The Investigation On The Infection Of Paramyxo Virus Tianjin Strain In Infants And Young Children, Construction And Screening Of Phage Antibody Library

Paramyxovirus Tianjin strain was isolated from common cotton-eared marmosetlung in 1999;it caused lethal infection on the marmoset.IgM against paramyxovirusTianjin strain in sera of infants and young children with acute respiratory tractinfection were detected,the positive rate was 36.9%.Taking into account closegenetic relationship between the marmoset and human,high IgM positive rate in seraof children with respiratory tract infection,we consider that paramyxovirus Tianjinstrain may be a common respiratory virus in human and animal.Objective:To assess the infection status of paramyxovirus Tianjin strain in the crowd,andaccurately understand the relationship between human and paramyxovirus Tianjinstrain;To prepare monoclonal antibodes and genetic engineering antibody,provide abasis for further study on paramyxovirus Tianjin strain,including diagnosis,therapy,host range changes,and so on.Methods:Monoclonal antibodies agaist paramyxovirus Tianjin strain were preparedthrough hybridoma technique and phage antibody library technology.A moresensitive detection methods was established,and paramyxovirus Tianjin strain wasdetected directly from clinical specimens.Results:1.Twenty-three hybridoma cell strains were obtained.Three hybridoma cellstrains G7H4D3,G7D9G3 and G7G7E7 among them,show specificity forparamyxovirus Tianjin strain.The three mAbs secreted by these three hybridoma cellsshowed the ability of specific binding to paramyxovirus Tianjin strain,and nocross-reactions were evidently detected with influenza A and B,New castle diseasevirus (NDV),Human parainfluenza virus (hPIV) type 1 and 3,Mycoplasmapneumoniae.ELISA additivity tests indicated the three mAbs recognize the same orclosely adjacent epitope domains,and which were also immunodominant in theimmune response against paramyxovirus Tianjin strain by blocking assay.2.A sandwich ELISA assay is developed in which the rabbit polyclonal antibodies against paramyxovirus Tianjin strain are used as the capture antibody andmAb G7G7E7 as detection antibody.Paramyxovirus Tianjin strain in BALF samplesof the newborns and young children with severe lower respiratory tract infectionswere detected.The positive rate is 2.1% (11/523).The sandwich ELISA assay showsbetter sensibility and specificity than RT-PCR,hemagglutination inhibition assay (HI)and indirected ELISA.It is suitable for clinical diagnosis.The detection results alsosuggest that paramyxovirus Tianjin strain have a close relation to infants and youngchildren bronchitis and pneumonia,and may be one of causative agents in the lowerrespiratory tract infections.3.The Fab phage antibody library against paramyxovirus Tianjin strain wasconstructed,and biopanning and identifying of anti-paramyxovirus Tianjin strainantibodies were accomplished.We obtained seven positive phage antibody clonesagainst paramyxovirus Tianjin strain,and produced six soluble Fab antibodies.Among them,one positive clone binds with recombinant protein HN_{375aa～575aa},andtwo positive clones bind with recombinant protein HN_{253aa～452aa}.Sequence analysis ofpositive clone 20 showed that the light chain variable domain (V_K) gene belong toV_K9 subgroup,the heavy chain variable domain(V_H) gene belong to V_H7 subgroup,most high identity is respectively 94.87% and 97.85%.Conclusion:Three specific mAbs were obtained through hybridoma technique,and asandwich ELISA assay is developed.We detected lower respiratory tract infectionssamples by this method,get accurate data on relationship between the virus andhuman.An immune phage antibody library against paramyxovirus Tianjin strain wasconstructed,six mAbs was obtained,and to lay a foundation of further study ondiagnosis and pathogenic mechanism of paramyxovirus Tianjin strain.