抗促黄体激素单克隆抗体的研制：Ⅱ单克隆抗体的纯化及特性鉴定

在建立六株稳定分泌抗促黄体激素(LH)单克隆抗体的杂交瘤细胞株后,用羟基磷灰石(HAP)吸附层折技术从小鼠特异腹水中一步纯化抗LH单克隆抗体,抗体纯度可达85％—90％,在纯化抗体的聚丙烯酰胺凝胶电泳(PAGE)图谱上基本显示IgG一条带,每ml腹水回收IgG量为3.6mg,并保留大部分抗体活性。特性鉴定结果表明,在六种抗体LH单克隆抗体中,AL-02和AL-06两种抗体与促卵泡激素(FSH)不产生交叉反应,是抗LH-β亚单位上的抗原决定簇的:其它四种抗体与FSH产生完全的交叉反应,是抗LH-α亚单位上抗原决定簇的。用測定抗体与抗原相对结合力的方法估算了四种抗LH单克隆抗体的相对亲和力,结果是AL-01抗体相对亲和力提高,其后依次是AL-03,AL-04和AL-02抗体。     

高内涵筛选NF-kB信号通路的技术体系建立

NF-KB是参与免疫调节、炎症反应、肿瘤发生等过程重要的转录因子,在TNFa、IL-1β、LPS等因子诱导下NF-KB发生核转位并渺活其转录活性.尝试利用高内涵筛选(High Content Screerung,HCS)和RNA干扰(RNA interference,RNAi)技术建立NF-KB核转位高通量筛选体系,该体系的应用将有助于发现新的NF-KB信号通路调节因子,为免疫与肿瘤的机制研究提供线索.     

对虾白斑症病毒的分离纯化及形态结构观察

取患白斑症的濒死斑节对虾 (Penaeusmonodon)的表皮、鳃和胃 ,匀浆后通过蔗糖密度梯度离心和蔗糖垫层差速离心两种方案 ,提纯出WSSV ;两种方案比较 ,蔗糖垫层差速离心操作简便 ,不需使用超速离心机 ,所得病毒纯度较高 ,损失少 .负染观察发现 ,纯化的病毒核衣壳 2个末端结构不同 ,其中一端呈圆形 ,高约 2 1nm ,另一端较平 ,高约 15nm ;完整核衣壳的两个末端结构间的螺旋单位一般为 15圈 ,但也偶见个别较长的核衣壳 ,其螺旋单位数达 2 3圈 .     

Cis-platin去似织毛虫Histriculus similes 小核的研究

用4种浓度的cis-platin(顺式二胺二氯铂)处理似织毛虫的G1期细胞，共建立无小核细胞系7个。所有被处理的细胞都要经历一个生长抑制期，无小核细胞的获得率与cis-platin的浓度和处理时间有密切的关系。所有失去小核的细胞核，约70％的细胞的大核形态和数目异常。结果表明：似织毛虫的小核对于保持大核形态和数目的稳定性发挥着明显的作用。     

纤毛原生动物大核发育的多线染色体的超微结构观察

纤毛原生动物含有大核和小核。小核DNA分子量很大,组成染色体,在无性系生长期闯不产生RNA。小核在结合生殖时经过减数分裂而产生大核,大核只有无丝分裂,核内含400至10000碱基对DNA。这种DNA分子在结合生殖时通过大核发育而成。 Ammermann,Alonso et al,Golikova,最早发现腹毛类毛虫大核发育早期出现和双翅目幼虫相似的多线染色体。Kloetzel,Murti用电镜观察了游仆虫和棘尾虫的多线染色体,并指出多染色体在发育后期被纤维物质横贯形成染色体间带,进     

单抗亲和纯促卵泡激素（FSH）的制备及其特性研究

采用细胞杂交技术生产出抗猪促卵泡激素(pFSH)的单克隆抗体,以其中的AF05单克隆抗体(亲和力常数K=(1.14±0.28)×10~(-9)M)制作成单抗免疫亲和层析柱。以简便快速的一步纯化法从猪垂体浸出液中提取出高纯度pFSH。经小鼠卵巢增重法测定,该pFSH的生物学活性在30Armour单位/mg蛋白以上;聚丙烯酰胺凝胶电泳(pAGE)结果显示,该pF-SH的纯度超过美国Schering公司的pFSH产品。     

