CPSF在真核表达载体上的克隆与瞬时表达

目的：研究CPSF在真核表达载体上的克隆与瞬时表达。方法：以质粒pBS1761为模板扩增TAP-tag片段,PCR产物经纯化后克隆在真核表达载体pTRE2-hyg上。再以pUK-CPSF30k、73k、100k为模板扩增CPSF基因片段,将其克隆在质粒pTRE2-hyg-TAP-tag中TAP-tag片段的下游,并将重组质粒转化入细胞株Hela Tet-offS3细胞内。结果：细胞抽提液经SDS PAGE电泳后进行蛋白质印迹杂交,胶片上出现野生型的CPSF条带和分子量较大的滞后条带。后者经分子量与分子标记对照,确系重组体TAP-tag-CPSF所表达的蛋白条带。结论：重组质粒pTRE2hyg-TAP-tag-CPSF30k,73k,100k在Hela tet-offS3内完好表达。     

前体mRNA的选择性多聚腺苷酸化与人类疾病

真核细胞的前体mRNA必须经过复杂的加工过程才能成熟,包括5’端加帽、剪接和3’端加工,其中3’加工包括3’端的切割和多聚腺苷酸化.该过程由前体mRNA上的顺式作用元件以及多个蛋白质因子控制.组成哺乳动物前体mRNA3’端加工机器的核心蛋白质复合体有切割和多聚腺苷酸化特异性因子、切割刺激因子、切割因子Ⅰ和切割因子Ⅱ.其他因子包括poly(A)聚合酶、poly(A)结合蛋白、偶对蛋白(symplekin)等.哺乳动物基因通常含有多个ploy(A)位点,选择性多聚腺苷酸化不仅可产生具有不同长度3’UTR的mRNA异构体,还可能改变基因的CDS区.作为真核生物基因表达调控的关键机制,选择性多聚腺苷酸化在细胞生长、增殖和分化中起着重要作用.本文综述了哺乳动物前体mRNA的3’端加工过程,3’端加工机器的组成及功能,探讨了选择性多聚腺苷酸化在多种人类疾病中的作用机制,以期为读者带来一些新的见解.     

Dicer蛋白与Wig-1蛋白的相互作用的研究

Dicer是RNA干扰中起核心作用的一种dsRNase．Wig-1是受p53诱导的、与dsRNA特异结合的一种锌指蛋白。本研究初步证实了Dicer蛋白与Wig-1蛋白存在相互作用。GST pull-down实验表明Dicer蛋白能与GST—Wig-1结合,而Wig-1蛋白能竞争性干扰Dicer与GST—eIF2C1的结合：Far—western分析证实Dicer蛋白可以与结合在硝酸纤维素膜上的GST—Wig-1蛋白结合：进一步发现Wig-1能有效促进重组Dicer的dsRNase酶切活性。这些结果揭示Dicer蛋白和Wig-1蛋白很可能通过相互作用共同参与了dsRNA调控相关的生理过程。     

Oligo(dT)-纤维素与Oligo(U)-纤维素的合成及其应用

Oligo(dT)-纤维素与Oligo(U)-纤维素具有分离信使RNA(mRNA)的功能。这是由于大多数的mRNA 分子的3’-末端含有多聚腺嘌呤核苷酸的片段(亦有例外,如一类组蛋白的mRNA),在低温与高离子强度的条件下,此片段能与多聚脱氧胸腺嘧啶核苷酸或多聚尿嘧啶核苷酸形成互补的碱基配对,而核糖核蛋白体RNA(rRNA)与转移RNA(tRNA)缺少多聚腺嘌呤核苷酸的片段,不能被该树脂吸附,利用这一特性可将mRNA与其它RNA分离开来。     

SARS冠状病毒N蛋白和CYP4F3的相互作用

目的：研究SARS冠状病毒（SARS-CoV）N蛋白与CYP4F3的相互作用。方法：应用免疫共沉淀、GST—pull down、BIACORE实验验证SARS—CoVN蛋白与CYP4F3的相互作用。结果：SARS—CoVN蛋白与CYP4F3能够相互作用,BIA-CORE实验证实CYP4F3与SARS—CoVN蛋白的亲和常数为Ko=4．3×10^-11。结论：SARS—CoVN蛋白与CYP4F3在细胞内、外均能形成复合物,这为进一步探讨SARS—CoVN蛋白在SARS致病机理中的作用奠定了基础。     

