| Bovine parainfluenza virus 3(BPIV3)belongs to the Respirovirus genus within the family Paramyxoviridae and is the topic cause of respiratory disease in young calves, also known as the shipping fever virus. Clinical symptoms of BPIV3 infection vary greatly from asymptomatic infection to severe respiratory disease. Calf pneumonia is a common clinical disease which causes huge economic losses in cattle industry each year. This disease is popular in the world, especially in America and Asia. At present there is less research on this disease in our country, and no related diagnostic reagents.BPIV3 contains at least six proteins, namely nucleocapsid (NP) protein, phosphoprotein (P),matrix (M) protein, fusion (F) protein, haemagglutinin-neuraminidase (HN) and large (L) protein. The NP protein of BPIV3 contains the major genus strain antigenic determinants, which are able to induce strong immune response. According to the published complete genome sequences of BPIV3, two pairs of primers was designed on conserved region in the N gene coding for NP protein. With the RNA template extracted from BPIV3 strain SD0835 isolated in Shandong, the 1534bp N gene fragment was amplified by nested RT-PCR and cloned into pMD18-T vector. Sequencing results showed that the nucleotide sequence of N gene from the Chinese BPIV3 isolate SD0835 was only 84% highest homology with other reported BPIV3 isolates in this study. Nucleotide phylogenetic analysis of partial N gene for parainfluenza virus type 3 (PIV3) indicated that the Chinese BPIV3 isolate SD0835 was distinct from the previously reported genotype A (BPIV3a) and genotype B (BPIV3b) and should be classified into a new genotype C (BPIV3c).The recombinant plamid identified by sequencing was digested and the target fragment was harvested and cloned into the expression vector pET30a and transformed into E coli BL21, which was induced with IPTG and a recombinant NP protein was expressed in inclusion bodies. The recombinant NP protein was purified by Ni affinity chromatography under denaturing conditions and tested by indirect ELISA and Western blotting. The result showed that the purified recombinant NP protein has a good antigenicity and specificity. Antibodies against BPIV3 were detected in suspected bovine sera collected from a herd of cattle showing respiratory signs by using indirect ELISA with the purified recombinant NP protein as coating antigen. The agreement rate is 92.8% compared with the imported standard ELISA kit for BPIV3 infection. The polyclonal antibody derived from rabbit inoculated with the recombinant NP protein subcutaneouly and in the popliteal lymph node. The results of indirect ELISA and werstern blotting showed that polyclonal antibodies could identify the recombinant NP protein and the whole virus protein. An indirect ELISA showed that the reaction remained positive, when antibodies were diluted to 1:8192. Indirect immunofluorescence test showed that the antibody could identify the NP protein expressed by bovine parainfluenza virus type 3 in infected cells, even in the dilution of 1:2560. Viruses were reisolated from lungs, trachea, hearts, spleens, kidneys and livers collected from mice inoculated with BPIV3 for 2 days post inoculation. The virus isolates were identified as BPIV3 by indirect immunofluorescence test with the polyclonal antibody against the recombinant NP protein.In this study, the NP protein of BPIV3 was successfully expressed in E.coli. The purified recombinant protein has good antigencity, and can be used to develop diagnosis kits for detection of antibodies against BPIV3. The polyclonal antibody which derived from rabbit inoculated with recombinant NP protein has a good reactivity and can be used for the virus identification and related research on BPIV3.
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