人copineV蛋白多克隆抗体的制备

目的：制备兔抗人copineV多克隆抗体。方法：将copineV N端423bp（626-1048bp）构建到原核表达载体pET28a（＋）,转化大肠杆菌BL21（DE3）,在IPTG诱导下进行蛋白表达;以镍柱纯化后的蛋白为抗原,与等体积佐剂混合后免疫家免3次;用ELISA和Western印迹检测抗血清,用（NH4）2SO4沉淀法初步纯化抗体。结果：表达并纯化了copineV N端蛋白,ELISA检测表明抗血清具有高亲和性,Western印迹检测表明抗体能特异性识别内源性和过表达的copineV。结论：制备了具有高亲和性和特异性的抗人copineV多克隆抗体。     

COX-2的基因克隆和多克隆抗体制备

环氧合酶-2（COX-2）是一种具有多种功能的神经调节物质,它的许多功能细节仍不十分清楚,还需要进一步深入研究.用PCR技术从大鼠脑cDNA文库中,扩增到正常成年大鼠COX-2的cDNA序列,测序结果与已发表的序列一致.通过PCR扩增和基因重组,分别构建COX-2编码基因全长和羧基端部分编码序列的表达载体,并导入大肠杆菌DH5α中,通过IPTG诱导表达重组融合蛋白,结果导入全长编码基因表达载体的DH5α无COX-2融合蛋白表达,而羧基端部分基因编码的COX-2融合蛋白在DH5α中以包涵体的形式进行表达.经SDS-聚丙烯酰胺凝胶电泳分析,在相对分子质量（M_r）为44 000处有1条特异的蛋白质条带.对以包涵体形式表达的蛋白质进行变性、重折叠及纯化后,得到了高纯度融合蛋白.以重组融合蛋白免疫新西兰种大白兔,制作了抗COX-2多克隆抗体,经酶联免疫吸附测定、蛋白质印迹和免疫细胞化学方法检测,此抗体具有较高效价和特异性.COX-2基因和多克隆抗体的获得,为进一步研究COX-2的功能创造了条件.     

斑马鱼ANF基因的克隆及多克隆抗体的制备

心房钠尿肽ANF是钠尿肽家族中的一员,是心肌细胞分泌的一种循环激素,主要在心房组织分泌。生物信息学分析表明,斑马鱼ANF基因开放阅读框全长321bp,编码106个氨基酸,PCR扩增获得斑马鱼ANF基因全长。将所得的PCR片段插入原核表达载体pGEX-4T-1中,并将重组质粒(pGEX-4T-1-ANF)转化大肠杆菌BL21。通过IPTG诱导表达GST-ANF融合蛋白,经GST亲和层析法纯化后,免疫新西兰大白兔制备了多克隆抗体,Western blotting分析表明,制备的抗体具有良好的高效价性和特异性。     

SDHB蛋白的表达、纯化及多克隆抗体制备

SDHB(succinate dehydrogenage complex,subunit B)基因可能介导呼吸链生物功能和调控细胞生长.采用PCR技术扩增出SDHB基因,并将其连接到pGEX-4T-1原核表达载体中,经酶切及测序鉴定后,转化BL21细菌,并用IPTG诱导表达融合蛋白,谷胱甘肽琼脂糖珠亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体.获得了SDHB原核表达重组融合蛋白及高效价的特异性兔抗SDHB多克隆抗体,为SDHB进一步的功能研究奠定了基础.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

多疣壁虎tubulin beta3基因克隆和多克隆抗体制备

Tubulin beta3(TUBB3)是特异表达于神经元的微管蛋白tubulin beta家族成员,被认为在维持轴突正常状态起着重要作用,是神经元特异的标志蛋白。该研究旨在获得多疣壁虎TUBB3全长cDNA序列并制备其多克隆抗体,为进一步研究多疣壁虎断尾再生提供基因和抗体工具。根据多疣壁虎中枢神经组织cDNA文库中TUBB3的EST片段序列,采用RACE-PCR方法,获得了全长cDNA,序列全长1790bp,编码450个氨基酸,与其他物种TUBB3蛋白高度同源;RT-PCR方法和Northern blotting检测了TUBB3组织表达谱及其转录本的大小;在GenBank登录号为JQ080315;NJ法构建的系统进化树分析表明,多疣壁虎的TUBB3蛋白与鸡的TUBB3蛋白在进化距离上较近;构建pET-32a-TUBB3原核表达载体,诱导表达并纯化TUBB3蛋白,获得兔抗壁虎TUBB3多克隆抗体,ELISA检测其效价大于1:65536,Western blotting方法证实所制备的多克隆抗体可特异识别多疣壁虎神经组织的的TUBB3蛋白。     

