抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.     

抗肠炎沙门氏菌单链抗体制备及其特异性分析

目的:利用基因工程技术制备抗肠炎沙门氏菌的单链抗体.方法:从抗肠炎沙门氏菌单克隆抗体的杂交瘤细胞中纯化RNA,反转录后扩增出抗体的重链可变区(VH)和轻链可变区(VL)基因片段,采用重叠延伸的方法,用柔性多肽Linker接头(Gly4 Ser)3按VL-Linker-VH方式将VH基因和VL基因拼接成单链抗体基因片段后,连接到pGEX-4T-1载体上,进行重组转化.挑取阳性克隆,经IPTG诱导后,通过GST柱进行亲和层析,最后利用ELISA检测抗体的活性.结果:成功构建了表达抗肠炎沙门氏菌单链抗体的基因工程菌株,经SDS-PAGE和ELISA检测结果表明,诱导表达的单链抗体scFv分子量约为60 kDa,其能特异与肠炎沙门氏菌结合,但与副甲伤寒沙门氏菌、鸭沙门氏菌、鼠伤寒沙门氏菌有轻度交叉反应.结论:成功构建了抗肠炎沙门氏菌单链抗体的表达菌株,表达的单链抗体scFv可作为沙门氏菌的检测的候选抗体分子.     

一株针对人EGFR的单链抗体克隆与哺乳细胞表达

目的:制备特异性抗人表皮生长因子受体(EGFR)的单链抗体(sc Fv),鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法:从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5'RACE技术扩增轻链和重链可变区(VL、VH)基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果:获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论:成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。     

卵巢癌抗独特型单链抗体的人源化——抗独特型微抗体的构建与表达

为了降低人抗鼠抗体反应 ,获得满意的免疫原性 ,将模拟人卵巢癌抗原的抗独特型单链抗体人源化 .采用重叠PCR和基因工程的技术 ,将 6B11ScFv基因的轻链和重链颠倒 ,成为 6B11VL VH.再与人IgG1铰链区和CH3区的基因进行融合 (VL VH CH3) ,构建抗独特型微抗体的原核表达载体 .转化E .coliBL2 1(DE3)后用IPTG诱导表达 .经SDS PAGE分离显示 ,在 5 0kD左右处有一诱导蛋白带 .不连续非变性凝胶电泳显示 ,表达产物分子量为 10 0kD左右 .采用ELISA、竞争抑制实验、West ern印迹对其进行活性测定 .结果表明 ,人源化的抗独特型微抗体具有特异性双价结合卵巢癌单克隆抗体COC16 6 9和识别人免疫球蛋白IgG1的活性 ,成功地将鼠源的scFv的人源化     

Ⅱ型志贺毒素单克隆抗体可变区基因克隆及单链抗体的表达和功能鉴定

目的从分泌抗肠出血性大肠埃希菌Ⅱ型志贺毒素中和单克隆抗体杂交瘤细胞株S2C4中克隆抗体可变区基因,构建单链抗体（ScFv）,进行原核表达,并对其功能进行鉴定。方法从杂交瘤细胞株S2C4中提取总RNA,逆转录成cDNA。在cDNA3’-OH末端添加poly．G。PCR扩增包括5’非翻译区和信号肽序列在内的抗体重、轻链可变区基因VH和VL,PCR产物装入T—A载体测序。根据测序结果,设计引物分别扩增VH和VL编码区,再通过重叠PCR,在VH和VL．编码区基因之间引入连接链,构建Scn基因,并克隆到表达载体pComb3xSS中。重组载体导入E．coliTop10F’进行表达,重组蛋白经纯化后,分别用ELISA和动物保护性实验鉴定其生物学活性。结果VH和VL编码区基因全长分别为396bp和378bp,ScFv基因能在大肠埃希菌中高效表达,表达产物的分子量为34000,用NiSO4亲和层析柱成功纯化。功能性实验表明纯化的重组蛋白可以与Stx2毒素有效结合,能保护动物抵御毒素分子的攻击。结论成功地克隆S2C4单抗可变区基因,并构建、表达其单链抗体ScFv,为下一步进行该抗体人源化奠定实验基础。     

