稳定携带H5亚型禽流感病毒候选DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-HA扩增出禽流感病毒JSGO(H5N1)株的血凝素(HA)基因,将其克隆入真核表达质粒pmcDNA3.1 中,获得重组表达质粒pmcDNA3.1-HA。通过电穿孔转化法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207*,构建成功携带DNA疫苗的重组沙门氏菌SL7207*(pmcDNA3.1-HA)。经体内体外试验证实,重组质粒pmcDNA3.1-HA在沙门氏菌中的稳定性显著高于pcDNA3.1-HA。将重组菌SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)分别以2×109CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对禽流感病毒HA蛋白的黏膜抗体。重组菌以5×109CFU剂量两次口服免疫试验鸡,免疫鸡的小肠样品中可测到针对禽流感病毒HA蛋白的黏膜抗体,且SL7207*(pmcDNA3.1-HA)免疫组的抗体效价高于SL7207*(pcDNA3.1-HA)免疫组。免疫保护试验结果显示,SL7207*(pmcDNA3.1-HA)和SL7207*(pcDNA3.1-HA)免疫组的免疫保护率均与空载体组之间存在显著性差异(P<0.05),且SL7207*(pmcDNA3.1-HA)免疫组的保护率较SL7207*(pcDNA3.1-HA)免疫组提高了22.6%,说明稳定携带H5亚型禽流感病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性。     

携带新城疫病毒DNA疫苗的鼠伤寒沙门氏菌及其安全性与免疫效力

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计一对引物,通过RTPCR扩增出鹅源新城疫病毒分离株JS5F基因,测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1F。将pVAX1F转化减毒鼠伤寒沙门氏菌SL7207,构建成携带DNA疫苗的重组沙门氏菌SL7207(pVAX1F)。重组菌以不同剂量口服免疫1日龄雏鸡,结果表明,细菌对雏鸡具有良好的安全性,且不影响鸡的增重。将SL7207(pVAX1F)分别以108CFU和109CFU的剂量3次口服免疫1日龄商品代伊莎褐蛋鸡,抗体检测结果显示,在三免后1周,SL7207(pVAX1F)109CFU剂量组的血清抗体效价与空载体组之间存在显著性差异(p<0.05)。重组菌两个剂量组在首免后2周开始出现粘膜抗体,并于二免后2周和3周达到较高水平。免疫保护试验结果显示,SL7207(pVAX1F)109CFU剂量组的保护率为77.27%,与空载体组之间存在显著性差异(p<0.05)。     

减毒鼠伤寒沙门氏菌运送的鸡传染性支气管炎病毒s1基因DNA疫苗及其免疫效力研究

为研制鸡传染性支气管炎新型基因工程疫苗,构建和表达了运送鸡传染性支气管炎病毒(IBV)DNA疫苗的重组减毒鼠伤寒沙门氏菌。首先将鸡传染性支气管炎病毒M41株的全长s1基因插入到嵌合CpG DNA的真核表达质粒pVAX1中,构建出重组真核表达质粒pVAX1-S1-CpG。然后将重组质粒转化减毒鼠伤寒沙门氏菌SL7207,构建出运送DNA疫苗的重组减毒沙门氏菌SL7207(pVAX1-S1-CpG)。将重组菌SL7207(pVAX1-S1-CpG)分别以1×109CFU,5×109CFU,1×1010CFU的剂量滴鼻和口服接种4日龄雏鸡,2周内所有试验鸡均无任何不良反应,试验结果表明重组菌SL7207(pVAX1-S1-CpG)具有良好的安全性。在免疫后35 d,商品鸡体重测定结果表明重组菌免疫不影响鸡体增重。以5×109CFU重组菌滴鼻和口服两次接种4日龄商品代伊莎褐蛋鸡,二免3周后,血清抗体和小肠黏膜抗体测定结果表明,SL7207(pVAX1-S1-CpG)免疫组抗体水平与空白对照组、空载体对照组之间分别存在极显著性差异(P<0.01)。攻毒试验结果表明,重组菌SL7207(pVAX1-S1-CpG)免疫组有较高的免疫保护效力,与灭活苗和减毒苗效力相当,显示出一定的应用前景。     

