抗CEA单链抗体与链亲和素融合基因的表达

克隆分泌CEA杂交瘤细胞重链可变区（V_H）和轻链可变区（V_L）,以Linker连接V_H及V_L构建抗CEA单链抗体.同时以Spacer连接单链抗体和链亲和素,构建成功单链抗体和链亲和素融合基因,克隆该融合基因至原核表达载体,pET21a(+),经IPTG诱导表达出该双特异性融合蛋白.活性鉴定表明该融合蛋白具有结合CEA及生物素的双特异性.该融合蛋白在生物领域中有较广阔的应用前景.     

抗肿瘤血管三结构域单链抗体VH/L的构建与表达

以本室研制的一株抗肿瘤血管单克隆抗体AA98为基础,采用PCR扩增抗体AA98基因的重链可变区(V_H)和轻链(L),以重链恒定区1(C_H1)5′端12个氨基酸的序列作为连接肽,并将连接肽中的Lys突变为Ser,构建VH连接肽-L三结构域单链抗体。重组VH/L单链抗体在大肠杆菌中得到了高效表达,其表达量占菌体总蛋白质的20%。表达的蛋白质在菌内形成包含体,经凝胶过滤法复性,获得了有抗原结合活性的V_H/L。该三结构域单链抗体的成功构建和复性,为重组抗体片段的研制提供了借鉴。     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体(IgG)的完整的恒定区连接起来,构建全抗型抗CD3嵌合抗体,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植,减少受体产生免疫排斥,提高移植器官的存活率。利用PCR方法从抗CD3 ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD₃抗体的轻链和重链可变区,将轻链和重链可变区组装到含有人抗体（IgG）恒定区的表达载体中,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN100和PG1D105,并用脂质体法共转染CHO细胞。结果证明,抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符,ELISA和Western blot检测结果证实转染细胞的培养上清中含有抗CD₃嵌合抗体IgG的表达,表达产物能与Jurkat细胞结合,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性,³H-TdR掺入实验表明, 抗CD3嵌合抗体与亲代抗体HIT3a一样,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达,表达产物有较好生物活性,具有潜在的临床应用价值。     

抗CD20嵌合抗体片段Fab′突变体的表达和活性研究

利用PCR方法从抗CD20单链抗体（ScFv）表达载体上扩增抗CD20抗体轻链可变区基因（V^L）、重链可变区基因（V^H）,同时在抗体的可变区引入突变,然后将V^H、V^L基因重组到Fab′表达载体pYZF1中,构建抗CD20嵌合抗体Fab′片段表达载体,并在大肠杆菌16c9中进行高效可溶性分泌表达。经大量的筛选,获得一个产量和活性均有所提高的突变克隆。其突变位点在轻链可变区的CDR1区,即G77→A（Ser→Asn）。突变的抗体的表达量为每克干菌3.8 mg,而未突变抗体的表达量为每克干菌1.3 mg。突变体的亲和力常数K_a为2.2×10⁹ L/mol,约为突变前的2倍。竞争性免疫荧光抑制实验表明,突变的Fab′片段能竞争性抑制鼠源性抗CD20抗体HI₄₇和CD20表达细胞Raji细胞的结合,使HI₄₇的结合阳性率由98%下降至37.55%,体外细胞生长抑制试验亦证明突变的Fab′片段的抑制活性明显高于未突变的抗体。     

抗松材线虫纤维素酶单链抗体库的构建及筛选

构建鼠源性松材线虫纤维素酶（Bursaphelenchus xylophilus cellulase, BXC）的噬菌体单链抗体库,从中筛选特异性BXC的单链抗体。以BXC为抗原免疫BALB/C小鼠,从脾脏提取总RNA,用RT-PCR技术扩增小鼠抗体重链（V_H）和轻链(V_L)可变区基因。经重叠PCR(SOE-PCR)在体外将V_H和V_L连接成单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,电转化至大肠杆菌TG1,经辅助噬菌体超感染,成功构建了库容为5×10⁴的Anti-BXC单链抗体库,并从该抗体库中初步筛选到了特异性识别BXC的噬菌体单链抗体scFv。将表面展示单链抗体的单克隆噬菌体转化大肠杆菌HB2151进行可溶性表达,SDS-PAGE及Western blot分析结果显示,可溶性scFv获得表达,且与BXC具有结合活性,为松材线虫的检验检疫以及病理学研究奠定了基础。     

