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猪塞内卡谷病毒VP1蛋白原核表达及多克隆抗体制备
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作者 莫红芳 石宗承 +6 位作者 陆晶山 何东贤 杨延辉 梁淑芳 俸祥仁 钱平 韦巧燕 《南方农业学报》 CAS CSCD 北大核心 2023年第7期2175-2183,共9页
【目的】原核表达猪塞内卡谷病毒(Seneca valley virus,SVV)VP1蛋白,制备SVV-CH-HB2016 VP1蛋白多克隆抗体并分析其抗原性,为进一步研发SVV病毒诊断试剂盒和亚单位疫苗提供参考依据。【方法】对SVV-CHHB2016 VP1基因序列进行密码子优化... 【目的】原核表达猪塞内卡谷病毒(Seneca valley virus,SVV)VP1蛋白,制备SVV-CH-HB2016 VP1蛋白多克隆抗体并分析其抗原性,为进一步研发SVV病毒诊断试剂盒和亚单位疫苗提供参考依据。【方法】对SVV-CHHB2016 VP1基因序列进行密码子优化,构建质粒pUCK/VP1-opt,双酶切后连接至pET-28a(+)载体,构建原核表达质粒pET-28a-VP1。将重组质粒pET-28a-VP1转化大肠杆菌BL21(DE3)分子伴侣感受态细胞,经IPTG诱导表达获得重组VP1蛋白。采用Western blotting方法分析VP1蛋白的反应原性,并以包涵体变性和复性方式纯化。随后将VP1蛋白免疫新西兰白兔,分离血清获得重组VP1蛋白的多克隆抗体。以间接酶联免疫吸附试验(ELISA)法测定抗体的效价水平,以Protein A+G Agarose抗体纯化试剂盒纯化多克隆抗体。通过Western blotting和间接免疫荧光抗体试验(IFA)法验证多克隆抗体与SVV-CH-HB2016毒株反应的特异性。【结果】成功构建原核表达质粒pET-28a-VP1,在IPTG终浓度为0.1 mmol/L、37℃诱导4 h条件下,重组VP1蛋白成功表达并主要以包涵体形式存在;Western blotting分析结果显示,重组VP1蛋白可与His-Tag Mouse mAb发生特异性结合,具有良好的反应原性;经变性和复性纯化的重组包涵体蛋白纯度和浓度均较高;采用间接ELISA法检测多克隆抗体的效价为1∶32000;纯化后的抗体无明显杂带,纯度大于90.0%;Western blotting和IFA鉴定结果表明,多克隆抗体可特异性识别SVV-CH-HB2016毒株,重组VP1蛋白具有较好的免疫原性。【结论】通过原核表达SVV VP1蛋白获得的重组VP1蛋白具有较好的抗原性,制备的多克隆抗体特异性良好,可作为诊断试剂盒及亚单位疫苗研制的候选抗原。 展开更多
关键词 猪塞内卡谷病毒 VP1蛋白 原核表达 多克隆抗体
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REYNOLDS NUMBER EFFECTS IN TALL BUILDING WIND TUNNEL TESTS 被引量:2
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作者 Cheng Zhao shi zong-cheng Zhang Feng(State Key Lab. for Disaster Reduction in Civil Eng., Tongji Univ Shanghai 200092, P.R. China) 《Journal of Hydrodynamics》 SCIE EI CSCD 1996年第4期56-62,共7页
In wind tunnel tests of tall building models comprised of rounded or streamlined surfaces, Reynolds Number Effects (RNE) may be remarkable. In most researches and projects, owing to the presence of sharp edged corners... In wind tunnel tests of tall building models comprised of rounded or streamlined surfaces, Reynolds Number Effects (RNE) may be remarkable. In most researches and projects, owing to the presence of sharp edged corners at model, RNE was often treated as negligible, and data colleCted in subcritical flow regime were applied directly to designs which were sure to be supercritical without any modification. But it was proved necessary to take RNE into consideration when a model had predominant rounded smooth surface(s). Some research activities were devoted to the mentioned condition and two experimental techniques accompanied by two wind tunnel model tests were introduced in the article. The authors also presented some amusing phenomenon such as extremely low pressure coefficients caused by the separating bubbles when now past sharp edged corners, unsymmetrical pressure coefficient distributions commenced by biased gap f'low (seen Figs. 3, 4) while models were placed symmetrically in the approaching flow. 展开更多
关键词 Reynolds Number Effect wind tunnel simulation nows around building
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