SFTSV糖蛋白Gn重组质粒的构建及其体液免疫原性研究
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国家重大科技专项(卫生部十二五支撑计划)(2013ZX10002005)


Construction of recombinant plasmids expressing SFTSV glycoprotein Gn and immunogenicity study
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    摘要:

    目的:研究严重发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus,SFTSV)糖蛋白Gn的体液免疫原性-方法:将PCR方法扩增的SFTSV糖蛋白Gn基因和密码子优化后Gn基因克隆入载体pJW4303,构建Gn野生型和双优化重组质粒;经酶切和测序鉴定确认为目的质粒后,分别转染HEK293T细胞,Western blot检测糖蛋白Gn的表达;用Gn重组表达质粒及空载体分别免疫BALB/c小鼠,ELISA检测免疫后小鼠血清抗Gn特异性IgG抗体-结果:成功构建Gn重组野生型质粒pJW4303-WSP-Gn和双优化质粒pJW4303-tPA-Gn-opt;Western blot证实pJW4303-WSP-Gn编码Gn可在HEK293T细胞内表达,pJW4303-tPA-Gn-opt编码Gn在HEK293T细胞内表达并分泌到细胞外;ELISA检测免疫后小鼠血清抗Gn特异性IgG抗体证实各重组质粒均能诱导特异性IgG抗体产生,双优化质粒较野生型质粒诱导产生的IgG时间更早-滴度更高-结论:SFTSV的糖蛋白Gn具有良好的免疫原性;和野生型质粒相比,双优化质粒更有利于糖蛋白Gn的表达和分泌,并且具有更好的体液免疫原性-

    Abstract:

    Objective:To explore the immunogenicity of severe fever with thrombocyte-penia syndrome virus(SFTSV)glycoprotein Gn. Methods:SFTSV glycoprotein Gn gene and optimal Gn gene were amplified by polymerase chain reaction(PCR)respectively. The gene product were cloned to pJW4303 to construct recombinant plasmids and all constructs were identified by sequencing,and then transiently transformed into HEK293T cells to measure the Gn expression by Western blot. We immuned the BALB/c mice with recombinant plasmids and blank vector,and verified its immunogenicity by enzyme-linked immunosorbent method. Results:The recombinant plasmids pJW4303-WSP-Gn and pJW4303-tPA-Gn-opt were constructed success-fully. The pJW4303-WSP-Gn transiently expressed Gn antigen in cell lysates of HEK293T cells in vitro,and pJW4303-tPA-Gn-opt in supernatants as same as cell lysates. The specific IgG antibodies induced by pJW4303-tPA-Gn-opt was earlier and higher than that of pJW4303-WSP -Gn. Conclusion:SFTSV glycoprotein Gn showed good immunogenicity. The recombinant plasmid pJW4303-tPA-Gn-opt promoted the synthesis and secretion of Gn,and showed better humoral immunogenicity than pJW4303-WSP-Gn.

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徐菱遥,韩亚萍,周宜庆,金 柯,艾宇洁,黄祖瑚,李 军. SFTSV糖蛋白Gn重组质粒的构建及其体液免疫原性研究[J].南京医科大学学报(自然科学版),2016,(5):554-558

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  • 收稿日期:2016-01-19
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  • 在线发布日期: 2016-05-23
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