基因工程二硫键抗体

二硫键抗体(dsFv)的概念最早出现于1990年,它是将抗体重链可变区(V_H)和轻链可变区(V_L)的各一个氨基酸残基突变为半胱氨酸,通过链间二硫键连接抗体可变区(Fv)的片段抗体.通用的突变位点是重链的44位和轻链的100位或重链的105位和轻链的43位.dsFv最显著的优点是生化性质稳定,能够耐受环境条件的剧烈作用,在血液中的半衰期长达14 d以上,符合临床给药要求.动物实验显示,dsFv-毒素在不对动物造成毒副作用的情况下,可完全抑制肿瘤生长.     

抗人CD3单链抗体与改形单域抗体的表达

设计并化学合成含有适当酶切位点及连接肽的寡核苷酸序列,与一定的背景载体连接并改造成适用于单链抗体表达的载体:外分泌型pWAI80和融合蛋白型pROH80从分泌抗人CD3单克隆抗体的杂交瘤细胞UCHT1中,经PCR扩增出轻、重链可变区基因V_H和V_K,并插入上述表达载体中构建成单链抗体基因.通过对鼠OKT3结合位点的结构模拟,并比较人、鼠抗体家族性保守序列,设计出改形OKT3的基因序列.化学法部分合成8个寡核苷酸片段,应用重叠PCR技术扩增出完整改形重链基因V_H,并克隆、酶切和测序鉴定.将所克隆V_H基因插入表达载体pCOMB3和 pGEX-4T-1中进行表达.经 IPTG诱导表达,对表达产物进行SDS-PAGE和 Western blot分析以及 ELISA检测,结果发现分泌型表达产物及 M13基因Ⅲ-V_H改形单域抗体融合蛋白具有与CD3单抗竞争抑制的活性;而融合型单链抗体及改形单域抗体表达产物主要以包涵体形式存在,占细菌总蛋白的 30％左右.     

抗人黑色素瘤单链抗体基因在大肠杆菌中的融合表达

应用RT-PCR技术从分泌抗人黑色素瘤单克隆抗体的杂交瘤细胞HB8760中克隆了抗体轻、重链可变区基因,用(Gly₄Ser)₃连接肽基因将V_H、V_L连接成ScFv基因,并进行了序列测定。ScFv基因全长927bp,其中VH基因长360bp,编码120个氨基酸,V_L基因长324bp,编码108个氨基酸。在大肠杆菌融合表达载体pGEX-4T-1中表达了GST-ScFv融合蛋白,表达产量占菌体总蛋白的29%。凝血酶消化后的产物具有黑色素瘤细胞结合活性。     

抗人CD19单链抗体基因的构建、表达及功能测定

采用RT-PCR方法从分泌抗人类白细胞表面分化抗原CD19单克隆抗体的杂交瘤细胞中克隆出V_H和V_L可变区基因,再通过重叠延伸拼接（spliceoverlap extension）PCR方法在V_H和V_L可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗人CD19单链抗体（抗CD19-ScFv）基因。将其克隆至表达载体PET28a并在大肠杆菌中表达。SDS-PAGE和Westernblot分析结果表明,抗CD19-ScFv在BL21（DE3）菌中获得表达,重组蛋白的相对分子量为27kD,表达产物以不溶性包涵体形式存在,经过溶解包涵体,镍柱亲和层析纯化和体外复性过程,获得了高纯度的单链抗体片段。流式细胞分析结果证实抗CD19ScFv可与人类白细胞表面的分化抗原CD19结合,保留了鼠源性单抗与CD19结合活性。抗人CD19-ScFv的构建与表达,为下一步针对B淋巴系统恶性肿瘤的靶向治疗奠定了基础。     

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.