G3P[3]型别多见于猫/狗A组轮状病毒(Group A rotavirus,RVA)。本研究前期工作中从广西农村一名腹泻儿童粪便标本中检测到1例在人的感染中少见的G3P[3]型RVA(命名M2-102),但经初步基因分析提示其VP7和VP4基因均同猫/狗G3P[3]RVAs进化距离远。为了解M2-102的种属来源,本研究对其全基因组11条基因进行了RT-PCR扩增、测序和序列分析。使用RotaC分型工具对所获得的M2-102全基因组基因序列进行分型。结果显示RVA/Human-wt/CHN/M2-102/2014/G3P[3]具有G3-P[3]-I3-R3-C3-M3-A9-N3-T3-E3-H6基因组型。序列分析进一步显示M2-102基因组中有5条基因(VP7、VP1、VP2、NSP2和NSP3)同云南蝙蝠MYAS33株亲缘关系近,处于相同的进化树支;另有5条基因(VP4、VP3、NSP1、NSP4和NSP5)同猿猴RRV株进化距离近,位于相同的进化树支;仅有1条基因VP6同人AU-1样RVAs处在相同进化树支,并具有较高核苷酸同源性。推测M2-102是一例由人AU-1样病毒同蝙蝠株MYAS33及猿猴株RRV类似病毒经基因重配而产生的多种属来源的重配毒株。
Human metapneumovirus(hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms.Matrix protein(M) is one of the most important structural proteins.For further studying of hMPV,the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCm-M1817 were cloned by PCR and sub-cloned into the pET30a(+) vector,which is a prokaryotic expression vector,after dual-enzyme digestion with Bam HI and Xho I.The positive recombinated plasmids were transformed into E.coli BL21(DE3) and expressed under the inducing of IPTG.Target proteins were characterized by SDSPAGE and Western blotting.In this article,we’ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6·His-N were highly produced after inducing by 1mmol/L IPTG at 37℃.A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE.Most of the target protein existed in inclusion body.Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV.So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities.It can be used for further studying of hMPV infections in Beijing.
