硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

针对ＨＢＶ的５个基因位点作为靶序列,设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段,观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达,及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用,且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性,在浓度为２０μｍｏｌ／Ｌ时,对细胞无明显杀伤作用,对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物,但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．     

牛疱疹病毒Ⅰ型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活作用

由含有ＢＨＢＶ－１（ＢｏｖｉｎｅＨｅｒｐｅｓＶｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＢＩＣＰＯ（ＢＨＶ－１ＩｎｆｅｃｔｅｄＣｅｌｌＰｒｏｔｅｉｎＯ）的ＤＮＡ序列至表达载体ｐＳＶＫ３,构建质粒ｐＳＶ２．９。将该质粒与ｐＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶＬＴＲ启动子控制下的荧光素酶基因的表达。根据ｐＳＶ２．９与含有ＢＩＶＬＴＲ不同区段缺失的质粒ｐＤ－３１９－Ｌｕｃ、ｐＤ－１１５－Ｌｕｃ、ｐＤ－５２－Ｌｕｃ共转染小牛肺细胞的实验结果,推测ＢＩＶＬＴＲ－３１９位上游区的ＤＮＡ序列影响ＢＩＣＰＯ基因产物对ＢＩＶＬＴＲ表达的激活作用。     

大肠杆菌脂蛋白在CTB—pres2抗原基因的融合及表达

首次采用基因融合方式,在乱毒素Ｂ亚基－乙型肝炎病毒Ｐｒｅｓ２抗原融合基因的５‘端引入编码大肠杆菌脂蛋白信号肽及Ｎ端九个氨基酸的核苷酸序列,分别置于ｃｔｂ及ｌａｃ启动子下在大肠杆菌中获得分泌性表达,表达的融合蛋白均定位于膜上,并且可以和ＧＭ１、抗－ＣＴＢ抗体及ＨＢＶＰｒｅＳ２单克隆抗体结合说明该融合蛋白保留了ＣＴＢ的基本高级结构及ＣＴＢ、ＰｒｅＳ２抗原的抗原性,＾３Ｈ－棕榈酸标记实验证实该融合蛋白发     

重组人血小板生成素在大肠杆菌中表达的研究

采用化学法全合成了编码人血小板生成素（ｔｈｒｏｍｂｏｐｏｉｅｔｉｎ,ＴＰＯ）成熟肽Ｎ端１５３氨基酸的基因序列,构建基于该合成基因的表达质粒,结果以谷胱甘肽转硫酶－ＴＰＯ１５３（ＧＳＴ－ＴＰＯ１５３）融合蛋白的方式获得了占全菌蛋白４０％的高效表达．进一步采用ＰＣＲ方法分别对ＴＰＯ合成基因及ＴＰＯｃＤＮＡ的翻译起始区（ＴＩＲ）序列进行定点突变,以降低这一区域的Ｇ－Ｃ含量．将突变序列分别插入到ｐＢＶ２２０表达载体中,重组质粒在转化大肠杆菌ＪＭ１０９后,均获得了表达,其中ＴＩＲ区突变后的合成基因表达产物约占全菌蛋白的１５％．为研究基因下游结构对表达的影响,在不改变氨基酸组成的基础上,构建了ＴＰＯ合成基因与ＴＰＯｃＤＮＡ的杂合序列表达质粒．研究结果表明翻译起始效率是影响ｒｈ－ＴＰＯ在大肠杆菌中表达的重要因素之一,同时基因下游序列的组成对表达水平也会产生影响．     

