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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p JW4303,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Ig G抗体。结果:成功构建Gn重组野生型质粒p JW4303-WSP-Gn和双优化质粒p JW4303-t PA-Gn-opt;Western blot证实p JW4303-WSP-Gn编码Gn可在HEK293T细胞内表达,p JW4303-t PA-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Ig G抗体证实各重组质粒均能诱导特异性Ig G抗体产生,双优化质粒较野生型质粒诱导产生的Ig G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。
Objective: To study the humoral immunogenicity of severe fever with thrombocytopenia syndrome virus (SFTSV) glycoprotein Gn. Methods: Gn gene of SFTSV and codon-optimized Gn gene amplified by PCR method were cloned into vector pJW4303 to construct Gn wild-type and double-optimized recombinant plasmids. After confirmed by enzyme digestion and sequencing, The HEK293T cells were stained with HEK293T. The expression of glycoprotein Gn was detected by Western blot. The BALB / c mice were immunized with Gn recombinant plasmid and empty vector respectively. The serum anti-Gn specific Ig G antibody was detected by ELISA. Results: The recombinant wild type plasmid pJW4303-WSP-Gn and the double optimized plasmid pJW4303-t PA-Gn-opt were constructed successfully. Western blot confirmed that pJW4303-WSP-Gn-encoding Gn was expressed in HEK293T cells. PJW4303- Gin-opt encoding Gn was expressed in HEK293T cells and secreted to the outside of the cell; ELISA detection of serum anti-Gn-specific Ig G antibody in mice confirmed that all recombinant plasmids can induce specific Ig G antibody production, double optimized plasmid IgG produced earlier than the wild-type plasmid time, titers higher. CONCLUSIONS: Gn of SFTSV has good immunogenicity. Compared with wild-type plasmid, double-optimized plasmids are more favorable for the expression and secretion of glycoprotein Gn, and have better humoral immunogenicity.