干扰素a-2b的聚乙二醇修饰

采用分子量为20 kD的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰重组人干扰素a-2b(IFN a-2b), 建立了修饰反应及分离纯化工艺。考察了修饰反应各因素对单修饰转化率以及单修饰产物体外活性的影响, 获得了优化的修饰反应条件, 即在pH 6.5, 20 mmol/L的磷酸氢二钠-柠檬酸缓冲溶液中, 干扰素a-2b的浓度为4 mg/mL, PEG与IFN a-2b的摩尔比为8:1, 4oC时反应20 h; 在优化的反应条件下, 单修饰PEG-IFN a-2b的转化率达到55%。并且, 采用离子交换层析对修饰产物进行分离纯化, 单修饰产品纯度达到97%, 体外活性保留达到未修饰干扰素a-2b的13.4%, 其在SD大鼠体内的循环半衰期得到了较大的延长, 且具有较好的水溶液稳定性。     

干扰素α-2b的聚乙二醇修饰

采用分子量为20 kD的单甲氧基聚乙二醇丙醛(mPEG-ALD)修饰重组人干扰素α-2b(IFN α-2b),建立了修饰反应及分离纯化工艺.考察了修饰反应各因素对单修饰转化率以及单修饰产物体外活性的影响,获得了优化的修饰反应条件,即在pH 6.5,20 mmol/L的磷酸氢二钠-柠檬酸缓冲溶液中,干扰素α-2b的浓度为4 mg/mE,PEG与IFN α-2b的摩尔比为8:1,4℃时反应20 h;在优化的反应条件下,单修饰PEG-IFN α-2b的转化率达到55%.并且,采用离子交换层析对修饰产物进行分离纯化,单修饰产品纯度达到97%,体外活性保留达到未修饰干扰素α-2b的13.4%,其在SD大鼠体内的循环半袁期得到了较大的延长,且具有较好的水溶液稳定性.     

聚乙二醇单修饰重组人粒细胞集落刺激因子的研究

制备单修饰的PEG蛋白偶联物,对获得重复性好的修饰产品,减少后续分离步骤具有重要的意义。用N-羟基琥珀酰亚胺活化法对单甲氧基聚乙二醇 (mPEG,分子量20000) 进行活化,红外光谱分析, 并考察了其水解动力学性质。对重组人粒细胞集落刺激因子(rhG-CSF)进行化学修饰,通过正交试验结合SDS-PAGE电泳检测建立了单条PEG链修饰rhG-CSF的条件,单修饰PEG-rhG-CSF的收率为90%。离子交换层析对修饰产物进行分离纯化,高效凝胶过滤色谱(SEC-HPLC)检测纯度达到97%。     

集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

目的：设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰,以获得高活性的长效干扰素分子。方法：利用蛋白质分子同源模建,选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化,与单甲氧基聚乙二醇（mPEGMAL）定点偶联。修饰产物经纯化后,以SDSPAGE考察其纯度,用WISHVSV系统进行生物活性测定。结果：IIFN72C以包涵体形式表达,表达量占菌体总蛋白的30%以上,比活性与突变前相当;修饰产物大多数为单修饰体,纯化后纯度大于98%,比活性保留约为修饰前的8%。结论：成功设计并表达IIFN72C用于PEG定点修饰,修饰产物活性保留得以提高。     

胸腺素α1与复合α干扰素融合蛋白的表达及其生物学活性

实验旨在获得具有双重生物学活性的重组胸腺素α1(Thymosin alphal,TM-α1)与复合α干扰素(IFNα-con)融合蛋白.选择大肠杆菌偏爱的密码子.将合成的TM-α1与IFNα-con编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b( )、在宿主茵BL21(DE3)-Codon plus-RP-X中成功表达了可溶性融合蛋白(TM-α1.IFN-con).表达量占总蛋白的20%以上.通过硫酸铵沉淀、疏水层析,阴离子交换层析、阳离子交换层析,分子筛层析后,产品纯度达到96%以上.采用细胞病变抑制法测定融合蛋白的抗病毒活性,采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响.结果表明.融合蛋白的抗病毒活性优于市售的IFNα1b和IFNα2a.对小鼠脾淋巴细胞增殖的影响与市售的合成胸腺素α1相同.已有研究证实.该融合蛋白具有良好的体外抗HBV作用.其体外抗HBV活性比联合应用TM-α1和干扰素α强,且细胞毒性明显低于联合应用TM-α1和干扰素α,以上结果表明,通过大肠杆菌表达的可溶性融合蛋白(TM-α1.IFN-con).既具有良好的干扰素α抗病毒作用.也具有胸腺素α1促淋巴细胞增殖作用.     

