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Tropic1808基因的原核表达及其表达产物的生物活性   总被引:8,自引:0,他引:8       下载免费PDF全文
Tropic180 8基因是新近获得的一个鼠源性的cDNA .Tropic180 8基因开放阅读框架片段通过PCR方法从质粒中扩增后 ,重组入表达载体pET 2 1a中 ,转化大肠杆菌BL2 1(DE3 ) .用IPTG诱导目的蛋白的表达 ,SDS PAGE并凝胶图象分析确定目的蛋白表达水平占细菌总蛋白的 14%以上 .表达蛋白在N端融合有 16个氨基酸 ,将表达蛋白电转移至PVDF膜 .氨基酸序列分析表明 ,其N端第 17~ 2 5位氨基酸序列与Tropic180 8基因编码序列一致 .利用融合部分含T7·Tag ,通过亲和层析纯化表达蛋白 ,经Westernblot检测为目的蛋白 ,加入到无血清培养的新生SD大鼠背根神经节 (DRG)中 ,观察到表达蛋白对DRG具有促进存活和促进突起生长的作用 .  相似文献
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神经诱向生长因子18kD蛋白的纯化   总被引:6,自引:0,他引:6       下载免费PDF全文
 提取与纯化诱向生长因子是神经生物化学研究中的一个重要课题.采用自然系统凝胶电泳,分离周围神经损伤过程中产生的具有诱向生长作用的18kD蛋白,再以等电聚焦凝胶电泳与神经组织联合培养,揭示了pI为5.2的18kD蛋白具有诱神经生长的作用.经高效液相色谱仪分析获得较纯的18kD诱向因子.蛋白/多肽测序仪检测,18kD蛋白N端氨基酸序列为:PEPAWSAPAP.  相似文献
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本研究在前期工作基础上,用CHO细胞表达的含PreS1+S融合抗原的新型基因工程HBV颗粒疫苗(HBSS1)与Al(OH)3、CpG及CpG+Al(OH)3等佐剂配伍,在Balb/C小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFNELISpot检测)。结果表明:CpG佐剂结合HBSS1颗粒疫苗可快速诱导(单针免疫)高水平的抗PreS1及S抗体,IgG2a/IgG1比率>1,同时可诱导较高抗原特异的细胞免疫应答;Al(OH)3+CpG双佐剂组一次免疫后可诱导产生最高的抗S抗体滴度(1:105),其产生的抗体亚类包括IgG1、IgG2a与IgG2b;在S抗原N端(13~49aa)存在优势CTL表位。结论:CpG佐剂结合HBSS1颗粒疫苗应是发展新型治疗性乙肝疫苗的较佳选项。  相似文献
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张柯  谭文杰  邓瑶  李津  吴小兵  阮力 《病毒学报》2008,24(4):287-294
In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.  相似文献
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正Dear Editor,Since September 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) cases have been reported in more than 27 countries, and more than 2,000 cases have been confirmed in the laboratory (http://www.who.int/emergencies/mers-cov/en/). MERS-CoV causes an acute and severe respiratory illness with a high mortality rate(~35%) in humans (Shi et al., 2017, Zaki et al., 2012).Neutralizing antibodies targeting the spike of MERS-CoV have been shown to be a therapeutic option for treatment of lethal disease (Agrawal et al., 2016, Ying et al., 2014).  相似文献
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正Dear Editor,Branched-chain amino acids (BCAAs) are essential amino acids that must be obtained from the diet for humans and animals. Branched-chain amino acid transaminases (BCATs)catalyze a key step in the BCAA synthesis pathway, however, little is known about their biological roles in planta. In this study, we functionally characterized two BCATs, OsBCAT4 and OsBCAT5, in rice and showed that they regulate  相似文献
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The northern pig-tailed macaque(NPM,Macaca leonina) has become a widely used animal model in biomedical research. In this study, we measured serum immunoglobulin IgG, IgM, IgA, complement C3, C4 and CRP levels in 3-11 year old captive northern pig-tailed macaques using HITACHI 7600-20 automated chemistry analyzer in order to determine the influences of age and gender on these items. The results showed that serum IgA, IgM, C3 and C4 levels were not correlated with age(P0.05), while serum IgG levels increased progressively with age(r=0.202;P=0.045). Serum IgG, IgA, IgM and C3 levels were higher in females than in males(P0.05). Moreover, serum C3 concentration was both positively and strongly correlated with that of C4(r=0.700; P0.0001). This study provides basic serum immunoglobulin and complement data of captive northern pig-tailed macaques, which may prove useful for future breeding efforts and biomedical research.  相似文献
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