论文部分内容阅读
目的对2009年浙江省义乌市暴发流行的登革3型病毒进行系统进化分析并初步研究其E蛋白的免疫原性。方法 RT-PCR扩增义乌分离株全基因组,利用MEGA 4分别构建E及NS1区域核苷酸及氨基酸系统发生树,进行系统进化分析。构建重组质粒JESS-ywD3prM/E397JEV-pcDNA5,IFA及Western Blot检测E蛋白在293T细胞中的表达及分泌情况。将纯化后的分泌性E蛋白免疫BALB/c小鼠,通过ELISA、中和试验等方法对体液免疫进行测定。结果登革3型病毒义乌分离株与广州GZ1D3株及印度GWL-25株核苷酸及氨基酸序列同源性均达到99%以上;其分泌性E蛋白可以刺激小鼠产生登革特异性IgG抗体及针对同型病毒的中和抗体。结论义乌分离株属于登革3型病毒亚型Ⅲ,其分泌性E蛋白具有一定的免疫原性。
OBJECTIVE: To systematically analyze the evolutionary dengue 3 virus that is epidemic in Yiwu City, Zhejiang Province in 2009 and study its immunogenicity of E protein. Methods The whole genome of Yiwu isolate was amplified by RT-PCR. The phylogenetic tree of nucleotides and amino acids of E and NS1 regions was constructed by MEGA 4, and phylogenetic analysis was performed. Construction of recombinant plasmid JESS-ywD3prM / E397JEV-pcDNA5, IFA and Western Blot detection E protein in 293T cells in the expression and secretion. BALB / c mice were immunized with purified secreted E protein and humoral immunity was determined by ELISA, neutralization assay and other methods. Results The nucleotide and amino acid sequence homologies of dengue 3 virus Yiwu isolates with GZ1D3 strain of Guangzhou and GWL-25 strain of India reached more than 99%. The secreted E protein could stimulate the mice to produce dengue specific IgG antibody And neutralizing antibodies against the same virus. Conclusion The isolates belonging to Yiwu Dengue virus type Ⅲ Ⅲ, the secreted E protein has some immunogenicity.