聚乙二醇修饰重组溶葡球菌酶的初步研究

目的:探讨聚乙二醇(PEG)修饰重组溶葡球菌酶(lysostaphin)的反应条件以及修饰后产物的纯化方法.方法:采用超声波细胞粉碎机进行菌体破碎,阳离子交换层析、疏水层析进行蛋白纯化;在不同条件下,将活化的单甲氧基聚乙二醇琥珀酰亚胺丙酸酯(mPEG-SPA)与纯化后的lysostaphin反应,以单个PEG-Lysostaphin的比例为指标,用SDS-PAGE、MALDI-TOF-MS方法确定其在修饰产物中的所占比例;采用Sephacryl S-200分子筛凝胶层析法对修饰产物进行分离纯化.结果:mPEG-SPA修饰lysostaphin的反应条件为pH 8.0,温度4℃,lysostaphin与mPEG-SPA的质量比为1∶5,反应时间2.0h;反应产物经一步Sephacryl S-200分子筛凝胶层析纯化后,初步实现分离.结论:初步确定了聚乙二醇修饰lysostaphin的反应条件及修饰产物的纯化方法.     

胸腺素a1与复合a干扰素融合蛋白的表达及其生物学活性

实验旨在获得具有双重生物学活性的重组胸腺素a1(Thymosin alpha1, TM-a1)与复合a干扰素(IFNa-con)融合蛋白。选择大肠杆菌偏爱的密码子, 将合成的TM-a1与IFNa-con编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b(+)、在宿主菌BL21(DE3)-Codon plus-RP-X中成功表达了可溶性融合蛋白(TM-a1-IFN-con)。表达量占总蛋白的20%以上。通过硫酸铵沉淀、疏水层析、阴离子交换层析、阳离子交换层析、分子筛层析后, 产品纯度达到96%以上。采用细胞病变抑制法测定融合蛋白的抗病毒活性, 采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响。结果表明, 融合蛋白的抗病毒活性优于市售的IFNa1b和IFNa2a。对小鼠脾淋巴细胞增殖的影响与市售的合成胸腺素a1相同。已有研究证实, 该融合蛋白具有良好的体外抗HBV作用, 其体外抗HBV活性比联合应用TM-a1和干扰素a强, 且细胞毒性明显低于联合应用TM-a1和干扰素a。以上结果表明, 通过大肠杆菌表达的可溶性融合蛋白(TM-a1- IFN-con), 既具有良好的干扰素a抗病毒作用, 也具有胸腺素a1促淋巴细胞增殖作用。     

重组人粒细胞集落刺激因子的表达、纯化以及PEG修饰

重组人粒细胞集落刺激因子(rhG-CSF)经大肠杆菌温度诱导表达后,其表达产物以包涵体形式存在,包涵体经过变性、复性和分离纯化等步骤处理后得到纯化的rhG-CSF.在一定的实验条件下用单甲氧基聚乙二醇活性酯(mPEG20k-NHS)对rhG-CSF进行化学修饰,所得修饰产物经分离纯化后获得PEG20K-rhG-CSF偶联物.与修饰前的rhG-CSF相比较,尽管修饰后的rhG-CSF体外生物学活性下降至修饰前的20%左右,但其在体内的作用时间能够得到显著的延长,药效有了明显提高.     

重组肿瘤坏死因子-α衍生物的制备及聚乙二醇修饰

应用搭桥PCR技术,将肿瘤坏死因子-α基因的前4位氨基酸的编码序列删除,对hTNF-α的第8/9/10/29/31/157位氨基酸的密码子进行定点突变,将突变后的cDNA插入到pBV220载体中构建重组质粒pBV220-tnf-αD4。将重组质粒转化大肠杆菌DH5α,筛选获得了高效表达TNF-αD4突变体的工程菌,表达的重组蛋白约占菌体蛋白总量的45%左右,经硫酸铵沉淀和阳离子交换层析纯化得到纯度达90%以上的重组目的蛋白,比活性达到8×107。用单甲氧基聚乙二醇-丁醛（mPEG-ButyrALD）对TNF-αD4进行修饰,经阳离子交换层析纯化得到纯的mPEG-TNF-αD4,纯度达85%以上,比活性达到8.6 ×107,系统毒性也有了明显的降低。此项工作通过应用PEG修饰肿瘤坏死因子-α,为降低其毒性,增加其活性进行了有益的尝试,为其进一步研究与开发奠定了基础。     

人Leptin基因在大肠杆菌中的高效表达、纯化及其生物学活性研究

将人Leptin表达质粒pBV220-OB转化E.coliJM109,经热诱导获得了目的蛋白的表达。经SDS-PAGE鉴定分析,表达产物以包涵体形式存在,目的蛋白表达量占菌体总蛋白的40%以上。通过包涵体分离,Sephacryl S200HR凝胶和DEAE52离子交换层析及Hypersil C18柱反相色谱纯化,获得纯度在95%以上,内毒素含量小于10EU/mg的高纯度的重组人Leptin。Western-blot鉴定表明,纯化表达产物能和抗Leptin抗体特异性结合;蛋白质N端15个氨基酸序列分析结果和预期的序列一致。纯化产物经复性处理,其分子中Cys96和Cys146形成二硫键。体内活性检测显示,纯化和复性的rh-Leptin明显抑制BALB/c小鼠的进食和体重增长,提示其具有明显的生物学活性。     

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.     

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.     

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。