用单价STV制备单分子蛋白质-DNA复合物的研究

将生物素结合位点失活的链霉亲和素(streptavidin,STV)变异体变性后的亚基与野生型STV变性后的亚基以摩尔比3∶1的比例混合,然后重折叠复性,镍柱层析分离得到只有一个有活性生物素结合位点的突变体STV,即单价STV。将单价STV与500bp一端生物素标记的双链DNA(biotin-DNA)按不同摩尔比混合孵育,探讨使用单价STV制备1STV-1DNA复合物的可行性,并筛选制备1STV-1DNA复合物的最佳混合摩尔比。结果表明,在所用的摩尔比梯度中,单价STV与biotin-DNA混合的最佳摩尔比为1∶1,与使用野生型STV制备方法相比,使用单价STV更为简便、高效,为单分子DNA操纵、单分子DNA-蛋白质相互作用研究以及单分子DNA芯片的制备等提供了便捷的方法。     

适体SELEX筛选中单链DNA的制备

探讨建立适体SELEX筛选中单链DNA(ssDNA)的制备方法,生成适体筛选的次级库,为适体SELEX筛选奠定基础.将PCR扩增后带生物素标记的双链DNA(dsDNA)连接在链霉亲和素标记的凝胶上,用NaOH变性解链制备ssDNA.利用荧光法分析dsDNA与凝胶结合所需的时间及NaOH对生物素与链霉亲和素标记凝胶结合的...     

2种不同方法制备玉米赤霉烯酮单克隆抗体适配子的ssDNA文库的比较

体外合成全长为71个碱基的随机ssDNA文库,采用SELEX技术,以玉米赤霉烯酮单克隆抗体为靶蛋白,微孔板为筛选介质进行筛选。通过对比不对称PCR法和生物素-链霉亲和素磁珠2种分离方法制备ssDNA文库的效果,确定了以生物素-链霉亲和素磁珠分离方法制备ssDNA为优选方法。ssDNA文库扩增为双链DNA(dsDNA)文库的PCR反应条件为:退火温度50℃,循环数20次。不对称PCR退火温度为50℃,最佳循环数为30次,引物Ⅰ与引物Ⅱ的比例为80∶1。此反应条件下,ssDNA文库PCR扩增的条带清晰、稳定、特异性高。生物素-链霉亲和素磁珠法为有效筛选特异性强的玉米赤霉烯酮单克隆抗体适配子的优选技术方案,为适配子SELEX筛选奠定基础。     

以金磁微球为载体的H5N1 AIV免疫PCR检测方法的建立

【目的】将高特异性的抗原-抗体反应与高灵敏性的PCR扩增技术相结合,建立快速检测低浓度H5N1禽流感病毒(Avianinfluenzavirus,AIV)的免疫PCR方法。【方法】以pUC19为模板,采用5′端标记有生物素的引物进行PCR扩增,制备含有生物素的报告DNA分子。以金磁微球为固相吸附载体,通过亲和素-生物素的桥联作用,将含有生物素的报告DNA分子与生物素标记的抗AIVH5N1血凝素蛋白的检测抗体分子连接,H5N1AIV与检测抗体结合后,体外扩增报告DNA,间接放大低含量病毒信号,建立可有效检测微量H5N1AIV的免疫PCR方法。对建立的免疫PCR方法的最适报告DNA浓度、最适链亲和素工作浓度、灵敏度和特异性进行确定和评价。【结果】建立的免疫PCR方法最适报告DNA质量浓度为1ng/mL,最适链亲和素工作质量浓度为20ng/mL,该方法可成功检测到10-4EID50/mL的H5N1AIV,而且特异性良好。【结论】建立的以金磁微球为吸附载体的免疫PCR是一种灵敏度高、特异性好的检测微量H5N1AIV的方法。     

一种适用于SELEX技术的单链DNA制备方法

为了建立一种简单、高效制备高纯度单链DNA方法,用于SELEX技术筛选过程中次级文库的筛选,对制备链霉亲和素磁珠的实验条件进行了优化,确定了对称PCR产物与链霉亲和素磁珠偶联的最佳时间和饱和度,并验证了不同浓度NaOH对解链效果的影响,建立了适于SELEX技术的单链DNA制备体系。利用荧光法和聚丙烯酰胺凝胶电泳对本实验所建立方法及传统不对称PCR法制备单链DNA的回收效率和纯度进行了比较,结果表明在最优实验条件下用磁性分离法制备的单链DNA纯度及回收效率均显著高于不对称PCR法。     

