高致病性禽流感病毒H5N1中和抗体的单链抗体构建与活性鉴定

在本实验室研制出的多株针对H5N1血凝素的鼠单抗中,10F7对34株H5N1病毒株都有血凝抑制和中和活性,具有特异性高、反应性强、识别谱广的特点。通过基因工程构建10F7单链抗体(scFv)表达重组质粒,在大肠杆菌中表达并纯化scFv,经血凝抑制实验及中和实验检测其活性。结果在针对3株病毒的血凝抑制实验中,10F7scFv蛋白对其中2株H5N1病毒均显示出结合活性,而对H9毒株没有反应。在针对7株H5N1病毒的中和实验中,10F7scFv对5株病毒具有较好的中和能力。H5N1广谱中和抗体10F7的单链抗体构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础

噬菌体抗体库固相筛选条件的初步研究

目的:探讨噬菌体抗体库的固相筛选条件,为筛选方案的设计提供实验依据。方法:利用多种针对HEVNE2蛋白的特异性噬菌体人源抗体和非特异性噬菌体人源抗体,对噬菌体抗体与抗原的结合时间、抗原包被的浓度、洗涤强度和洗脱方式等多种筛选的条件进行初步探索。结果:阳性噬菌体抗体与抗原反应1min,就可较好结合,洗涤次数为20～30次、洗涤液的pH为5时,筛选得到的阳性率最高。包被抗原的浓度对筛选的阳性率没有明显影响,用10mg/L抗原竞争洗脱60min,可得到较高的阳性率。结论:噬菌体抗体库的筛选是一个非常复杂的过程,其中的各个条件之间有着密切的联系,应该根据具体情况进行调整

多样性人源天然噬菌体抗体库的构建及初步应用

目的:构建多样性良好的人源天然噬菌体抗体库。方法:从正常人外周血中分离淋巴细胞,以RT-PCR和半巢式PCR扩增重链可变区VH基因和轻链可变区VL基因,以重叠延伸PCR将VH、VL组装成scFv基因,并将其克隆入噬菌粒载体pCANTAB-5E中。以pCANTAB-5E电转化大肠杆菌TG1,构建人源天然噬菌体抗体库,测序分析抗体基因的家族信息和多样性,并用多种抗原对其进行筛选。结果:获得了库容为2×108的人源天然噬菌体抗体库。分别用5种抗原对其进行筛选,均可获得特异性噬菌体抗体的富集。结论:成功地构建了一个多样性良好的人源天然噬菌体抗体库,可用于制备具有应用前景的人源抗体

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体

分子对接预测H5亚型禽流感病毒的广谱中和表位

H5N1禽流感病毒已经在亚洲、欧洲和非洲广泛传播,造成了巨大损失.最近我们鉴定出一株对多种来源的H5N1代表株均有良好中和活性的、识别H5亚型血凝素(hemagglutinin,HA)的广谱单克隆抗体8H5,它对寻找克服禽流感高变性的广谱治疗性抗体、疫苗和药物具有重要价值.本研究应用分子模拟技术,采用"典范结构"方法对8H5抗体Fab片段进行结构模建,并通过能量分析、SAS值分析、"拉曼强传图"检验、profile-3D分析等理论验证,获得较为合理的8H5Fab的三维空间结构.8H5Fab与3种HA蛋白晶体结构的分子对接结果表明,8H5抗体与HA蛋白的作用模式与HA宿主来源无关,但与HA结构的亚型相似性相关.综合抗原同源比对结果,推测8H5抗体识别的广谱中和表位是由HA上的Asp68,Asn72,Glu112,Lys113,Ile114,Pro118,Ser120,Tyr137,Tyr252不连续氨基酸残基组成的构象表位.这一模型将为H5亚型禽流感病毒广谱疫苗和治疗性药物的分子设计提供依据

Study on the Transfection Efficiency of the New Cationic Polymer Transfection Reagent: Sofast

