目的对1株在九江地区分离的柯萨奇A6病毒(CoxA6)VP1区域进行克隆,并对其编码蛋白的结构、功能及B细胞表位进行分析和预测,为CoxA6疫苗制备和诊断方法的研究提供理论基础。方法采用RT-PCR法对CoxA6分离株VP1区进行扩增和克隆、序列分析,...目的对1株在九江地区分离的柯萨奇A6病毒(CoxA6)VP1区域进行克隆,并对其编码蛋白的结构、功能及B细胞表位进行分析和预测,为CoxA6疫苗制备和诊断方法的研究提供理论基础。方法采用RT-PCR法对CoxA6分离株VP1区进行扩增和克隆、序列分析,应用SigaIP、TMPRED、TMHMM、Big-PI Predictor、Cell-Ploc、PSORT、NetNES、Netphos、SOPMA、NetNGlyc、MotifScan、InterProscan、SMART、PROSITE、GOR4、Bepipred Linear Epitope Prediction等生物信息学方法预测其VP1基因编码蛋白特性和潜在的B细胞表位。结果该CoxA6分离株VP1基因编码305aa的多肽,分子量为33.5kDa。该蛋白无信号肽、跨膜区,是亲水性蛋白;二级结构主要以无规则卷曲为主,其次为α-螺旋及β-片层。该蛋白共有7个可能的B细胞抗原表位,位于151~173aa区域内的表位分值最高为2.774。结论成功克隆了1株CoxA6的VP1基因,并对其进行序列分析、蛋白质结构和B细胞表位预测,为制备CoxA6疫苗和开发诊断方法提供了分子生物学基础。展开更多
Many studies point to an association between Helicobacter pylori(H.pylori)infection and inflammatory bowel diseases(IBD).Although controversial,this association indicates that the presence of the bacterium somehow aff...Many studies point to an association between Helicobacter pylori(H.pylori)infection and inflammatory bowel diseases(IBD).Although controversial,this association indicates that the presence of the bacterium somehow affects the course of IBD.It appears that H.pylori infection influences IBD through changes in the diversity of the gut microbiota,and hence in local chemical characteristics,and alteration in the pattern of gut immune response.The gut immune response appears to be modulated by H.pylori infection towards a less aggressive inflammatory response and the establishment of a targeted response to tissue repair.Therefore,a T helper 2(Th2)/macrophage M2 response is stimulated,while the Th1/macrophage M1 response is suppressed.The immunomodulation appears to be associated with intrinsic factors of the bacteria,such as virulence factors-such oncogenic protein cytotoxin-associated antigen A,proteins such H.pylori neutrophil-activating protein,but also with microenvironmental changes that favor permanence of H.pylori in the stomach.These changes include the increase of gastric mucosal pH by urease activity,and suppression of the stomach immune response promoted by evasion mechanisms of the bacterium.Furthermore,there is a causal relationship between H.pylori infection and components of the innate immunity such as the NLR family pyrin domain containing 3 inflammasome that directs IBD toward a better prognosis.展开更多
The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the ...The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.展开更多
根据已发表的鸭甲肝病毒(Duck hepatitis A virus,DHAV)疫苗株A66株基因组序列,利用生物学分析软件DNAStar进行VP1蛋白的二级结构和抗原域预测,确定其主要抗原域(major antigen domain of VP1,VP1M),设计并合成一对可扩增VP1M的特异性引...根据已发表的鸭甲肝病毒(Duck hepatitis A virus,DHAV)疫苗株A66株基因组序列,利用生物学分析软件DNAStar进行VP1蛋白的二级结构和抗原域预测,确定其主要抗原域(major antigen domain of VP1,VP1M),设计并合成一对可扩增VP1M的特异性引物,PCR扩增获得长为327 bp的VP1M基因片段。将VP1M基因片段定向克隆至p GEX-4T-1表达载体中,鉴定正确后进行IPTG诱导表达,获得了以包涵体为主的重组蛋白。重组蛋白纯化后,经过Western blot检测表明重组蛋白具有良好的抗原性。以该蛋白作为包被抗原,成功建立了检测DHAV-1的间接ELISA方法。该方法具有良好的特异性、敏感性和重复性,为DHAV的快速诊断、流行病学调查和免疫鸭群的抗体检测提供了快速实用的检测手段。展开更多
文摘目的对1株在九江地区分离的柯萨奇A6病毒(CoxA6)VP1区域进行克隆,并对其编码蛋白的结构、功能及B细胞表位进行分析和预测,为CoxA6疫苗制备和诊断方法的研究提供理论基础。方法采用RT-PCR法对CoxA6分离株VP1区进行扩增和克隆、序列分析,应用SigaIP、TMPRED、TMHMM、Big-PI Predictor、Cell-Ploc、PSORT、NetNES、Netphos、SOPMA、NetNGlyc、MotifScan、InterProscan、SMART、PROSITE、GOR4、Bepipred Linear Epitope Prediction等生物信息学方法预测其VP1基因编码蛋白特性和潜在的B细胞表位。结果该CoxA6分离株VP1基因编码305aa的多肽,分子量为33.5kDa。该蛋白无信号肽、跨膜区,是亲水性蛋白;二级结构主要以无规则卷曲为主,其次为α-螺旋及β-片层。该蛋白共有7个可能的B细胞抗原表位,位于151~173aa区域内的表位分值最高为2.774。结论成功克隆了1株CoxA6的VP1基因,并对其进行序列分析、蛋白质结构和B细胞表位预测,为制备CoxA6疫苗和开发诊断方法提供了分子生物学基础。
文摘Many studies point to an association between Helicobacter pylori(H.pylori)infection and inflammatory bowel diseases(IBD).Although controversial,this association indicates that the presence of the bacterium somehow affects the course of IBD.It appears that H.pylori infection influences IBD through changes in the diversity of the gut microbiota,and hence in local chemical characteristics,and alteration in the pattern of gut immune response.The gut immune response appears to be modulated by H.pylori infection towards a less aggressive inflammatory response and the establishment of a targeted response to tissue repair.Therefore,a T helper 2(Th2)/macrophage M2 response is stimulated,while the Th1/macrophage M1 response is suppressed.The immunomodulation appears to be associated with intrinsic factors of the bacteria,such as virulence factors-such oncogenic protein cytotoxin-associated antigen A,proteins such H.pylori neutrophil-activating protein,but also with microenvironmental changes that favor permanence of H.pylori in the stomach.These changes include the increase of gastric mucosal pH by urease activity,and suppression of the stomach immune response promoted by evasion mechanisms of the bacterium.Furthermore,there is a causal relationship between H.pylori infection and components of the innate immunity such as the NLR family pyrin domain containing 3 inflammasome that directs IBD toward a better prognosis.
文摘The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.