蚯蚓纤溶酶总RNA的提取及电泳分析

分离提取纯净完整的RNA对于分子克隆实验是很重要的,而且是进行基因表达分析的基础。本实验通过总RNA纯化试剂盒(V-gene Total RNA Purification Kit)从蚯蚓体内提取蚯蚓纤溶酶总RNA,为下一步进行RT-PCR等分子克隆实验打下了基础。     

CR/VAC/MMA/AA四元接枝共聚物合成及接枝率的研究

以醋酸乙烯酯(VAC)、甲基丙烯酸甲酯(MMA)、丙烯酸(AA)为接枝单体,以过氧化二苯甲酰(BPO)为引发剂,在氯丁胶(CR)上进行接枝共聚,制备出CR/VAC/MMA/AA四元接枝共聚物。探讨了提取剂和提取时间对四元接枝共聚物的接枝率测定的影响,并讨论了MMA/VAC配比、AA用量对接枝率和聚合转化率的影响。用红外光谱(IR)和DSC对接枝共聚物进行了表征。结果表明,选用丙酮作提取剂,提取时间为48h,接枝率数据较理想;试验中BPO质量为CR的1.2%-1.5%,CR/VAC/MMA/AA的质量配比为100/45/15/10时,其接枝率和聚合转化率最佳。     

小鼠肥大细胞蛋白酶4的原核表达和多克隆抗体制备

文章构建小鼠肥大细胞蛋白酶4（mMCP-4）原核表达体系,获得原核表达蛋白,并制备兔抗mMCP-4多克隆抗体,提取小鼠脾细胞总 RNA ,运用 RT-PCR技术获得目的基因 mMCP-4,构建重组原核表达载体pET28a-mMCP-4,将其转化到大肠杆菌BL21中诱导表达。mMCP-4融合蛋白经Ni亲和层析法纯化后,作为抗原免疫兔子获得mMCP-4多克隆抗体。采用ELISA法测抗体效价,western blot检测抗体特异性。结果表明,构建重组表达质粒pET28a-mMCP-4,经大规模原核表达获得大量mMCP-4融合蛋白,制备的兔抗血清效价达1∶128000,并表现出较好特异性。研究结果为进一步研究mMCP-4生物学功能奠定了基础。     

高迁移率蛋白HmgB3和组蛋白Mlh1维持了四膜虫小核稳定性

高迁移率族蛋白(High Mobility Group protein,HMG)和组蛋白H1结合染色质DNA,在维持染色质的高级结构、基因组基因表达调控以及DNA修复等方面发挥重要作用。嗜热四膜虫细胞含有一个体细胞系的大核和一个生殖系的小核,小核特异定位的高迁移率蛋白HmgB3或组蛋白Mlh1的缺失并未引起生长期细胞的异常表型。本研究通过同源重组的方法构建了HMGB3和MLH1的双敲除细胞突变株ΔHMGB3ΔMLH1。突变细胞在营养生长期能够正常增殖,但对甲基磺酸甲酯较为敏感。缺对PCR检测发现突变株中小核染色体有缺失现象,并且不能完成有性生殖。有性生殖过程中,减数分裂后的小核异常降解。结果表明高迁移率蛋白HmgB3和小核组蛋白Mlh1共同维持了四膜虫小核的稳定性,并可能具有功能上的冗余性。     

2,4-二氯苯甲醛缩氨基硫脲Cu(I)、Fe(Ⅱ)、Zn(Ⅱ)配合物的直接电化学合成

本文在以2,4一二氯苯甲醛缩氨基硫脲(HL)为配体的非水溶剂中,用Cu,Fe,Zn金属做阳极,首次用电化学金属阳极氧化法合成2,4一二氯苯甲醛缩氨基硫脲(HL)与Cu(I)、Fe(Ⅱ)、Zn(Ⅱ)的金属配合物,通过元素分析、红外光谱、紫外光谱、摩尔电导等对配合物进行了表征。     