采用DNA延滞实验研究新一代阳离子聚合物梭华 Sofast转染试剂与DNA的结合能力,以不同报告基因(绿色荧光蛋白基因、β 半乳糖苷酶基因和荧光素酶基因),分析比较梭华 Sofast与阳离子脂质体Lipofectamine2000和阳离子聚合物JetPEI、Superfect转染HEK293细胞转染效率和细胞毒性,研究梭华 Sofast转染试剂转染效率.实验结果表明梭华 Sofast与DNA有很强的结合能力;以绿色荧光蛋白基因为报告基因,梭华 Sofast转染率最高,约60%左右,而且在单个细胞中表达的GFP量多;转染细胞荧光素酶活性检测结果表明,当梭华 Sofast与DNA质量比为16时,转染率最高,高于109RLU/mg蛋白,分别是JetPEI,Superfect和Lipofectamine2000转染细胞最高活性的1.6,2.3和5倍;梭华 Sofast转染β 半乳糖苷酶的染色结果显示95%以上的阳性细胞转染率;比较基因转染效率最高时的细胞毒性,在最佳转染剂量时,几种试剂的细胞存活率均在83%以上,而梭华 Sofast为94.23%.在目前市场上销售的转染试剂对HUV EC细胞的转染效果均不理想情况下,梭华 Sofast仍具一定的转染效率,阳性细胞约占20%.研究结果表明梭华 Sofast是一种高转染效率,低细胞毒性的转染试剂.To study the transfection efficiency of the new cationic transfection reagent: Sofast. DNA arrearage assay was firstly applied to determine the capacity of combination between transfection reagent Sofast and DNA. Then several different report genes (GFP gene , β-galactosidase gene and luciferase gene) were adopted to compare the transfection efficiency to HEK293 cells of Sofast and the toxicity with those of cationic liposome Lipofectamine 2000, cationic polymer Jet PEI and Superfect . In our study, DNA arrearage assay showed the strong combination between Sofast and DNA; Using GFP as report gene, we found that the highest transfection efficiency can be acquired from Sofast among the four transfection reagents, it is about 60%, moreover GFP expressed largely in single cell; Activiation luciferase detection suggested that when the weight ratio of Sofast to DNA was about 16, its transfection efficiency got over 10~9RLU/mg and this was higher than that of the other three. Furthermore, this efficiency is 1.6, 2.3 and 5 folds of the highest efficiency of Jet PEI, Superfect and Lipofetamine 2000, respectively. According to β-galactosidase staining, we found that the positive cell transfection ratio of Sofast got over 95%. On the other hand, the cytotoxicity assay showed that over 83% cell livability could be achieved by Lipofectamine 2000, Jet PEI and Superfect , while 94.23% cell livability by Sofast. Specially, to HUV-EC cell line, Sofast could still maintain about 20% transfection efficiency, though that of all the other reagents available in market now was low.Sofast is a high efficiency but low cell toxicity transfection reagent

限量饮食对致癌物活化代谢及 DNA 断裂的影响

目的: 研究限量饮食(DR)对小鼠肝代谢活化致癌物以及致癌物导致的 DNA 链断裂程度的影响。方法: 用32P后标记和高效液相色谱法测定致癌物-DNA 主要加合物生成量; 用随机寡核甘酸引物合成法(ROPS)测定 DNA 链断裂程度; 用体外代谢酶活性测定法检测有关代谢酶的活性。结果:①限量饮食减少小鼠肝中黄曲霉毒素 B1(AFB1) -和 2-乙基氨基芴 ( 2-AAF) -DNA 加合物(AF-C 8-dG)的生成,同时也减少致癌物导致的 DNA 链断裂; 限量饮食增加苯并[ a] 芘( BaP) -DNA 加 合物( BaP- N 2-dG)和 6-硝基苯甲萘( 6 -NC) -DNA 总加合物的生成, 也增加致癌物导致的 DNA 链断裂。 ②在小鼠肝中, 致癌 物-DNA 加合物的生成量与致癌物导致的 DNA 链断裂程度密切相关;③限量饮食诱导某些与致癌物代谢有关的酶。结论: 限 量饮食特异地改变小鼠肝代谢酶的活性继而影响致癌物的代谢活化, 这种作用在致癌过程的起始阶段可能起着重要的作用