CPSF6在胶质母细胞瘤进展中的作用及相关调控机制研究*

目的: 研究mRNA前体切割和多聚腺苷酸化特异性因子6(polyadenylation specific factor 6,CPSF6)对人胶质母细胞瘤(glioblastoma,GBM)细胞系U87和U251的增殖、迁移、侵袭以及ATP水平的影响,进一步探究其相关调控机制。方法: 通过Western blot和免疫组化检测CPSF6在GBM组织中的表达水平,利用在线数据库分析CPSF6在GBM组织和配对的非肿瘤组织中的表达水平,同时分析CPSF6与GBM的组织学级别和患者预后的关系。构建敲低CPSF6的U87和U251稳定表达细胞株,并采用RT-qPCR和Western blot方法分别验证U87和U251细胞中CPSF6的敲低效率;利用CCK8和Transwell实验分别检测CPSF6敲降对细胞增殖、迁移和侵袭能力的影响;ATP实验检测细胞内的ATP水平变化,确定CPSF6在GBM中的致癌作用。通过RNA-seq分析敲低CPSF6后GBM内mRNA 3'UTR变化情况,KEGG富集分析差异靶基因相关的信号通路。在富集出的信号通路指示下,利用透射电镜和Western blot实验进一步验证敲低CPSF6后GBM自噬的发生情况。 结果: CPSF6在GBM组织中呈现出高表达,其表达水平随组织学级别的增加而升高,且与患者不良预后相关。在U87和U251中敲低CPSF6后,细胞的增殖、迁移及侵袭能力均明显降低,细胞内ATP水平下降。对RNA-seq结果分析表明,敲低CPSF6后发生3'UTR缩短事件的基因远多于3'UTR延长事件的基因;KEGG富集到自噬信号通路与肿瘤进展密切相关,透射电镜和Western blot实验验证敲低CPSF6可以促进自噬通路的激活。结论: CPSF6在GBM中高表达,且与GBM的组织学级别和患者不良预后呈正相关,CPSF6可能通过抑制自噬通路的激活来促进U87和U251细胞的增殖、迁移、侵袭以及ATP的生成,进而促进GBM发生、发展。     

重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

多聚磷酸相关蛋白结构及生物学功能

多聚磷酸（polyphosphate,polyP）是由几个到数百个磷酸基通过高能磷酸酐键连接而成的链状多聚体,存在于所有细胞生物中.多聚磷酸相关蛋白包括多聚磷酸相关酶和多聚磷酸结合蛋白.多聚磷酸相关酶如多聚磷酸激酶（polyphosphate kinase,PPK）催化polyPn生成polyPn+1的可逆反应;外切聚磷酸酶（exopolyphosphatase,PPX）、内切聚磷酸酶（endopolyphosphatase,PPN）能将polyP水解成磷酸残基;多聚磷酸依赖的激酶将polyP的磷转移到生物小分子上,如葡萄糖和烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide,NAD）,使其分别磷酸化为6 磷酸葡萄糖和烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate,NADP）.多聚磷酸结合蛋白可与多聚磷酸结合,发挥各种生物学功能.本文将简要介绍多聚磷酸相关蛋白的结构与主要生物学功能,以阐述多聚磷酸参与的细胞内生化过程.     

中国家蚕抗菌肽CBM1、CBM2的克隆和表达研究

根据所测定的中国产家蚕CBM1和CBM2 cDNA序列,设计成熟肽的特异性引物,将此基因与凝血因子Xa（FXa）的切割序列以PCR方法连接起来,克隆至pGEX—KG表达质粒中。该载体为改进的GST融合表达质粒,所携带的6个Gly有助于提高FXa的切割效率。克隆得到的融合载体在大肠杆菌JM109中扩增和筛选。经DNA测序,证实为含有正确序列的乙酰化的pGEX—KG—FXa—NCBM1（pKG—FXa—NCBM1）、非乙酰化的pGEX—KG—FXa—CBM1（pKG—FXa—CBM1）和乙酰化的pGEX—KG—FXa—NCBM2（pKG—FXa—NCBM2）、非乙酰化的pGEX—KG—FXa—CBM2（pKG—FXa—CBM2）。将该载体在BL21中诱导表达,得到GST-FXa切割位点NCBM1、CBM1、NCBM2、CBM2等4种融合蛋白。经SDS—PAGE,考马斯亮兰染色后,Lab—Work扫描,其表达产物达到15％～20％,每升培养液平均可得20mg融合蛋白。菌体总蛋白经GST-亲和层析柱得到纯的GST—FXa—NCBM1、GST—FXa—CBM1、GST—FXa—NCBM2、GST—FXa—CBM2融合蛋白,再经FXa酶切后,透析过柱得到重组的NCBM1和CBM1。平板抑菌试验证明,4种融合蛋白均有明显的生物学活性,且没有显著的差异。     

人PIRH2b与ARF4相互作用的初步研究

目的 利用酵母双杂交技术筛选PIRH2b的相互作用蛋白。方法 以PIRH2b为诱饵蛋白,利用酵母双杂交技术筛选人胎肝cDNA文库,用GST—pull down验证PIRH2b与ARF4在体外的相互作用,并用绿色荧光蛋白标记PIRH2b,红色荧光蛋白标记ARF4,观察两者在肝癌细胞株Hep3B中的亚细胞定位。结果利用酵母双杂交筛选到一个能与PIRH2b相互作用的蛋白ARF4,GST—pull down验证了两者在体外的相互作用,荧光标记共定位结果显示两个蛋白共定位于Hep3B细胞的核周区域。结论首次发现并证实了PIRH2b与ARF4的相互作用,PIRH2b对ARF4的功能可能有重要影响。     

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。     

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.     

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.     

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。     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.