tRNA-包埋核酶在HepG2细胞中的抗HBV活性

已知 ｔ Ｒ Ｎ Ａ 包埋核酶比裸露核酶在胎牛血清和 Ｈｅｐ Ｇ２ 细胞抽提液中有较高的稳定性 构建了 ｈ Ｃ Ｍ Ｖ 启动子驱动下的抗 Ｈ Ｂ Ｖ（ａｄｒ 亚型）的 ｔ Ｒ Ｎ Ａ 包埋核酶基因质粒,与携带 Ｈ Ｂ Ｖ 基因的ｐ１２Ⅱ质粒共转染 Ｈｅｐ Ｇ２ 细胞,用 Ｇ４１８ 筛选抗性细胞 分析稳定表达细胞中的 Ｈ Ｂ Ｖ Ｒ Ｎ Ａ, Ｈ Ｂ Ｖ 抗原合成和新生 Ｄ Ｎ Ａ 合成,表明ｔ Ｒ Ｎ Ａ 包埋核酶比裸露核酶有较高的抑制 Ｈ Ｂ Ｖ 活性．ｔ Ｒ Ｎ Ａ 包埋核酶和裸露核酶分别使靶 Ｒ Ｎ Ａ 减少 ８２％ ～８７％ 和 ７５％ ～８１％ ,抗原减少 ７３％ ～８０％ 和７０％ ～７４％ 以及新生 Ｄ Ｎ Ａ 减少 ７４％ ～７６％ 和 ６７％ ～７１％ 结果指出,核酶,特别是 ｔ Ｒ Ｎ Ａ 包埋核酶,对 Ｈｅｐ Ｇ２ 细胞中 Ｈ Ｂ Ｖ 表达和复制有明显抑制作用,可能作为 Ｈ Ｂ Ｖ 基因治疗的手段之一      

反义寡核苷酸对丙型肝炎病毒调控基因表达的体外抑制活性研究

为探讨反义寡核苷酸对丙型肝炎病毒的抑制活性,研究和开发新型抗ＨＣＶ药物。采用ＨＣＶ５’ＮＣＲ调控荧光素酶基因的稳定表达细胞株ＨｅｐＧ２．９７０６,评价了３条针对ＨＣＶ调控基因的ＡＳＯＤＮ,即ＨＣＶ３６３,ＨＣＶ３４９及ＨＣＶ２７９。将Ｌｉｐｏｆｅｃｔｉｎ包封的ＡＳＯＤＮ与ＨｅｐＧ２．９７０６细胞株每天作用５小时,连续３天后检测荧光素酶活性。     

甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

EGFR反义脱氧寡核苷酸及其硫代修饰片段抑制人肝癌细胞系BEL…

本文旨在研究针对ＥＧＦＲ ｍＲＮＡ的反义寡核苷酸片段对ＢＥＬ－７４０４细胞ＥＧＦＲ基因表达及其对细胞生长的影响。合成了互补于ＥＧＦＲ基因５’起始编码区的２１聚脱氧寡核苷酸（ＯＤＮｓ）。实验结果显示,３．２ｕｍｏｌ／Ｌ ＯＤＮｓ明显抑制ＢＥＬ－７４０４细胞生长,「３Ｈ」Ｔｄｒ掺入抑制试验显示其对细胞ＤＮＡ合成的抑制作用表现剂量依赖性;密度扫描分析显示ＯＤＮｓ处理６、２４ｈ后,肝癌细胞ＥＧＦＲ ｍＲＮ     

牛疱疹病毒I型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活…

由含有ＢＨＶ－１（Ｂｏｖｉｎｅ Ｈｅｒｐｅｓ Ｖｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＩＣＰＯ（ＢＨＶ－１Ｉｎｆｅｃｔｅｄ Ｃｅｌｌ Ｐｒｏｔｅｉｎ Ｏ）的ＤＮＡ序列至表达载体ｐＳＶＤ３,构建质粒ｐＳＶ２．９。将该质粒与ＰＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶ ＬＴＲ启动子控制下的荧光素酶基因的表达。根据ＰＳＶ２．９与含有Ｂ     

丙型肝炎病毒核心区优势抗原表位基因的化学合成及其表达产物的抗原性分析

依据丙型肝炎病毒（ＨＣＶ）多蛋白核心区Ｎ端氨基酸序列和密码子简并性,人为设计并采用半化学半酶促的方法合成了一个ＤＮＡ片段。经核酸杂交检测以及ＤＮＡ序列分析证实,该片段的核苷酸序列与设计完全一致。将合成的ＤＮＡ片段插入融合表达载体ｐＧＥＸ－２Ｔ中,表达产物经亲和层析纯化后进行Ｗｅｓｔｅｒｎ免疫印迹实验和间接ＥＬＩＳＡ分析,结果表明,融合蛋白具有ＨＣＶ核心区抗原的免疫反应性,可望用于ＨＣＶ抗体的检测．此外,编码ＨＣＶ优势抗原表位的化学合成基因有可能为ＨＣＶ嵌合抗原的研究提供一条捷径。     

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.