单粒微米珠-单根DNA复合物的微流控分离初步研究

定点单分子DNA芯片有重要用途,但其制备极具挑战性,用光镊、磁镊及原子力显微镜等方法分离单分子DNA,为此建立的平台设备昂贵,且分离效率太低,难以满足实验要求。本研究采用微流控技术,在层流基础上电泳分离与富集单粒微米珠-单根DNA的复合物,旨在为基于微米孔阵列承载这种复合物的定点单分子DNA芯片的制备提供样本。此技术的完善将为单分子DNA芯片制作开辟新的途径。     

梅花鹿微卫星DNA富集文库的构建与鉴定

梅花鹿基因组DNA经限制性内切酶MboI酶切,用1.5%的琼脂糖凝胶电泳回收400～1000 bp的DNA片段,与MboI连接接头相连接,连接产物与生物素标记的(AC)15微卫星探针变性及退火,再通过链亲和素偶联磁珠亲和捕捉,经吸附,洗涤及洗脱,然后以洗脱产物为模版,以其中一个接头为引物进行PCR扩增,将扩增产物回收纯化并与pMD18-T载体相连接,转化大肠杆菌DH5a,构建梅花鹿(AC)n微卫星DNA富集文库,经测序分析表明该文库含重组克隆约为1 380个,其中计算得到阳性克隆率为82.9%。梅花鹿微卫星文库的建立将为一步进行梅花鹿基因组结构的分析、梅花鹿遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。     

高灵敏小鼠免疫球蛋白IgG的ELISA检测方法的构建

目的为从天然化合物中高效筛选具有体液免疫抑制活性的先导化合物,本研究将构建高灵敏且价格低廉的小鼠免疫球蛋白IgG的ELISA检测方法。方法利用山羊抗小鼠IgG的Fc段抗体、生物素标记的山羊抗小鼠IgG的F(ab’)2段抗体、辣根过氧化物酶标记的链霉亲和素构建小鼠免疫球蛋白IgG的ELISA检测方法;通过对包被抗体浓度、检测抗体浓度和链霉亲和素浓度的优化,使检测方法灵敏度达到最高;再利用该检测方法检测体外培养小鼠B细胞上清中IgG浓度验证方法的有效性。结果在山羊抗小鼠IgG的Fc段抗体浓度达到5μg·mL~(-1),生物素标记的山羊抗小鼠IgG的F(ab’)2段抗体浓度达到0.25μg·mL~(-1),辣根过氧化物酶标记的链霉亲和素浓度达到0.5μg·mL~(-1),小鼠免疫球蛋白IgG的ELISA检测方法灵敏度达到最高(0.05ng·mL~(-1)),线性范围在0.1~10ng·mL~(-1);该检测方法可以高灵敏检测体外培养LPS刺激小鼠B细胞分泌的IgG抗体。结论本研究成功构建高灵敏且价格低廉的小鼠免疫球蛋白IgG的ELISA检测方法。     

罗非鱼消化道5-HT内分泌细胞的免疫组织化学研究

用链霉亲和素-生物素化过氧化物酶复合物(Strept Avidin Biotion -peroxidase Complex SABC )免疫细胞化学方法,对罗非鱼消化道内分泌细胞进行了免疫组织化学定位.结果表明:5-羟色胺细胞在消化道各段都有分布,其中胃体部密度最大,其次是幽门和喷门处,后肠的密度最小.本文主要讨论了5-HT内分泌细胞在罗非鱼消化道免疫细胞化学定位,可为鱼类的内分泌学提供主要依据.     