犬腺病毒2型抗原的制备及间接ELISA的建立

用MDCK细胞增殖犬腺病毒2型弱毒疫苗株,病毒培养上清液经差速离心和酒石酸钾密度梯度离心浓缩、纯化后作为诊断抗原,建立了犬腺病毒2型抗体检测的间接ELISA。确定最佳抗原包被量为0.168μg/孔,待检血清最佳稀释倍数为1:100,作用时间为60 min,羊抗犬酶标抗体稀释倍数为1:2000,作用时间为60 min,底物作用时间为37℃,10 min。判定标准为S/P值≥0.351者判为阳性,S/P值≤0.318者判为阴性。该抗原不与犬瘟热病毒、犬细小病毒、犬波特氏杆菌等3种犬常见传染病的阳性血清反应,具有良好的特异性;批内、批间重复试验,变异系数小于15%,显示具有较好的重复性;检测血清样品56份,与病毒中和试验结果比对的符合率为93.75%。本研究结果为实现犬腺病毒感染监测,进行犬腺病毒流行病学调查提供了一种简便的血清学诊断方法。     

抗棉铃虫组织蛋白酶B单克隆抗体的制备及鉴定

分别以棉铃虫组织表达的组织蛋白酶B和基因重组的大肠杆菌表达的组织蛋白酶B为抗原免疫BALB／c小鼠，选择血清抗体滴度高的免疫小鼠，取其脾细胞和Sp2／0骨髓瘤细胞进行细胞融合．经ELISA法筛选阳性克隆和有限稀释法克隆细胞，共获得分泌抗组织蛋白酶B的单克隆抗体杂交瘤细胞3株，分别命名为9D5，100E2和100H6．以ELISA法和Western blotting法对3株杂交瘤细胞株分泌的单克隆抗体进行了特异性鉴定，其中9D5和100E2能够同时识别两种不同来源的组织蛋白酶B，而100H6只能识别基因重组的大肠杆菌表达的组织蛋白酶B．     

Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytochemical method.

Current immunocytochemical techniques detect all antibodies that react with tissue. Unfortunately, some of these antibodies may react with antigens other than those intended. Such problems are minimised by using sets of antibodies detecting different regions of the desired antigen. However, as immunocytochemical methods can now detect very low antibody concentrations, the purity of antisera is critical. Furthermore, although antisers may be purified by affinity chromatography, difficulties in recovering high-avidity antibodies cause most affinity-purified antisera to be enriched in low-avidity antibodies, which may be dislodged during staining. We have therefore developed, and describe here, a new ultrastructural post-embedding staining technique, based on the divalency of IgG molecules and using antigen-coated colloidal gold granules. Previously, colloidal gold-labelled antibodies have been used for post-embedding staining. Unlike our gold-labelled antigen detection (GLAD) technique, however, these methods do not differentiate between specific and nonspecific antibodies. The GLAD method detects only specific antibodies and does not select against high-avidity antibodies, and in this it resembles the radioimmunocytochemical method. However, the GLAD method differs from the latter in that it is useful for ultrastructural studies, does not require autoradiography and allows simultaneous detection of multiple antigens. Moreover, specific activity compared with background may be quantitated.     

大豆胰蛋白酶抑制剂的制备与性质测定

用DEAE-纤维素柱层析分级洗脱,从大豆中分离出三种胰蛋白酶抑制剂。以BAEE作底物,测得三种组份对胰蛋白酶的抑制程度为16.6％、25.0％和100％。三种抑制剂的分子量分别为20000、21000和12000,等电点是pH3.4、pH2.7和pH4.5。     