减毒沙门氏菌介导的小鼠肝炎病毒S1基因DNA疫苗的免疫特性分析

根据GenBank公开序列自行设计一对引物,通过RT-PCR扩增出小鼠肝炎病毒的全长S1基因,并将其插入真核表达质粒pVAX1中,构建出重组真核表达质粒pVAX1-S1。将重组质粒转染COS-7细胞,采用间接免疫荧光检测出S1蛋白的体外表达。将重组质粒转入减毒鼠伤寒沙门氏菌SL7207中,构建出运送DNA疫苗的重组沙门氏菌SL7207(pVAX1-S1)。分别以5×108CFU、1×109CFU、2×109CFU剂量的重组菌口服接种6周龄BALB/c小鼠,试验结果表明,重组菌对小鼠具有良好的安全性。以1×109CFU剂量的重组菌口服免疫小鼠,抗体检测结果显示,在二免后两周和三免后两周,重组菌免疫组的血清抗体水平与SL7207(pVAX1)空载体免疫组间分别存在显著性差异(P<0.05)和极显著性差异(P<0.01)。在三免后两周重组菌免疫组出现了较高水平的肠黏膜抗体。     

稳定携带新城疫病毒DNA疫苗减毒沙门氏菌的构建及其免疫原性

采用PCR技术从重组质粒pVAX1-F中扩增出新城疫病毒JS5株的融合蛋白(F)基因,将其克隆入真核表达质粒pmcDNA3.1 中,获得重组表达质粒pmcDNA3.1-F.通过电穿孔转化法将重组质粒转入减毒鼠伤寒沙门氏菌SL7207,构建成功携带DNA疫苗的重组沙门氏菌SL7207(pmcDNA3.1-F).体内、体外试验结果表明,重组质粒pmcDNA3.1-F在沙门氏菌中的稳定性显著高于pcDNA3.1-F.将重组菌SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)分别以1×109 CFU剂量两次口服免疫BALB/c小鼠,免疫小鼠可产生针对新城疫病毒F蛋白的血清抗体和黏膜抗体.重组菌以5×109 CFU剂量两次口服免疫4日龄SPF鸡,免疫鸡产生的针对新城疫病毒F蛋白的血清抗体和小肠黏膜抗体效价水平与空载体组之间存在显著性差异(P＜0.05).免疫保护试验结果显示,SL7207(pmcDNA3.1-F)和SL7207(pcDNA3.1-F)免疫组的免疫保护率均与空载体组之间存在显著性差异(P＜0.05),且SL7207(pmcDNA3.1-F)免疫组的保护率较SL7207(pcDNA3.1-F)免疫组提高了20.0%,说明稳定携带新城疫病毒DNA疫苗的减毒沙门氏菌具有良好的免疫原性和免疫保护性.     

携带TGEV DNA疫苗减毒沙门氏菌的构建及安全性与免疫性分析

【目的】探讨以减毒沙门氏菌为载体,进行TGEV DNA疫苗口服免疫可行性。【方法】通过RT-PCR扩增TGEV四川株（SC-H）S基因5’端约2.1 kb的主要抗原位点片段,将其插入真核表达载体pVAX1,构建重组质粒pVAX-S,体外转染COS7细胞,间接免疫荧光检测S基因表达。通过电转化将pVAX-S转入减毒鼠伤寒沙门氏菌SL7207,构建SL7207（pVAX-S）重组菌,并在体外感染小鼠腹腔巨噬细胞,以RT-PCR、间接免疫荧光检测细胞内S基因的转录与表达情况。将SL7207（pVAX-S）重组菌以5×108、1×109、2×109CFU剂量口服接种BALB/c小鼠,分析其安全性,并以1×109CFU剂量的重组菌3次免疫BALB/c小鼠,通过间接ELISA检测免疫小鼠的血清IgG与肠道粘膜IgA抗体。【结果】成功构建重组质粒pVAX-S,且重组质粒能在COS7细胞中表达。重组菌SL7207（pVAX-S）感染巨噬细胞后检测到目的基因的转录、表达。小鼠口服接种不同剂量重组菌,具有良好的安全性。免疫小鼠于二免后两周可检测到针对TGEV S蛋白的特异性血清IgG与肠道粘膜IgA抗体,且三免后两周与SL7207（pVAX1）空载体免疫组间分别存在显著性差异（P<0.05）和极显著性差异（P<0.01）。【结论】携带TGEV DNA疫苗的减毒沙门氏菌小鼠试验显示了良好的免疫原性与安全性。     

