| The fatal respiratory infection of common cotton-eared marmosets broke out in the Experimental Animal Center in June, 1999. Subsequently, a strain of virus with high hemagglutination titer was isolated from the lungs of the dead common cotton-eared marmosets in Dept. Microbiology, Tianjin Medical University. It was found that the entire faculty working in the Experimental Animal Center have detectable antibody against this deadly virus, even people who had never contacted with the marmosets. It suggested that the virus may have a close relationship with human, and might be a common respiratory virus that causes both human and marmoset disease. The virus exhibits the typically viral structure of the family paramyxoviridae under the electron microscope and was designated paramyxovirus Tianjin strain.Up to now we have finished whole genomic sequencing of paramyxovirus Tianjin strain. The results of the cross-hemagglutination inhibition test disclosed that it has high homology with Sendai virus. Although paramyxovirus Tianjin strain has closer relationship to Sendai virus BB1 strain than the other strains, the phylogenetic tree analysis indicates it forms an isolated evolution branch. Therefore, we presume the paramyxovirus Tianjin strain might be a new genotype of Sendai virus.To further illuminate the evolution position of paramyxovirus Tianjin strain in the family Paramyxoviridae and the pathogenesis on the molecular level, the prokaryotic plasmids pMal-M, pET-28a-HN1, pET-28a-HN2 and pET-28a-HN3 were constructed to express the full-length gene of M protein and the extracellular domain of envelop glycoprotein HN. The recombinant proteins HN1 (aa61-aa260), HN2 (aa253-aa452) and HN3 (aa376-aa575) contain 200 amino acids partially overlapping in the extracellular domain of the HN protein. We also preliminarily analyze the biologic activity and immunologic competence of the recombinant proteins.First of all, we extracted RNA of paramyxovirus Tianjin strain, then designed the primers specific to HN1, HN2, HN3 and M gene, respectively, on the basis of the known genomic sequence of paramyxovirus Tianjin strain. The target gene fragments were amplified by RT-PCR, subcloned to prokaryotic expression vectors pET-28a (+) and pMAl-c2. The recombinant plasmids were transformed into the Escherichia coli TOP10, and then into Escherichia coli BL21 (DE3) after the identification by PCR, restriction enzyme digestion and sequencing analysis. The recombinant proteins were induced by IPTG for expression. The recombinant proteins HN1, HN2 and HN3 with 6×histidine tags could be purified by nickel-chelating affinity chromatography, whereas the M protein fused with MBP was purified by the amylose resin. We analyzed immunologic competence of the recombinant proteins by ELISA, Dot Blot and Western Blot. The results showed the diverse antigenicity in different proteins. The antigenicity ranks as HN2＞HN3＞N1 (precedence order). The recombinant M protein also shows the natural antigenicity. We preliminarily analyzed the biologic activity of the recombinant proteins HN1, HN2 and HN3 and their antiserums by hemagglutination test and hemagglutination inhibition assay, respectively. The results indicated that HN3 possesses the stronger hemagglutination than HN2, but HN1 has poor hemagglutination. Otherwise, we found that the HN1, HN2 and HN3 have positive reaction with the standard sera of influenza virus type A and B from WHO and Tianjin CDC.In summary, we have successfully constructed recombinant expression plasmids pET-28a-HN1, pET-28a-HN2, pET-28a-HN3 and pMal-M and recombinant proteins have been highly expressed and purified. They have natural bioactivity and antigenicity. The three truncated HN proteins and entire M protein have been applied to prepare their McAbs which are necessary in reconstruction of the paramyxovirus Tianjin strain by the reverse genetics technique and might helpful to set up the more specific detection method to investigate the epidemic of paramyxovirus Tianjin strain in human.
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