途径之一 .     

敲除Meq基因的重组鸡马立克氏病毒与CVI988/Rispens疫苗株免疫保护效力的比较

【目的】比较敲除meq基因的马立克氏病毒(MDV)与标准疫苗株CVI988/Rispens对MDV超强毒GX0101攻毒的免疫保护作用。【方法】本实验将1日龄SPF鸡120只随机分成4组,每组30只,分别饲养在正压过滤空气的SPF动物饲养隔离罩内。1日龄时,第1组鸡以2000PFU/只的剂量颈部皮下接种SC9-1;第2组鸡以2000PFU/只的剂量颈部皮下接种CVI988/Rispens;第3、4组为不免疫攻毒对照组。免疫接种后5 d后,第1、2、3组分别以2000PFU/只的剂量腹腔接种MDV GX0101。饲养至90日龄,记录死亡情况,对死亡鸡只剖检,并取疑似马立克特有病变脏器做病理切片。期间,检测不同免疫状态下病毒GX0101的增殖动态以及禽流感、新城疫灭活苗在鸡体诱导产生抗体的水平。对含有MDV母源抗体的海蓝褐鸡的试验方案与SPF鸡一致。【结果】SC9-1株免疫对感染MDV GX0101攻击SPF鸡、海兰褐鸡均提供100%的免疫保护作用;CVI988/Rispens对SPF鸡、海兰褐鸡分别提供86.7%、93%的免疫保护作用。未免疫SPF鸡攻毒组死亡率为53.3%,肿瘤率为16.7%;未免疫海兰褐鸡攻毒组死亡率为36.7%,肿瘤率为6.67%;相比,空白对照组鸡只没有任何病变及死亡。荧光定量结果显示,淋巴细胞和羽毛囊DNA中,SC9-1免疫组鸡体内GX0101的病毒拷贝数显著低于CVI988/Rispens免疫组。血凝抑制试验结果显示,SC9-1免疫攻毒组鸡的产生的AIV、NDV抗体水平高于CVI988/Rispens免疫攻毒组。【结论】SC9-1株免疫无论在SPF鸡还是含有MDV母源抗体的海兰褐鸡均能提供比CVI988/Rispens更好的免疫保护效果。     

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.     

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.     

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.