脱落酸人工抗原合成及多克隆抗体制备

目的:为了对植物细胞中的脱落酸(ABA)进行定量和定位分析,研究了脱落酸人工抗原的合成以及多克隆抗体的制备。方法:用重氮化法将ABA分别与牛血清白蛋白(BSA)、卵清白蛋白(OVA)联结,得到ABA的免疫抗原和包被抗原,并采用紫外全波长扫描和SDS-PAGE对合成的抗原进行了鉴定。以经过鉴定的抗原免疫白兔,制备出ABA的多克隆抗体;采用间接酶联免疫法(ELISA)对抗血清进行效价检测,通过离子交换层析法获得纯化的抗体。结果:ABA与BSA的平均偶联比为5.3∶1,抗血清效价为1∶16000。结论:人工抗原和多克隆抗体制备成功,为采用ELISA和免疫胶体金技术(ICG)检测ABA提供了有效工具。     

斑马鱼Lefty1特异性多克隆抗体的制备

Lefty蛋白是TGFβ大家族的配体,通过与Nodal以及Nodal的受体结合而抑制Nodal信号转导。本文采用PCR技术扩增出斑马鱼lefty1基因（z—lefty1）的部分编码区,将其插入到pGEX-4T-1原核表达载体中。所构建的重组质粒（pGEX／z-Lefiy1）转化入BI21大肠杆菌,通过IPTG诱导表达GST—z—Lefty1融合蛋白。蛋白经尿素洗涤分离后用于免疫新西兰大白兔以制备多克隆抗体。Western blotting检测结果表明获得了高效价的特异性兔抗z—Lefiy1多克隆抗体。这为进一步研究斑马鱼心脏发育的分子机制创造了条件。     

PBP2a多克隆抗体制备及其乳胶凝集检测法的建立

目的制备兔抗青霉素结合蛋白2a(penicillin binding protein 2a,PBP2a)抗体,建立检测PBP2a的乳胶凝集法。方法以重组PBP2a转肽酶区蛋白免疫家兔制备多克隆抗体,ELISA和Western blot法检测所制备的抗血清效价和特异性,用纯化的多抗建立乳胶凝集法。结果纯化的重组蛋白免疫家兔能有效地刺激特异性抗体的产生,抗血清的效价达1∶25 600,Western blot显示该抗体能有效识别原核表达及MRSA临床分离株中的PBP2a,建立了乳胶凝集法,敏感性及特异性良好。结论成功制备了抗PBP2a抗体血清,初步建立了检测PBP2a的乳胶凝集法,为有效制备高特异的单克隆抗体进而研制MRSA快速鉴定试剂盒奠定了良好的基础。     

一种新的制备多克隆抗体的方法

用亲和色谱纯化的rhEPO作为抗原免疫KM小鼠和Bal b/c小鼠,然后腹腔注射S180细胞或SP2/0细胞。S180细胞可刺激KM小鼠产生腹水,腹水量大,抗体效价高。而直接注射SP2/0细胞既未能使KM小鼠产生腹水,也未能使Bal b/c小鼠产生腹水。实验提供了一种新的简便、快速、经济地制备高效价多克隆抗体的方法。     

A procedure for arterial blood sampling in the rabbit

The ear vessels in the rabbit appear to be easily accessible for blood puncturing. Several measures are described to overcome problems of vascular spasm and uncontrolled movements of the animal during injection.     

A method for the production of highly specific polyclonal antibodies

Two polyclonal antibodies directed against paramyosin and tropomyosin from Owenia fusiformis (a marine polychete annelid) were obtained using a new method of immunization. After purification by two-dimensional gel electrophoresis, proteins were transferred onto a nitrocellulose sheet using the Western blot technique. The proteins bound to their cellulose support were injected into rabbits without Freund's adjuvant and without solubilization of nitrocellulose with dimethyl sulfoxide. Highly specific polyclonal antibodies were generated.     

