Assembly And Release Of Peste Des Petitis Ruminants Virus-like Particles

Peste des petits ruminants (PPR) is an acute, febrile, highly contagious viral disease of smallruminants such as goats, sheep and wild small ruminants. PPR is caused by Peste des petitusruminants virus (PPRV) and set a notifiable terrestrial animal disease by Office International DesEpizooties (OIE) and a Grade I animal disease in Category List of Legal Animal Disease regulatedby Ministry of Agriculture of the People’s Republic of China. An attenuated vaccine against PPR,Nigeria75/1, is an effective means of prevention and control of the disease. But it has poor thermostability which often causes immune failure and is at a considerable risk of spread because the virushas potential to rebuild infectious ability. The Virus Like Particles (VLPs) that was supposed to be astructure of a hollow particle or envelope particle constituted with one or several structural proteinsof a virus was developed in the1980s’. As an alternative vaccine candidates, VLPs are not only ofsafety but also of good immunogenicity, so many researchers pay close attention to it. There were noreports on PPRV VLPs by now. In order to develop a new efficient vaccine for prevention andcontrol of PPR, we cloned four structural proteins genes (N, M, F and H) of PPRV, constructedrecombinant eukaryotic expression plasmid respectively, designed experiments on VLPs assembling,and investigated the key factors during the process of VLPs assembling and releasing. The resultswere presented as follows:1. The recombinant plasmids prokaryotic expression of F gene marked pET-30a/F was constructed,the polyclonal antibody against F protein was prepared through inoculated rabbit with the expressedprotein and the serum antibody titer was1:1.1×105.2. Four different plasmids expressing N, M, F and H gene were constructed based on pCAGGSvector frame. These plasmids were improved efficiently in Vero cells by Indirect Immunofluorescentand Western blotting assays.3. The four recombinant plasmids were co-transfected into Vero cells, and the PPRV VLPs wasfirstly assembled and released successfully. To research the function of the plasmid combinations,the four recombinant plasmids and the combination were transfected in Vero cells, and the resultsshowed that M protein could make assembly and releasing alone.4. The sequence of M protein was separated into11parts and mutants were constructed based onpCAGGS/M by deletion PCR. These mutants were transfected into Vero cell respectively, and thekey sequence in the M protein to the VLPs assembly and releasing was confirmed by electronmicroscope. 5. The transmembrane(TM) region sequence of F protein was separated into10parts and mutantswere constructed based on pCAGGS/F by Alanine Scaning mutated. These mutants wereco-transfected with recombinant plamids of other three structural protein genes into Vero cellsrespectively, and the result demonstrated that the TM region of F protein did not affect VLPsassembly and releasing.Success in VLPs assembly provids a new way to develop the vaccines against PPRV, and open awindow for research in the function of M protein.