以减毒沙门氏菌为载体的小鼠肝炎病毒DNA疫苗的构建及其免疫原性

目的：构建以减毒沙门氏菌为载体的小鼠肝炎病毒DNA疫苗,研究该疫苗的免疫原性。方法：以小鼠肝炎病毒S1基因的重组真核表达质粒pVAX1—S1免疫BALB／c小鼠,ELISA检测其诱导抗体产生情况;再将重组质粒pVAX1—S1电转化到减毒鼠伤寒沙门氏菌SL7207中,构建运送S1基因的重组减毒沙门氏菌SL7207（pVAX1—S1）,口服免疫BALB／c小鼠,间接免疫荧光试验鉴定减毒沙门氏菌运送的DNA疫苗的免疫原性。结果：与pVAX1空载体对照组相比,重组真核表达质粒pVAX1—S1免疫组二免及三免后抗体水平分别存在显著性差异（P〈0．05）和极显著性差异（P〈0．01）。减毒沙门氏菌运送的DNA疫苗SL7207（pVAX1—S1）诱导小鼠产生了特异性的血清抗体。结论：构建的重组减毒沙门氏菌SL7207（pVAX1—S1）具有良好的免疫原性,可诱导小鼠产生特异性的体液免疫应答。这为进一步研制冠状病毒新型基因疫苗奠定了基础。     

以减毒沙门氏菌运送的H5亚型禽流感病毒DNA疫苗的构建及其对小鼠的免疫原性

根据GenBank中已发表的H5亚型禽流感病毒HA基因序列,设计一对引物,通过RTPCR扩增鹅源H5亚型高致病力禽流感病毒HA基因,测序确认后,将其克隆入真核表达载体pVAX1和asdpVAX1得到重组表达载体pVAX1HA和asdpVAX1HA。将重组质粒转染P815细胞,经间接免疫荧光试验证实,HA基因在细胞内得到了瞬时表达。进一步将重组质粒转化减毒鼠伤寒沙门氏菌X4550得到两种运送DNA疫苗的重组沙门氏菌X4550（pVAX1HA）和X4550（asdpVAX1HA）,以1×109CFU/只的剂量两次口服免疫BALB/c小鼠,免疫小鼠不仅可以检测到HA特异性的血清抗体应答,而且还能抵抗稳定表达H5亚型禽流感病毒HA基因的P815肥大细胞瘤的攻击,说明该运送DNA疫苗的减毒沙门氏菌系统在体内能够成功释放所携带的质粒,并且能够刺激机体产生保护性免疫应答。     