A new approach for producing polyclonal antibodies using impure antigens

A new convenient approach has been designed to produce polyclonal antibodies (PcAb). The approach is based on the principle of the immunoglobulin (Ig) class switch in the immune response. We produced six different antibodies (Ab) against calcineurin A subunit (CNA). CNA, His-tagged calcineurin A subunit (His-CNA), single chain calcineurin (CNB-CNA) and single chain calcineurin-calmodulin complex (CaM-CNB-CNA) were expressed in Escherichia coli (E. coli) BL21 strain, and they were used to immunize male BALB/c mice. These Ab were examined by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The results clearly demonstrated that the specificity of the Ab produced by different protein samples was much higher than that of the Ab produced by a single sample. We used CNA, CaM-CNB-CNA and His-CNA to immunize mice in turn and obtained monospecific PcAb against CNA fortunately by our new approach. Remarkably, our approach not only offered a simple and general alternative to other methods for producing PcAb described previously, but also disclosed a novel process of immunization that could be used to produce monoclonal antibodies (mAb).     

A new approach for producing polyclonal antibodies using impure antigens

A new convenient approach has been designed to produce polyclonal antibodies (PcAb). The approach is based on the principle of the immunoglobulin (Ig) class switch in the immune response. We produced six different antibodies (Ab) against calcineurin A subunit (CNA). CNA, His-tagged calcineurin A subunit (His-CNA), single chain calcineurin (CNB-CNA) and single chain calcineurin–calmodulin complex (CaM-CNB-CNA) were expressed in Escherichia coli (E. coli) BL21 strain, and they were used to immunize male BALB/c mice. These Ab were examined by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The results clearly demonstrated that the specificity of the Ab produced by different protein samples was much higher than that of the Ab produced by a single sample. We used CNA, CaM-CNB-CNA and His-CNA to immunize mice in turn and obtained monospecific PcAb against CNA fortunately by our new approach. Remarkably, our approach not only offered a simple and general alternative to other methods for producing PcAb described previously, but also disclosed a novel process of immunization that could be used to produce monoclonal antibodies (mAb).     

Solid-phase adsorption method for removing undesired antibodies from polyclonal antiserum

We have developed a novel and simple method, requiring only a small amount of antigen, for removal of undesired antibodies from antiserum. The method was established using a well-characterized antiserum against rat luteinizing hormone (anti-rLH). Wells of polystyrene tissue culture plates were coated with rat LH (rLH). Anti-rLH diluted 1:3000 was added to rLH-coated wells and shaken to remove LH antibodies. Control anti-rLH was treated in a similar manner in non-rLH-coated wells. Both antisera were tested by immunocytochemistry on rat pituitaries. Antiserum from rLH-coated wells stained no cells, whereas the control serum stained cells that were morphologically typical of LH cells. The effectiveness of this antibody removal was also confirmed in a modified ELISA. In another experiment, anti-rLH and anti-hTSH beta sera were mixed. The final dilution of both antisera was 1:10,000. Anti-rLH was removed by the purification method described. Completeness of antibody removal was confirmed by a double-immunohistochemical staining of rat pituitary in which sections were first stained by the PAP method and then stained with an immunofluorescence procedure after elution of the first antigen-antibody complex. The mixed antiserum incubated in rLH-coated wells did not stain LH cells. There was no co-localization between the LH immunopositivity demonstrated by an anti-rLH serum using immunofluorescence and cells immunostained with the purified antiserum using the PAP method. As indicated in ELISA, the titer of the TSH beta antiserum was not decreased compared to that of the untreated, mixed control antiserum, and the LH antibodies were eliminated by the treatment. This new purification method has four distinct advantages: (a) antiserum is not treated chemically; (b) it requires only a small amount of antigen compared with the amount required for affinity chromatography; (c) neither the undesired antigen-antibody complex(es) nor an excess amount of antigen is present in the purified antiserum; and (d) removal of undesired antibodies can be monitored by ELISA.