Research On Eukaryotic Expression And Reverse Genetics Of Paramyxovirus Tianjin Strain

Paramyxovirus Tianjin strain is a new genotype of Sendai virus.Sendai virus causes outbreaks of high lethal pneumonia in mouse colonies.In 1999,the acute respiratory infectious disease outbroke among the common cotton-eared marmosets colonies in animal experiment center of Tianjin Medical University and most of marmosets died in 5 to 10 days even if promptly treated with kinds of antibiotics.We could see lung tissue's focal necrosis in autopsy analysis.However,the experimental mice in the same animal laboratory had not been suffering from respiratory disease during the epidemic period of respiratory infectious disease.Paramyxovirus Tianjin strain had been detected the in the bronchoalveolar lavage fluid(BALF) samples from the newborns and young children with severe lower repiratory tract infections by Sandwich ELISA assay.The positive rate was about 1.92%(2/104).All of these suggest that paramyxovirus Tianjin strain possess the unique characteristics in pathogenicity and host tropism,and it maybe the important pathogen of young children.More and more people pay attention to emerging pathogens,particularly Viruses coming from animals which cause emerging infectious diseases of human beings.It is important to make clear the mechanism of high pathogenicity and interspecies transmission of virus for prevention.Under natural condition,the interspecies transmission of paramyxovirus is limited by its host specificity.Although there are many factors influencing host specificity,the HN protein is important for recognizing and absorbing cell receptor.Therefore,we focus on paramyxovirus Tianjin strain by means of eukaryotic expression system to provide the basis for disclosing the mechanism of high pathogenicity and interspecies transmission of paramyxovirus Tianjin strain.We also do preliminary study on paramyxovirus Tianjin strain in reverse genetics to understand the interaction between Tianjin strain and host cells.In the experiment,we expressed nucleoprotein(NP) and hemagglutinin-neuraminidase (HN) of paramyxovirus Tianjin strain in eukaryotic cells,analyze the antigenicity and hemagglutination activity of HN and three truncated fragments HN1, HN2,HN3. First,HN gene and its segments HN1,HN2 and HN3 of paramyxovirus Tianjin strain was constructed into the eukaryotic expression vector pEGFP-C2-HN,pEGFP-C2 -HN1,pEGFP-C2-HN2,pEGFP-C2-HN3 and transfected into HEK293 cells, which is human embryonic kidney cells.The expressed proteins are identified by Western blot assay,in situ ELISA and hemadsorption assay.The results present the cells which were transfected by pEGFP-C2-HN,pEGFP-C2-HN1,pEGFP-C2-HN2 and pEGFP-C2-HN3 showed the green fluorescence.The expression of HN,HN1, HN2 and HN3 can be detected by Western blot assay.HN,HN1,HN2 and HN3 showed the diverse antigenicity in transfected HEK293 cells.The antigenicity ranks as HN＞HN2＞HN1＞HN3(precedence order).We also observed that HN and HN3 proteins could effectively adsorb the fresh chicken red blood cells,whereas HN1 and HN2 proteins were negative in the hemadsorption test.Second,we do further research of paramyxovirus Tianjin strain by reverse genetics. Paramyxovirus Tianjin strain was propagated in chick embryo allantoic liquid.We extracted RNA of paramyxovirus Tianjin strain and amplified NP gene and P gene by RT-PCR,then constructed the eukaryotic expression vectors which were named pcDNA3.1(+)-NP,pcDNA3.1(+)-P.We also amplified L gene by fusion PCR method. The transient and stable expression of NP gene in HEK293 cells which were transfected with the constructed vector could be detected by immunofluoresence assay and western blotting.These tests lay the base for further studies on NP protein function and high pathogenicity,as well as unique host tropism of paramyxovirus Tianjin strain by reverse genetics.