N-对甲苯磺酰基-N，N-双（2-亚胺乙基）-氨-双氮杂桥富勒烯［60］的合成与表征

以三乙醇胺和对甲苯磺酰氯为原料，通过酯化、叠氮化的系列反应合成了N-对甲苯磺酰基-双（2-叠氮乙基）胺，并以此为中间体与富勒烯C₆₀发生1,3-偶极环加成反应，制备合成了N-对甲苯磺酰基-双（2-叠氮乙基）胺-富勒烯［60］，并用MS、FT-IR、¹³C-NMR、¹H-NMR、二维异核位移相关谱（HMBC、HSQC）、同核位移相关谱（COSY）等手段对产物结构进行了表征。     

T-cell responses to human AIDS virus in macaques immunized with recombinant vaccinia viruses

There is much interest in developing vaccines against acquired immune deficiency syndrome (AIDS), which is caused by a retrovirus termed human immunodeficiency virus (HIV). Isolates of this virus include human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), and AIDS-associated retrovirus (ARV). Several approaches towards the development of an AIDS vaccine result in the production of antibodies in subprimates. These methods involve the use of: antigens isolated from the AIDS virus; viral antigens expressed by transfected cells or by recombinant vaccinia viruses; and particular synthetic peptides of viral antigens. Because T-cell-mediated immunity (in addition to antibodies) is involved in resistance to diseases and death caused by various enveloped viruses, we sought to determine whether potential AIDS vaccines can induce T-cell responses against the AIDS virus. Here we report that immunization of non-human primates, Macaca fascicularis (macaques), with recombinant vaccinia viruses that express LAV envelope glycoproteins gp41 and gp110 results not only in the production of antibodies against the LAV envelope antigens but also in the generation of T-cells that proliferate and produce the lymphokine interleukin-2 (IL-2), in response to stimulation with purified LAV. We believe this is the first report demonstrating T-cell-mediated immunity to the virus that causes AIDS.     

Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes

Altered subcellular distribution and activity of protein kinase C (PKC) is associated with transmembrane signalling in a variety of systems in which receptor occupancy leads to increased hydrolysis of polyphosphoinositides. Here we report evidence that in B lymphocytes, cyclic-cAMP-generating signal transduction pathways can activate translocation of PKC from the cytosol to the nucleus. Elevated cAMP levels and translocation of PKC to the nucleus are induced by antibodies against Ia antigens in normal B lymphocytes. Further, cAMP analogues mediate the translocation of PKC to the nucleus of these cells. These findings suggest that in physiological situations, ligation of B-lymphocyte Ia molecules by helper T cells leads to increased cAMP production which in turn causes PKC translocation to the nucleus. In view of recent observations that antibodies against Ia antigens induce differentiation of B cells, we conclude that nuclear PKC may function in the regulation of gene expression.     

锰氧化物La_(0.55)Ca_(0.45)MnO_3输运性质的研究

本文中,利用交流磁化率、电阻率、热电势和ERP谱等多种测量手段对锰氧化物La_(0.55)Ca_(0.45)MnO_3多晶样品的输运性质和磁阻特性进行研究。实验结果表明,是双交换作用和小极化子效应的共同作用决定了CMR效应的特性。La_(0.55)Ca_(0.45)MnO_3与La_(0.7)Ca_(0.3)MnO_3样品的电阻率在T<相似文献   

Activation at M-phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2+

In both starfish and amphibian oocytes, the activity of a major protein kinase which is independent of Ca2+ and cyclic nucleotides increases dramatically at meiotic and mitotic nuclear divisions. The in vivo substrates of this kinase are unknown, but phosphorylation of H1 histone can be used as an in vitro assay. We have purified this kinase from starfish oocytes. The major band in the most highly purified preparation contained a polypeptide of relative molecular mass (Mr) 34,000 (34K). This is the same size as the protein kinase encoded by cdc2+, which regulates entry into mitosis in fission yeast and is a component of MPF purified from Xenopus. Here, we show that antibodies against p34 recognize the starfish 34K protein and propose that entry into meiotic and mitotic nuclear divisions involves activation of the protein kinase encoded by a homologue of cdc2+. Given the wide occurrence of cdc2+ homologues from budding yeast to Xenopus and human cells, this activation may act as a common mechanism controlling entry into mitosis in eukaryotic cells.