大容量人天然抗体库的构建、鉴定及初步应用

从未经主动免疫的健康志愿者的外周血淋巴细胞中提取总RNA,用RT-PCR扩增人抗体重链(VH)和轻链(VL)可变区基因,得到了6种VH家族基因,11种VL家族基因,这些抗体基因家族覆盖了人抗体基因多样性的95%以上。采用改进的SOEPCR法将VH基因和VL基因连接成人单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,将连接产物电转化大肠杆菌TG1,经辅助噬菌体M13KO7超感染,构建了库容为5.58×109的噬菌体单链抗体库。采用BstNI酶切法证明,构建的噬菌体单链抗体库具有良好的多样性。以TNF-α为靶,从该抗体库中筛选到了抗TNF-α抗体,这说明该抗体库可用于人源抗体的筛选。     

天然兔源噬菌体单链抗体库的构建与鉴定

目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。     

抗人血管内皮生长因子单链抗体基因的构建、表达及活性鉴定

鼠单克隆抗体E11能与人血管内皮生长因子(VEGF)特异结合,已用于临床检测恶性肿瘤细胞VEGF的表达,并初步证明其体内抑瘤活性。为便于大规模生产,特进行基因工程改造,构建小分子的单链抗体(scFv)。首先通过逆转录及多聚酶链式反应(PCR),分离并克隆E11的可变区基因。经测序表明,E11轻链可变区(VL)基因全长333bp,编码111个氨基酸,归属小鼠轻链可变区基因第Ⅲ亚组。重链可变区VH基因全长369bp,编码123个氨基酸,归属小鼠重链可变区基因Ⅱ(A)亚组。然后用一编码亲水性多肽接头的DNA片断将E11单抗轻、重链可变区基因连接,构建表达质粒pET-15YV,在大肠杆菌BL21(DE3)中进行表达。表达产物(包含体)经变性及复性后,用免疫组化法检测该单链抗体结合抗原(颊癌)能力。对颊癌组织检测的结果表明,基因工程抗体scFv与亲代抗体一样,具有较高的组织特异性。本研究获得的抗人VEGF单链抗体具有潜在的临床价值,为肿瘤放射免疫显像及以血管为靶标的抗血管生成治疗奠定了基础。     

抗伏马菌素单链抗体与碱性磷酸酶的融合表达及活性鉴定

为原核表达抗伏马菌素单链抗体-碱性磷酸酶融合蛋白并分析其活性,本研究根据抗伏马菌素单链抗体H2的基因序列设计引物,PCR扩增获得目的基因,经限制性核酸内切酶SfiⅠ和Not Ⅰ的酶切位点克隆到pDAP2/S载体中,转化大肠杆菌(Eschrichia coli)菌株XL1-Blue并鉴定阳性转化子.IPTG诱导H2-AP融合蛋白基因的表达,利用Western blot检测其表达情况,AP显色反应和ELISA鉴定其活性.结果显示成功构建了 pDAP2/S-H2原核表达载体,融合蛋白在大肠杆菌中实现可溶性表达,并保留单链抗体和碱性磷酸酶的活性.因此,H2-AP融合蛋白通过原核表达后可用于发展伏马菌素的快速免疫检测方法.     

抗乙型肝炎表面抗原单链抗体(ScFv)的原核表达、纯化及鉴定

从抗HBsAg鼠单抗的杂交瘤细胞提取RNA,经反转录得到cDNA,进一步扩增得到鼠重链可变区基因(VH)和轻链可变区基因(VL),按VH-linker-VL的结构将VH、VL基因拼接成单链抗体(scFv)基因;经测序正确后进一步构建了表达重组体p26HBSc并在E.coli中表达,得到一约30kD的外源蛋白;纯化后经ELISA检测与HBsAg有较高的亲和力活性,为下一步的人源化改造奠定了基础。     

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.     

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.     

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.