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31.
目的通过构建原核表达载体,获得纯化的肺炎链球菌S.pn重组假想蛋白SPD0873,并制备多克隆抗体,进一步分析其在常见S.pn菌株中的保守性。方法分离培养D39型肺炎链球菌,获取其基因组DNA。利用PCR方法扩增去除信号肽的spd0873序列,采用基因体外重组法将spd0873序列克隆到原核表达载体pET-32(a)内,测序鉴定。将重组质粒转化到E.coli Rossetta(DE3)中,经IPTG诱导大量表达融合6个组氨酸标签的SPD0873重组蛋白,经Ni—NTA树脂纯化后,获得的重组蛋白用SDS—PAGE和Western印迹鉴定;将鉴定后纯化的蛋白免疫BALB/C小鼠制备多克隆抗体,并用间接ELISA检测多克隆抗体的效价,Western印迹方法分析多克隆抗体的特异性,同时,鉴定该蛋白在5种常见肺炎链球菌分离株的保守性。结果克隆的spd0873序列与GenBank中的数据相符,并实现了SPD0873蛋白高水平的可溶表达。纯化蛋白免疫BALB/C小鼠获得高滴度、高特异性的的多克隆抗体,Western印迹验证SPD0873蛋白在5株常见肺炎链球菌菌株中均有表达。结论成功制备了高滴度、高特异性的SPD0873蛋白多克隆抗体,同时,检测到SPD0873蛋白在5种常见的肺炎链球菌菌株中非常保守,为研究该蛋白的生物学功能及肺炎链球菌多肽联合疫苗的研制奠定了基础。 相似文献
32.
Rudenko NV Sinegina LL Arzhanov MA Ksenzenko VN Ivashina TV Morenkov OS Shaloiko LA Vinokurov LM 《Journal of biochemical and biophysical methods》2007,70(4):605-611
The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications. 相似文献
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目的:克隆水稻YTB osvdac5基因,原核表达后获得纯化的OSVDAC5蛋白,制备相应的抗体.方法:采用Trizol法提取水稻总mRNA,反转录为cDNA,通过PCR扩增得到该基因与原核表达载体连接,构建重组质粒pET-30a-osvdac5,并转入大肠杆菌进行原核表达,SDS-PAGE检测表达产物.通过镍柱纯化获得的单一目的蛋白用于抗体制备,用Western Blot检测抗体的特异性.结果:克隆到原核表达载体中osvdac5基因的ORF为813 bp,编码271个氨基酸.在大肠杆菌中15℃、0.7mmol/L的IPTG浓度诱导17 h是pET-30a-osvdac5融合蛋白表达的优选条件,表达的OSVDAC5蛋白属于包涵体蛋白.镍柱纯化后的OSVDAC5为30 kD左右的单一条带.Western Blot分析表明,抗体能够与30 kD处的OSVDAC5蛋白进行特异性结合.结论:成功克隆了水稻YTB osvdac5基因,原核表达蛋白OSVDAC5制备的多免隆抗体具有一定特异性,能与免疫抗原结合,这为进一步研究OSVDAC5蛋白在植物不同生长发育时期中的表达模式奠定了基础. 相似文献
37.
构建含有HPT的原核表达质粒,经诱导表达纯化后获得HPT抗体。从含有hpt的质粒pCambia1301上扩增目的基因,经SalⅠ和NotⅠ双酶切后与原核表达载体pET-28b(+)进行粘性末端连接,成功构建了pET-28b-hpt质粒,并转化到宿主细菌大肠杆菌Rosset(DE3)中进行蛋白表达。在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下,pET-28b-hpt原核表达载体在E.coli Rosset菌株中以包涵体的形式高效表达了HPT蛋白,并以纯化的目的蛋白为抗原免疫兔制备了多克隆抗体。利用本方法可以获得特异性强、灵敏度高的多克隆抗体。 相似文献
38.
Sharan RN Devi BJ Humtsoe JO Saikia JR Kma L 《Molecular and cellular biochemistry》2005,278(1-2):213-221
Poly-ADP-ribosylation (PAR) of cellular proteins has been shown to have decisive roles in diverse cellular functions including
carcinogenesis. There are indications that metabolic level of poly-ADP-ribosylated cellular proteins might indicate carcinogenesis
and, therefore, could be potentially used in cancer screening program. Keeping in mind the limitations of currently available
assays of cellular PAR, a new assay is being reported that measures the metabolic level of poly-ADP-ribosylated cellular proteins.
The ELISA based slot and Western blot immunoassay used polyclonal antibody against natural, heterogeneous ADP-ribose polymers.
It could be successfully employed to qualitatively and quantitatively assay metabolic levels of poly-ADP-ribosylated proteins
of spleen and liver tissues of normal mice or mice exposed to dimethylnitrosamine for up to 8 weeks; potentially PAR of cellular
proteins could be assayed in any tissue or biopsy. Implications of the results in cancer screening program have been discussed.
(Mol Cell Biochem 278: 213–221, 2005) 相似文献
39.
Recombinant polyclonal antibodies for cancer therapy 总被引:4,自引:0,他引:4
Although monoclonal antibodies are increasingly used for cancer therapy, remissions are only temporary due to emergence of tumor cell escape variants that are no longer affected by the antibody. The emergence of escape variants could be minimized by multi-targeting of tumor cells with polyclonal antibodies, which would also be more efficient than monoclonal antibodies at mediating effector functions for target destruction. A technology for generating recombinant polyclonal antibodies for cancer therapy has been developed based on the construction and selection of tumor-reactive Fab phage display libraries. The selected Fabs are mass-converted to full-length polyclonal antibody libraries (PCALs) of any isotype and any species. Prototypic PCALs generated against human colorectal cancer cell lines showed that libraries of diverse recombinant antibodies, enriched for reactivity to the cancer cells compared to normal human cells, can be obtained. The success of recombinant polyclonal antibodies as cancer therapeutics will depend on the ability to generate, characterize, and mass-produce PCALs with high ratios of cancer-to-normal reactivities that cross-react with many cancers of the same type. 相似文献
40.
A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) has been isolated from Zea mays by screening a cDNA library. The cDNA, designated ZmPLC, encodes a polypeptide of 586 amino acids, containing the catalytic X, Y and C2 domains found in all PI-PLCs from plants. Northern blot analysis showed that the expression of the ZmPLC gene in roots is up-regulated under conditions of high salt, dehydration, cold or low osmotic stress conditions. Recombinant ZmPLC protein was expressed in Esch- erichia coli, purified and used to produce polyclonal antibody, this polyclonal antibody is important for further studies to assess the ultimate function of the ZmPLC gene in plants. 相似文献