单分子DNA在亲水性基底表面的拉伸研究

报道了一种在亲水性表面拉伸裸露λDNA和DNA-微球复合物中λDNA的简单方法。将样品滴在水虎鱼溶液(piranha)和碱性过氧化氢溶液(RCA)清洗的盖玻片上,通过液滴展布与蒸发,利用静电力和表面张力相互作用,将DNA分子伸展并平铺在玻片表面上。激光共聚焦荧光显微镜成像表明,该方法可以较好地将这两种状态的DNA拉直,拉伸长度大于氨基硅烷修饰的表面拉伸方法,同时,也避免了以往一些分子梳过度拉伸而造成DNA变性的弊端。该方法为在单分子水平研究DNA的物理特性和DNA-蛋白质的相互作用提供了新的途径,也为大规模操纵单分子DNA提供了可能。     

沙丁胺醇免疫原的合成与鉴定

将沙丁胺醇(SAL)经过琥珀酸单酯化后,采用混合酸酐法与载体蛋白偶联,用紫外扫描(UV)和变性凝胶电泳(SDS-PAGE)方法检验偶联效果,推算分子结合比;制备全抗原后免疫BALB/c小鼠,间接ELISA测定多抗血清效价,阻断ELISA鉴定其敏感性。UV和SDS-PAGE分析结果表明,成功制备了BSA-HS-SAL,SAL-HS与BSA的分子结合比为11∶1;ELISA结果表明,获得了高效价、敏感的SAL多克隆抗体,为SAL残留免疫学检测方法的建立奠定了基础。     

Morphine-3-succinyl--bovine serum albumin: an immunogenic hapten-protein conjugate

Morphine-3-hemisuccinate was synthesized by reaction of morphine with succinic anhydride. This compound was conjugated to bovine serum albumin by the mixed anhydride method, and the degree of conjugation was determined by base hydrolysis of the conjugate, extraction, and measurement of free morphine. An average of 6.5 molecules of morphine were conjugated to each molecule of protein. Eleven rabbits immunized with varying doses of the conjugate were producing antibody 8 weeks later, as determined by a modification of the ammonium sulfate method, which measures primary binding of antigen by antibody.     

Definition of a bacterial type IV secretion pathway for a DNA substrate

Bacteria use conjugation systems, a subfamily of the type IV secretion systems, to transfer DNA to recipient cells. Despite 50 years of research, the architecture and mechanism of action of the channel mediating DNA transfer across the bacterial cell envelope remains obscure. By use of a sensitive, quantifiable assay termed transfer DNA immunoprecipitation (TrIP), we identify contacts between a DNA substrate (T-DNA) and 6 of 12 components of the VirB/D4 conjugation system of the phytopathogen Agrobacterium tumefaciens. Our results define the translocation pathway for a DNA substrate through a bacterial conjugation machine, specifying the contributions of each subunit of the secretory apparatus to substrate passage.     

玉米和花生中黄曲霉素B_1酶联免疫快速分析方法

将黄曲霉素Ｂ１衍生成胺氧乙酸肟,然后分别同牛血清白蛋白和辣根过氧化物酶进行化学交联而制得结合物。前者用来免疫新西兰大白兔,后者用作免疫反应的示踪物。将所得抗体和示踪物在免疫反应中进行浓度优化,开发出了ＡＦＢ１酶联免疫分析直接竞争抑制法。用所建立的方法能够准确、快速地检测玉米和花生中黄曲霉素Ｂ１的含量。ＡＦＢ１在磷酸盐－吐温缓冲液中的最低检测限为０．４ｐｇ／ｍＬ;在玉米和花生中的最低检测限均为４０ｎｇ／ｋｇ。     

Role of a ubiquitin-like modification in polarized morphogenesis

Type I ubiquitin-like proteins constitute a family of protein modifiers. Here we report the identification of a posttranslational protein modifier from Saccharomyces cerevisiae, Hub1. Overexpression of Hub1 resulted in enhanced conjugate formation when its carboxyl-terminal residue was deleted, suggesting that mature Hub1 may be produced by proteolytic processing. In vivo targets of Hub1 conjugation included cell polarity factors Sph1 and Hbt1. In the hub1Delta mutant, the subcellular localization of both Hbt1 and Sph1 was disrupted, and cell polarization during the formation of mating projections was defective. Consistent with these polarization defects, the hub1Delta mutant was deficient in mating.     