携带HCV核心蛋白原核表达质粒与真核表达质粒的减毒伤寒沙门菌诱导小鼠免疫应答之比较

丙型肝炎病毒(HCV)核心蛋白是丙肝疫苗的重要候选抗原,然而,该蛋白因具有免疫调控作用而影响免疫应答的诱导。构建了HCV核心蛋白的两种表达质粒,一种是体内激活型原核表达质粒pZW-C,另一种是真核表达质粒pCI-C。将该两种质粒转化减毒鼠伤寒沙门菌SL7207,得到重组菌SL7207/pZW-C和SL7207/pCI-C,分别将重组菌口服接种小鼠,检测小鼠的免疫应答,结果发现:①SL7207/pCI-C免疫鼠的CD3 CD4 T细胞持续降低,而SL7207/pZW-C免疫鼠的CD3 CD4 T细胞无明显改变;②SL7207/pCI-C免疫只诱导低水平抗HCV核心蛋白抗体,加强免疫对抗体阳转率及抗体水平无明显影响,而SL7207/pZW-C免疫组所有小鼠均产生较高水平的抗核心蛋白抗体。③SL7207/pCI-C免疫鼠脾细胞的体外增殖活性、细胞毒性T细胞活性以及加强免疫对细胞免疫应答的增强作用均明显不及SL7207/pZW-C免疫鼠。结果提示:携带真核表达质粒pCI-C的沙门菌因在小鼠细胞内表达天然形式(结构以及磷酸化修饰)的HCV核心蛋白,可能通过对T细胞的免疫抑制作用而弱化免疫应答。而以携带原核表达质粒pZW-C的沙门菌免疫可避免这一问题,并具有接种方便,成本低廉等优点,从而可望作为基于HCV核心蛋白为靶抗原的HCV疫苗的候选免疫方式。     

表达禽流感病毒特异性siRNA分子减毒鼠伤寒沙门氏菌的构建

为了寻找一种新的短干扰RNA(short interfering RNA,si RNA)片段的表达和递送系统,并进行评价,根据禽流感病毒核苷酸序列,设计了3条分别针对A型禽流感病毒核衣壳蛋白、聚合酶A和H5亚型禽流感病毒血凝素的特异性si RNA分子,人工合成相应DNA片段,退火后形成双链,与阴性对照片段分别连接到构建的pSIREN-ASD ZsGreen载体鼠源U6启动子下游,测序鉴定证实获得了阳性质粒,分别命名为PAZ-NP、PAZ-PA、PAZ-HA和PAZ-NC,1ng的质粒可以完全抑制50个PFU的禽流感病毒在MDCK细胞上的复制,细胞不产生任何病变,细胞上清的血凝效价为20,而对照组细胞在病毒感染36~48h后细胞变圆、死亡、脱落,其细胞上清的血凝效价可达24。将重组质粒转化减毒鼠伤寒沙门氏菌X4550得到重组沙门氏菌X4550(PAZ-NP)、X4550(PAZ-PA)、X4550(PAZ-HA)和X4550(PAZ-NC)。对4日龄雏鸡口服1×109菌落形成单位(CFU)的重组菌后第1d、7d、15d用105ELD50的H5亚型AIV进行攻毒,最后一次攻毒后观察15d,实验结束时采泄殖腔棉拭子对存活鸡进行病毒分离。结果表明,表达不同的si RNA分子对雏鸡有一定的保护力,保护率为27%~50%不等,存活鸡泄殖腔棉拭子未分离到病毒,说明重组减毒沙门氏菌携带质粒在体内产生的si RNA能够对机体产生一定保护力。     

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.     

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.     

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.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.     

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。     

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.     

Scales of spatial patterns of distribution of intertidal invertebrates

Few comparative studies of spatial patterns at different scales have examined several species in the same habitat or the same species over a range of habitats. Therefore, variability in patterns among species or among habitats has seldom been documented. This study quantifies spatial patterns of a suite of intertidal snails and a species of barnacle using a range of statistical techniques. Variability in densities was quantified from the scale of adjacent quadrats (over a distance of centimeters) to tens of kilometers. Significant differences in abundances occurred primarily at two spatial scales. Small-scale differences were found at the scales of centimeters or 1–2 m and, for many species on many shores, these accounted for most of the variability in abundances from place to place. These are likely to be determined by behavioural responses to small-scale patches of microhabitat. Large-scale differences in abundance were also found in most species at the scale of hundreds of meters alongshore. These are likely to be due to variation in recruitment (and/or mortality) because of limited dispersal by adults of these species. There was little or no additional variation among shores, separated by tens of kilometers, than was shown among patches of shore separated by hundreds of meters. Identification of the scale(s) at which significant differences in abundance are found focus attention on the processes (and the scales at which these processes operate) that influence patterns of distribution and abundance. Some of the advantages and disadvantages of various procedures are discussed.