Correlation of C_3/Ig Two-Component-Determined Circulating Immune Complexes with Humoral Immunity in HBsAg Asymptomatic Carriers

目的　研究 HBs Ag无症状携带者 (ASC)的体液免疫反应。方法　采用直线相关分析法 ,较系统地研究ASC的 C3/ Ig双特异性循环免疫复合物 (C3/ Ig M- TCIC、C3/ Ig G- TCIC和 C3/ Ig A- TCIC)与 Ig M、Ig G、Ig A和 C3的相关性。结果　在健康对照组的诸种具有相关关系的指标中 ,ASC仅在 C3/ Ig M- TCIC与总和游离 Ig M以及 C3/ Ig G- TCIC与总和游离 Ig A具正相关与之一致。此外 ,ASC的 C3/ Ig G- TCIC与复合 Ig A呈显著负相关 ,而健康人与之无关。结论　 ASC确存在体液免疫调节紊乱。其中最主要的是具有重要中和作用的 Ig G类抗体产生不足Objective:To study humoral response in HBsAg asymptomatic carries(ASC).Methods:Association of C 3/Ig two component determined circulating immune complexes(C 3/Ig TCIC:C 3/IgM TCIC,C 3/IgG TCIC and C 3/IgA TCIC)with IgM,IgG,IgA and C 3 has been characterized by correlation analysis.Results:Compared with correlation markers from the healthy controls,only positive correlation of C 3/IgM TCIC with total and free IgM and C 3/IgG TCIC with total and free IgA occurred in ASC.Conclusion:There is indeed a regulation disorder of humoral immune in ASC,in which low neutralizing antibody of IgG class is a main problem.福建省自然科学基金重点资助项目 (C970 16

Isolation of Egg Cells, Zygotes and Proembryos from Allium tuberosum Roxb

卵细胞的分离是进行植物离体受精和研究卵细胞发育的基础。将韭菜的胚珠酶解30MIn后吸打可将其外珠被去掉。将胚珠转移到不含酶的相同溶液中,用解剖针将胚珠从中部切割,然后挤压胚珠的珠孔部位,卵器细胞从胚珠的切口处逸出。用显微操作仪将卵细胞和两个助细胞分开,达到分离韭菜卵细胞的目的。用同样的方法也成功地分离了韭菜的合子和早期原胚。韭菜分离的卵细胞可用于建立韭菜离体受精体系,而分离的合子及早期原胚则可用于探索其受精机制和研究其胚胎发育的过程。Egg cells, zygotes and proembryos of Allium tuberosum Roxb were isolated using an enzymatic digestion.The ovules were incubated in an enzymatic solution for 30 min to remove outer integument.Then they were transferred into an isolated solution without enzymes for mechanical dissection.The ovules were cut from their middle part, and squeezed gently on the micropyle, resulting in the liberation of the egg, zygote and proembryo from the ovules of different stage.About 5-10 egg apparatus could be released from 30 ovules within 1 h.Viable egg cell could be separated with two synergids and collected into a pure population using a micromanipulator.Viable zygotes and proembryos were isolated using the same method.Coupled with our successful isolation of mature sperm cells, the isolation of egg cells of Allium tuberosum Roxb will make in vitro fertilization possible in this plant.The isolation of viable zygotes and proembryos can be used to explore its embryonic development.国家自然科学基金资助项目(No.30670126)---

Some Observations on the Asymmetric Reductive Alkylation of Cych'c Imides

苹果酰亚胺的不对称还原烷基化是合成γ-内酰胺的有效方法,为扩大该法的应用范围,本文研究了多种环状酰亚胺的还原烷基化反应。探讨了影响还原烷基化反应的因素并提出相关的解释。总结出3-取代和3,4-二取代琥珀酰亚胺可成功地进行还原烷基化的规律,并提出了相应的解释。国家杰出青年基金(29625204);国家自然科学基金(29832020);高等学校骨干教师资助计划;厦门大学资