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2010年12月15日

【期刊论文】肺炎衣原体CPn0308 的基因克隆及其内源性蛋白定位的研究

刘殿武, 贾天军, 罗建华, 张庶民, 钟光明

卫生研究,2008,37(2):219~222,-0001,():

摘要

目的 克隆肺炎衣原体基因CPn0308,表达融合蛋白,并制备抗体对其表达的内源性蛋白进行初步定位。方法 根据STD基因库提供的信息设计引物,聚合酶链反应(PCR)克隆目的基因,用BamHI/NotI对克隆的目的基因和pGEX-6P2 载体进行酶切,T4 连接酶连接后,重组质粒经42℃转化XL1-blue 细菌:用PCR进行初步筛选,交叉PCR对提取质粒进一步确定,最后对插入片断进行序列分析:用IPTG诱导阳性克隆的细菌使其表达GST融合蛋白,纯化后免疫小鼠制备抗体,使用IFA 对内源性蛋白进行定位分析。结果 克隆出肺炎衣原体基因CPn0308,全长为366bp,编码121个氨基酸;并表达了融合蛋白GST-CPn0308,分子量约为39kD;制备了抗体,IFA实验发现该蛋白初步定位于肺炎衣原体包涵体膜蛋白上。结论 成功克隆肺炎衣原体基因CPn0308,其内源性蛋白初步定位于肺炎衣原体包涵体膜上。

关键词: 肺炎衣原体, 基因克隆, 内源性蛋白

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2011年01月04日

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2010年08月30日

【期刊论文】Molecular cloning and immune responsive expression of MDA5 gene, a pivotal member of the RLR gene family from grass carp Ctenopharyngodon idella

苏建国, Jianguo Su*, Teng Huang, Jie Dong, Jianfu Heng, Rongfang Zhang, Limin Peng

Fish & Shellfish Immunology 28(2010)712-718,-0001,():

摘要

The cytoplasmic helicase protein MDA5 (melanoma-differentiation-associated gene-5) recognizes long molecules of viral double-stranded RNA (dsRNA) and single-stranded RNA with 50 triphosphate and mediates type I interferon secretion. In the present study, the first MDA5 gene in fish was cloned and characterized from grass carp Ctenopharyngodon idella. The full length of the C. idella MDA5 (CiMDA5) cDNA is 3233 nucleotides in length and encodes a polypeptide of 961 amino acids. The deduced amino acid sequence contained six main structural domains: a CARD (caspase activation and recruitment domain), a DEXDc (DEAD/DEAH box helicase domain), a ResIII (conserved restriction domain of bacterial type III restriction enzyme), two HELICc (helicase superfamily c-terminal domain) and an RD (regulatory domain). The CiMDA5 mRNAwas widespread expression in the tested tissues, was high level in spleen, skin and gill tissues, and was up-regulated by GCRV injection by semi-quantitative RT-PCR assay. The CiMDA5 transcripts in spleen were significantly up-regulated at 12 h (1.80 folds, P< 0.05), reached the crest at 24 h (7.48 folds, P<0.05), and then recovered to normal level at 48 h post-injection (P>0.05) of grass carp reovirus (GCRV). In liver, the temporal expression of CiMDA5 mRNA was significantly increased at 48 h (5.00 folds, P<0.05) and returned to control level at 72 h (P>0.05) after GCRV challenge. These data implied that CiMDA5 involved in the early stage of antiviral innate immune defense to GCRV in grass carp, and provided new insight into the evolution research of RLR (RIG-I like receptor) gene family.

关键词: Grass carp (, Ctenopharyngodon idella), ,, MDA5,, RLR,, Gene cloning,, mRNA expression,, ., Grass carp reovirus

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2010年08月30日

【期刊论文】Genomic organization and expression analysis of Toll-like receptor 3 in grass carp (Ctenopharyngodon idella)

苏建国, Jianguo Su a, *, Songhun Jang b, Chunrong Yang a, Yaping Wang b, Zuoyan Zhu b

Fish & Shellfish Immunology 27(2009)433-439,-0001,():

摘要

investigate grass carp immune system responding to GCRV (grass carp reovirus) infection, the full-length cDNA sequence and genomic organization of grass carp TLR3 (CiTLR3) was identified and characterized. The full-length genome sequence of CiTLR3 is composed of 5668 nucleotides, including five exons and four introns. The full-length of CiTLR3 cDNA is 3681 bp in length and encodes a polypeptide of 904 amino acids with an estimated molecular mass of 102,765 Da and a predicted isoelectric point of 8.35. Analysis of the deduced amino acid sequence indicated that CiTLR3 has four main structural domains, including a signal peptide sequence, 14 LRR (leucine-rich repeat) motifs, a transmembrane region and a TIR (Toll/interleukin-1 receptor) domain. It is most similar to the crucian carp (Carassius auratus) TLR3 amino acid sequence with an identity of 99%. Quantitative RT-PCR analysis showed that CiTLR3 transcripts were significantly up-regulated starting at day 1 and continued through day 7 following GCRV infection (P<0.05). These data implied that CiTLR3 is involved in antiviral defense, provide molecular and functional information for grass carp TLR3, and implicate their role in mediating immune protection against grass carp viral diseases.

关键词: Ctenopharyngodon idella,, Toll-like receptor 3,, Genomic organization,, Gene cloning,, Grass carp reovirus

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2009年01月19日

【期刊论文】A New Type of p-N-Acetylglucosaminidase from Hydrogen-Producing Clostridium paraputrfmm M-2 1

李华钟, HUAZHONG LI, KENJI MORIMOT, TETSUYA KIMURA, KAZUO SAKKA, AND KUNIO OHMIYA*

JOUFWAL OF BIOSCIENCE AND BIOENGINEERING, 2003, 96, 268-274,-0001,():

摘要

暂无

关键词: gene cloning,, P-N-acetylglucosaminidase,, hydrogen,, Clostridiumparaputrijicum

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2005年03月08日

【期刊论文】旋毛虫抗原基因Ts88的克隆及鉴定*

诸欣平, 杨雅平, 杨静, 雷丽萍, Pascal Boireau

中国人兽共患病杂志,2004,20(1):19~21,-0001,():

摘要

目的 获取旋毛虫具有抗原性的新基因。方法 应用旋毛虫免疫血清和人工感染血清对旋毛虫成虫cDNA文库约3×105重组噬菌斑进行筛选。基因克隆、DNA测序及5’-RACE 获cDNA全长;采用ESEE、DNAstar软件及核苷酸序列数据库(GenBank)进行cDNA序列分析。结果 与结论免疫筛选成虫cDNA文库,获得3个阳性克隆。其中的Ts88克隆cDNA全长为1966bp,编码484个氨基酸,含有一个PWWP功能结构域及多个抗原表位。Ts88cDNA是一个尚未见报道的旋毛虫抗原新基因。

关键词: 旋毛虫, cDNA文库, 基因克隆, 筛选, cDNA末端快速扩增

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