Studies On Gene Immunization Of Peste Des Petits Ruminants

Peste des petits ruminants is a acute or subacute,highly contagious diease of domestic and wild small rumianats,especially sheep and goats. It is caused by Peste des petits ruminants virus ( PPRV) that is also known as fever, oculo-nasaldischarges, stomatitis pneumo enteritis complex. Peste des petits ruminants virus is paramyxovirus classified in the Morbillivirus genus along with rinderpest virus(RPV), phocine distemper(PDV) measles virus(MV) and dolphin morbillivirus(CDV) for all these viruses are closely related. It has six components proteins: N/H/F/M/L/P. H and F gene are very important to induce response of protection host immune against the virus and most of the netutralising antibodies are product directly against H gene. However N is the most abundant and the most immunogenic protein of PPRV proteins, does not induce protective immunity against the virus. It has been used in the development of diagnostic tests.The disease was reported in 1942 firstly in Coted'Ivoire of WestAfrica. Then it has spread to sub-Saharan and north of the equator of most African countries in ten years, as well as the Middle East to Turkey and almost all countries. In 2003 it outbroke in the most countries which are border with china in the southwest, which caused enormous economic losses for the local people. In July 2007, Peste des petits ruminants outbroke in Tibet, China and caused enormous economic losses for the local people, Currently there are not an effective attenuated vaccine and recombinant vaccines that prevention and treatment of the disease be saled in our country and depend on import. Therefore, it is urgent to reserch an effective genetic engineering vaccine.In July 2007 RNA of PPRV was extracted from tissue of the disease sheep in Tibet and were been done the following research on gene immunization:Five primers were designed by Vector NTI software reference the sequences of H and Fgeneof Peste des petits ruminants published in Genbank, the two antigen epitopes of H gene and three antigen epitopes of F gene amplified by reverse transcription-polymerase chain reaction (RT-PCR) technology and were inserted expression vector PET-28b, after the recombinant plasmid were analysised by restriction enzyme digestion and DNA sequencing then were transferred into E.coil Rosetta, afterwards, the result of H and F gene antigen epitopes expression was analysised by SDS-PAGE and Western-Blotting.The result showed H and F gene antigen epitopes have been expressed. the proteins of H and F gene were purified which were regard as antigens of ELISA reaction can be detected in the animal virus antibody; Three primers were desigened by Vector NTI software reference the sequences of N,H and F gene of Peste des petits ruminants published in Genbank, the N, H and F gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR) technology and inserted eukaryotic expression vector pIRESineo after digestion. The recombinant plasmids were analysised by restriction enzyme digestion and DNA sequencing. The recombinant plasmid were transfected Vero cell, after 24 hours, to abserve its expression by fluorescence microscope. The result showed that the recombinant plasmid can be express in vitro; Four recombinant plasmids: pIRES1-N,pIRES1-H,pIRES1-F,pIRES1-NF and empty plasmid pIRESineo(negatie control) were extracted and were injected into the goats by muscle, 12 goats were choosen and were devided into six groups, these goats were immuned once per two weeks and were immuned five times in all, one goat was injected 1.5mL plasmid once time, every goat was taken a blood sample before immuned, The antibody of serum was detected by ELISA and Western Blotting.The result showed that in addition to the control plasmid (no virus genes), each of the plasmid was able to produce antibody.