| Peste des petits ruminants (PPR) is a acute infectious disease of small ruminants with a high infection and death rate caused by the PPR virus. It is characterized by high fever, necrotizing gastritis and pneumonia on clinical. The disease was first reported in West Africa, then, the Arabian Peninsula, Israel, Iraq, Jordan, Nigeria, Serbia and Ghana and other countries have reported the disease occurred. At present all countries around the world attach great importance to this disease, and the USA, South Korea, India and other countries are actively carrying on the related research work.In July,2007, PPR broke out at Ali soil, leather Kyrgyzstan, Zanda and other counties, causing a large number of the local sheep and goat death. Because the disease is new, there is little research on its diagnosis and detection method of infection. This study is to establish ELISA method by chemical synthesis of N protein. The main research includes:1The chemical synthesis of the Tibetan PPRV-N proteinFirstly, for the purpose of expressing in E.coli, simplifying the complex secondary structure, transforming the restriction enzyme cutting site, and optimizing the rare codons, an overall reconstruction of the Tibetan PPRV-N protein sequence published on GenBank took place by the software such as DNAstar and RNA structure. The homology between the renovated nucleotide sequences and the original sequences is71.9%, and the homology of the both amino acid sequences is100%. Then20pairs of primers were designed and synthesized on the current international advanced chemical synthesis method of gene. Most of the primers were60bp long, and the adjacency opposite primers had21bp repeat sequences. DNA sequences of404bp to414bp were synthesized by polymerase chain reaction (PCR) with5pairs of primers in the non-template condition. Then with the four DNA fragments above as templates, the lateral primers for amplification primers, the gene that expressed the Tibetan PPRV-N protein was synthesized by overlap-PCR method. After comparison, site-directed mutation that appeared in the synthetic process was made a specific modification. Finally the N gene was successfully cloned into the vector simple pMD-19T. The second results showed that the synthetic sequencing of genetic sequences was correct,1578bp length, as expected.2Cloning, expression, purification and identification of the antigenicity of N geneHomologous recombination is the most popular and convenient way of cloning, especially for the cloning of long-nucleotide fragment. So in our study this method was used to conduct the molecular cloning. First, the vector primers (15bp) at both ends of coincidence were added into the original gene sequence, and the exogenous genes required for cloning were obtained (Both ends of the original N gene were added into a15bp linearized vector sequence). Close behind acquiring the need genes, linearizing vector occurred, in the role of the homologous recombination enzyme, accompanied with the ribonucleotide sequence (about1600bp) coloned into pET-32a expression vector(5900bp). After sequencing, the correct clone was transformed into E. coli strains B121-DE3-plysS. E. coli bacteria were induced with IPTG to get the expression yields under the conditions of concentration, induction time, induction temperature and the others, and maximized the amount of soluble protein. Total expressed protein bonded to its N monoclonal antibody was detected by western-blot. Under the optimal induction conditions, the supernatant of the expressed product was purified by ion affinity chromatography, which optimized the imidazole content of eluent buffer. The result showed that:the N gene (1600bp) synthesized, was cloned into pET-32a expression vector (5900bp); the N protein could be correctly expressed by prokaryotic; expressed protein could be purified by HIS label, and the purity of purified product can be higher than95%; the expressed protein and its monoclonal antibody could be combined well.3Establishment of indirect ELISA, and compared with the commercial kitThis study was aimed to measure and ascertain the reaction in PPR indirect ELISA: concentration of coating antibody, dilution of serum, blocking solution, action time of serum, second antibody and substrate. Under different conditions, we chose the max ratio of positive serum OD and negative serum OD as the optimum test conditions to decrease the negative value and increase the positive value. In the optimal conditions we selected the average value of P/N among198negative serum plus two times of the standard deviation as the cut off value. Kit inter and intra coefficients of variation was less than10%, verifying the stability of this ELISA method. Compared with commercial competitive ELISA kit, sensitivity and specificity were96.7%and96.1%, which enrich the serological testing of domestic PPRV.4Prepration of single epitope peptide antiserum and immune characterization testingThe four major epitope peptides of PPR N protein were chemical synthesized, and the4peptides were used as antigen to be ELISA tested by PPRV-positive serum and RPV-positive serum. The results showed that the19amino acid peptide (RPGRPRGETPGQLLLEIMP) could discriminate the two kinds of serum (PPR and RP) wells. This peptide was used as antigen to immunize5rabbits to produce antibodies. The antibody level significantly raised after90days (titer1:64000). Finally, the antibody titer was verified on ELISA method, and the coated original nuclear proteins and peptides were determined on indirect ELISA. The results indicated that the antibody titers reached its maxlmum after90days, and the prepared antibody was suitable to bind with N protein through prokaryotic expression. This study established the foundation of the competitive ELISA.5Establishment of competitive ELISA method and comparison with commercial kitsThe Nucleocapsid (N) protein of Peste des petits ruminants virus (PPRV) was successfully expressed by prokaryotic as the capture antigen. One epitope (RSGKPRGETPGQLLPEIMQ) of the N protein was chosen and chemically synthesized to immunize rabbits, which showed much higher binding capacity with PPR antibodies than the rinderpest virus (RPV) antibody. The immunized serum was against the single epitope and the tested serum was competitive first antibody. We developed the competitive ELISA(C-ELISA) method to monitor the PPRV antigen in our country. The results of study showed that the optimal antigen concentration is32ng/well, the tested-serum dilution is1:5and the single epitope antibody dilution is1:1000. The cut off value was35, which was derived from mean of negative sera plus diploid standard deviation. Compared with the commercial competitive ELISA test, the sensitivity and specificity of this C-ELISA were96.18%and91.29%respectively by testing1039serum samples. 6Detection of PPR seraSince PPR outbreak in Tibet in China in July2007, a large number of goats were immunized in Tibet. Three main aspects of Tibetan serum samples were analyzed:Firstly, the competitive ELISA kits were used to test serum samples in some counties of Tibet and the positive rate was calculated, showing the immune effect. Secondly, the calculation of PI value showed PPRV antibody level. The result served for immunization programs in Tibet. |
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