人3型腺病毒GZ13株的分离鉴定及六邻体蛋白的分子进化研究

目的分离鉴定1株引起儿童急性支气管肺炎的腺病毒及分析其主要中和抗原六邻体蛋白的分子进化趋势。方法采用荧光定量PCR方法,对1例引起严重支气管肺炎的患儿咽拭子进行检测,并从中分离得到1株腺病毒(命名为GZ13)。利用型特异性引物PCR进行初步亚型鉴定;克隆该株腺病毒完整的六邻体蛋白基因,测序和序列比对,确定其型别;同时与GenBank上人3型腺病毒的六邻体蛋白进行同源性比对,分析预测六邻体蛋白的分子进化趋势。结果该临床标本经荧光定量PCR鉴定为腺病毒强阳性,型特异性引物PCR初步鉴定为3型;完整六邻体基因比对最终确定为人3型腺病毒;GZ13株与我们先前分离的GZ01、GZ02株及台湾地区分离株核苷酸及氨基酸的同源性均大于99%。结论引起该例儿童急性支气管肺炎的病原体为人3型腺病毒,目前在广州以及台湾地区流行的3型腺病毒仍属同一流行株,其主要中和性抗原六邻体蛋白未出现明显变异,这对今后开发研究人3型腺病毒疫苗及药物具有重要指导意义。

Isolation and identification of human adenovirus type 3 GZ13 strain and the molecular evolution of hexon protein

Objective To isolate and identify the human adenovirus(HAdV) from a child with an acute bronchopneumonia,and to analyze the molecular evolution of the neutralizing antigen hexon protein.Methods The adenovirus,named GZ13,was isolated from the throat swab of a child with severe acute bronchopneumonia by FQ-PCR.Type-specific primers were used to primary identification of virus type by PCR.The complete hexon gene was cloned and sequenced for confirmation.The hexon protein was also compared with the other HAdV-3 to predict its molecular evolution.Results The adenovirus specimen was confirmed by FQ-PCR,and classified as HAdV-3 by primary PCR typing,which was further confirmed with hexon gene blast.There was 99% homogeneity of nucleotide and amino acid between the GZ13 strain and GZ01,GZ02 or Taiwan strain according to alignment with all hexon proteins of adenovirus type 3.Conclusion HAdV-3 is associated with this acute bronchopneumonia.The prevalent type 3 adenoviruses,GZ13,Taiwan Strains and other Guangzhou strains,belong to circulating single strain.The stability and conservation of hexon is helpful in development of HAdV-3 vaccine in future.