Expression Of Paramyxovirus Tianjin Strain Nucleocapsid Protein And Application In Clinical Detection For Children With Lower Respiratory Infection

In 1999,a strain of virus was isolated from the lungs of common cotton-eared marmoset that died during an outbreak of severe respiratory infection in the marmoset colonies in an animal laboratory.The isolates exhibit the typical morphology of paramyxoviruses under the electron microscope.Electron microscopic observations, serodetection and hemagglutinin-neuraminidase(HN)nucleotide sequence analysis indicated that the virus belonged to the member of family Paramyxoviridae and has a close relationship to Sendai virus(SeV).Serodetection indicated that the majority of the personnel working in the Center have detectable antibodies against this virus, even people(14/40)who had never come in contact with marmosets.We also detected the antibodies in sera of young children with acute respiratory tract infection. The IgM positive rate was 30-37%.These results suggested that paramyxovirus Tianjin strain may have a close relationship with humans,and might be a common respiratory virus that infects both human and marmoset.More importantly,the common SeV usually cause lethal pneumonia in mice.However,experimental mice and rats bred in the same animal laboratory with the common cotton-eared marmosets had never suffered from the respiratory disease in 1999.Therefore,we theorize that the paramyxovirus Tianjin strain might be a new variant of SeV which have higher pathogenicity to primates than rodents.Up to now,the complete genome sequence of paramyxovirus Tianjin strain has been determined(GenBank accession number EF679198).Phylogenetic analysis proved that paramyxovirus Tianjin strain has high homology with SeV.It is most likely a new genotype of SeV.To further investigate the relationship between Tianjin strain nucleocapsid protein (NP)structure and function and make the diagnosis more convenient and specific, three pairs of oligonucleotide primers specific to NP1,NP2 and NP3 gene,which overlap the full-length of NP were designed and synthesized,respectively based on the genome of paramyxovirus Tianjin strain in GenBank.Amplified target DNA segments were ligated into pET28a vector between EcoR I and Sal I sites and the expression plasmids pET28a-NP1,pET28a-NP2 and pET28a-NP3 were then constructed.Subsequently,E.coli Top10 were transformed by the recombinant plasmids.After identification by PCR and double restriction digestion(EcoR I and Sal I),E.coli BL21(DE3)were transformed by the plasmids pET28a-NP1, pET28a-NP2 and pET28a-NP3.The recombinant NP1,NP2 and NP3 were induced by IPTG for expression.The rNP1,rNP2 and rNP3 with 6×histidine tags could be purified by nickel-chelating chromatography.Polycloned antibody(PcAb)against paramyxovirus Tianjin strain was produced routinely in Japanese White Rabbits and BALB/c mice by immunization with ultracentrifugation purified paramyxovirus Tianjin strain.Anti rNP1,rNP2 and rNP3 mouse polyclonal antisera were prepared in specific pathogen free(SPF)BALB/c mice by immunization with the purified rNP1, rNP2 and rNP3.ELISA,dot blots and western blots were applied to detect the antigenicity of rNP1,rNP2 and rNP3.Then IgG and IgM antibodies against paramyxovirus Tianjin strain in 186 serum samples from children with lower respiratory infection were detected.Monoclonal antibodies(McAb)against rNP1, rNP2 and rNP3 were prepared by routine hybridoma technique.The specificity, stability of secretary McAb,antigen-binding epitopes was analyzed by ELISA method.The results indicate that rNP1,rNP2 and rNP3 had the native antigenicity by binding to the specific rabbit anti-Tianjin strain PcAb,intensity ranking as NP3＞NP1＞NP2.PcAbs against rNP1,rNP2 and rNP3 don't show cross reactivity to influenza virus type A and B,Mycoplasma pneumonia and influenza virus type A(mouse FM1 strain),while PcAbs against rNP1,rNP3 have cross reactivity to parainfluenza virus typeⅠ,Ⅲand Newcastal disease virus(NDV),PcAbs against rNP2 only exhibits cross reactivity to parainfluenza virus typeⅢ.Moreover,recombinant proteins were more convenient to produce,more safe to use and more specific and sensitive than inactivated virus.The positive rates of IgG and IgM antibdy detection by Tianjin strain-ELISA is 30.65%(57/186)and 19.89%(37/186),respectively.IgG antibdy positive rates in rNP1,rNP2 and rNP3 as coating antigens were 24.19%,22.04%and 20.97%,respectively,while the IgM antibody positive rates were 18.82%,16.67% and 15.6%,respectively.A strain of hybridoma cell,G1B9D5 which could excrete McAb against rNP2 was obtained.The supernatant of cells could not bind to rNP1, rNP3 and other common respiratory pathogens.Therefore,antigen-binding epitopes of the McAb G1B9D5 might be located among aa202-aa324.The obtained McAb could facilitate to further comprehension of relationship between protein structures and functions of paramyxovirus Tianjin strain on the molecular level and lay base to establishment of the more sensitive,specific detection assay for paramyxovirus Tianjin strain.