| Parainfluenza virus type 3(PIV3), a member of the genus Respirovirus in the family Paramyxoviridae, is an enveloped virus with a non-segmented negative-sense RNA genome. The PIV3 is one of the most important viral respiratory pathogens for humans and for many animals, which includes human parainfluenza virus types 3(HPIV3) and bovine parainfluenza virus type 3(BPIV3). At present, there were many reports about HPIV3 and BPIV3, however, reports of PIV3 infection in goat/sheep are limited. In 2013, a novel member of the respirovirus genus in the Paramyxoviridae family was detected and identified from goats, and named JS2013. Etiology and serological detection methods were desired for epidemiological investigation in goat herds.The PIV3 genome encodes six structural proteins: the nucleocapsid(N), phosphoprotein(P), matrix(M), fusion(F), hemagglutinin–neuraminidase(HN), and large(L) proteins. The HN and F proteins are envelope glycoproteins, which are the major protective antigens, and there were at least six neutralization epitopes in HN. Strong hydrophobic was performed at amino terminal of HN, three neutralizing epitope was present in this terminal. Recombinan His-HN protein was expressed successfully using prokaryotic expression system, hybridoma technique was utilized to develop monoclonal antibodies(Mc Ab). The Mc Ab could be useful for developing the techniques and studying on the structural and functional characterization of CPIV3 HN protion.1. Cloning and prokaryotic expression of partial HN gene of CPIV3 JS2013 strainThe HN gene segment of caprine PIV3 strain JS2013 was partially amplified by reverse transcription polymerase chain reaction(RT-PCR) with a pair of primers, which was designed according to the genome sequence of BPIV3 NM09 strain(Gen Bank accession number: JQ063064). The obtained RT-PCR product was purified and sequenced, the gene shared the highest identity of 74.3% and 79.8% with HPIV3 and BPIV3 by Blast analysis, respectively. Phylogenetic analysis showed that JS2013 strain formed one unique branch, which clearly differed from HPIV3 and BPIV3 genotypes that have been reported in Gen Bank. The gained HN gene was cloned into p ET-32a(+) vectors and the expression plasmid(p ET-32a-HN) was successfully constructed. After transformation into BL21(DE3), the expressed recombinant His-HN protein with molecular weight of 46 ku was confirmed by SDS-PAGE and western blot with His Mc Ab; and mainly expressed in the inclusion bodies. BALB/c mice were immunized with the purified His-HN protein and the mouse polyclonal antibodies could specifically react with the protein in western blot. In addition, JS2013 infected MDBK cells were positively stained with the antibodies by indirect immunofluorescence(IFA). These results indicated that the expressed protein possessed good immunogenicity and reactionogenicity.2. Development of monoclonal antibodies against HN protein of CPIV3 strain JS20136 week-old female BALB/c mice were immunized with purified His-HN fusion protein for 3 times, spleen cells from immunized mice were fused with SP2/0 myeloma cells. Hybridoma cells were screened by indirect enzyme linked immunosorbent asssy(ELISA), and positive hybridoma cells were subcloned using limiting dilution method. Three positive clones named 1F8, 2D5, 4A7 was obtained through screening and subcloning three times. The titers of cell supernatant and ascites for 1F8, 2D5 and 4A7 were 29, 28, 28 and 106, 106, 105, respectively. These Mc Abs were identified, and belonged to Ig G2 b subtype, the light chains were classfied to ? type. Western blot analysis showed that Mc Abs were reacted with HN protein specifically, Dot-ELISA and indirect immunofluorescence showed that 3 Mc Abs recognized CPIV3 infected in MDBK cells. These Mc Abs would provide basic materials for the development of a CPIV3-testing method, which was specific and sensitive. |
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