Development of Anti-Isoproturon Polyclonal Antibody

A competitive enzyme-linked immunosorbent assay (ELISA) suitable for the determination of the urea herbicide isoproturon,3-(4-isopropylphenyl)-1,1-dimethylurea, in food and environmental samples was developed. Two haptens named 1-(3-carboxypropyl)-3-(4-isopropylphenyl)-1-methylurca (hapten 4C) and 1-(5-carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (hapten 6C) were synthesized. The haptens were coupled to bovine serum albumin (BSA) and ovalbumin(OVA), respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen,with which a high-titer anti-isoproturon polyclonal antibody (pAb) was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36:1 and 46:1, respectively, as calculated by a UV spectrophotometry.The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6×105. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg L-1 and 1.0 × 105, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding (IC50) was 0.07 mg mL-1.The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.     

氯霉素人工免疫原的合成与鉴定

将氯霉素(CAP)进行化学修饰引入羧基活性基团,合成具有半抗原结构特征的氯霉素半琥珀酸酯(CAP-HS);采用混合酸酐(MA)法将CAP-HS与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成人工免疫原BSA-CAP-HS和包被原OVA-CAP-HS,用红外(IR)、紫外(UV)、凝胶电泳(SDS-PAGE)进行鉴定;用BSA-CAP-HS免疫BALB/C小鼠,间接ELISA测定多抗(pAb)效价,阻断ELISA鉴定其敏感性,交叉反应试验鉴定其特异性。结果表明,CAP-HS与BSA合成成功,其分子结合比为15.6∶1,获得了高价、敏感、特异的CAP pAb,为CAP残留免疫学检测方法的建立奠定了基础。     

蜂王浆介导外源基因转移的初步研究

采用DNA延滞实验研究蜂王浆与DNA的结合能力,然后通过蜂群哺育和人工培育两种方式,对1日龄工蜂幼虫进行短期饲喂,初步研究蜂王浆介导绿色荧光蛋白基因的方法和转移情况.结果表明:在蜂群环境下,蜂王浆中的外源DNA短期内不会完全被分解;但用蜂王浆作媒介并未实现蜜蜂基因转染,蜜蜂能否通过蜂王浆介导实现基因转移,还需进一步探讨.     

Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens

Agrobacterium tumefaciens transfers part of its Ti plasmid, the transferred DNA (T-DNA), to plant cells during tumor induction. Expression of this T-DNA in plant cells results in their transformation into tumor cells. There are similarities between the process of T-DNA transfer to plants and the process of bacterial conjugation. Here, the T-DNA transfer machinery mediated conjugation between bacteria. Thus, products of the Vir region of the Ti plasmid of Agrobacterium tumefaciens, normally involved in transfer of DNA from bacteria to plants, can direct the conjugative transfer of an IncQ plasmid between agrobacteria.     

健康鸡源肺炎克雷伯菌检测到质粒介导喹诺酮耐药qnrA基因

通过PCR技术检测195株健康动物肠杆菌中qnrA基因的流行情况;琼脂稀释法对qnrA阳性菌株进行8种抗菌药物的药物敏感性试验;质粒接合转移试验研究qnrA的可转移性;Southern杂交对qnrA基因进行定位;PCR检测qnrA阳性菌中Ⅰ类整合酶intI基因的携带情况.结果表明,195株肠杆菌中检出1株qnrA阳性的肺炎克雷伯菌Klebsiella pneumoniae GDKA1,经Blast鉴定为qnrA1.药物敏感性试验表明GDKA1对磺胺甲恶唑、氨苄西林、大观霉素、氯霉素、四环素耐药,对环丙沙星(MIC=0.019 mg·L-1),头孢噻肟(MIC=0.047 mg·L-1)表现敏感,对萘啶酸(MIC=12 mg·L-1)表现为部分敏感.接合试验表明qnrA1位于可转移的质粒上,且可以通过接合试验水平传播.接合子TGDKA1对喹诺酮类药物的敏感性比大肠埃希菌Escherichia coli J53 AzR稍高,但远低于GDKA1·South-ern杂交进一步证实了qnrA1基因位于质粒上.TGDKA1的质粒扩增出intI1,表明qnrA1阳性菌株携带I类整合子.可见,qnrA1基因已在健康鸡源肺炎克雷伯菌中出现且同时携带I类整合子位于质粒上,整合子灵活多样的存在方式和传播方式为qnrA基因的传播提供了便利条件,整合子可能携带qnrA基因通过食物链传播给人,因此健康动物共生菌携带qnrA1基因值得注意.