Study On Developping Of Peste Des Petits Ruminants's ELISA And PCR Test

Peste des petits ruminants (PPR), is an acute contagious disease caused by Peste des petits ruminantsvirus (PPRV). PPRV is classified in the Morbillivirus genus of the Paramyxoviridae family. Characters of thedisease include fever, erosive stomatitis, conjunctivitis, gastroenteritis, and pneumonia. PPR was firstdescribed in Crte d'Ivoire, but it occurs in most African countries south of the Sahara and north of theequator, and in nearly all Middle Eastern countries up to Turkey. Recently the diffusing eastward of PPR ispersistence, around of china there have several countries like India and Nepal which PPR's outbreaks werereported.China has huge board area with several countries which were infected by PPRV like India and Nepal.There have plateaus, valleys and plains in this area, and several genus of wild small ruminant animal lived inthis board area. It will have a huge risk that PPRV transfer to China from board area when PPRV is outbreakin countries of China's neighbour. Livestock's amount of goat and seep in China is about 30 millions. IfPPRV is prevalence in china, it will damage china's economy. And there are a lot of wild small ruminantanimal including Tibet Antelope which is lucky animal of 2008 Beijing Olympic Game in board area. Thedirect contact or indirect contact with infected source all will result in outbreak of PPR in wild animal and willdamage them severely. For china, we should develop diagnostic technology of PPR to prevent the import ofPPRV.Diagnostic technologies of PPR include serological test and pathogeny test mainly. ELISA is the mostpopular serological test and PCR is the most popular pathogeny test in the diagnosis of PPR. This thesisdeveloped ELISA test which antigen is PPRV N protein and RT-PCR test which target gene is PPRV N gene.In this study, the content as follows:1 Synthesizing and expression of PPRV N gene Sequence of 16 strains PPRV's N gene weredownloaded. Evolutional relative of the genes were analysised by using MegAlign software. The Turkey 2000strain was selected and its sequence of N gene was downloaded. The codon wre modified basing favorabilityof E. coli, and composing of the alkaline base was changed on site of Ncoâ… and Hindâ…¢enzyme. Thesequence is synthesized by Shanghai Shengong Company after it is ensured, and then N gene was inserted toplasmid PUC18 and is transformed to E. coli JM109. PPRV N gene was amplified by using primer NP1 andNP2. pET32-H plasmid was extracted. Production of PPRV N gene and pET32-H were double digested byNcoâ… and Hindâ…¢enzyme. The target segments were extracted and purified. Purified PPRV N gene andpET32-H were sequenced by T4 DNA polymerase to construct recombinant vector pET-a-PPRV-N. And theproduction was transfered to competent cells of E. coli DH5α. The cells were inoculated to LB medium which include ampicillin. A protein which molecular weight is 6700 were expressed by inducing. The productionwas identified by SDS-PAGE test and Western-blotting test and it is PPRV N protein.2 Purifying of PPRV N protein and developing of I-ELISA E. coli pET-a-PPRV-N were incubated at 37℃with shaking. When OD value is 0.5-0.6 by measuring, IPTG were added until its final concentrationreached 1mol/L. The E. coli were induced to express for 4 hours at 37℃. Centrifuged sediment were re-solvedin 1×binding buffer. The solution was sonicated and centrifuged. Remain the supernatant and load it toprepared chromatography column, velocity of crossing should be 10 bed volumes per hour. Then wash thecolumn using 10 bed volume of 1xBinding, 6 bed volume of 1xWash Buffer, 6 bed volume of 1xELute Bufferseparately. Collect sample of 1xELute Buffer. Purified protein was concentrated and its salt is detached byCentrifugal Filter Device (the lowest molecular is 10000). The concentration of protein was measured,batched, and conserved.Using international positive PPRV sera to titer immuno-sera which were produced by inoculating rabbit,reference positive sera were prepared. Purified PPRV N protein was diluted (final concentration is 3ug/ml) byPBS and using it to coat ELISA plate. After incubating 3h at 37℃, the plate was coated overnight at 4℃. Theplate was blocked by 0.1% BSA for one hour at 37℃. Sample were diluted 1/40 by 0.1%BSA-PBST andadded to plate to incubate for 40min at 37℃. Enzyme conjugates protein G were dilute 1/10000 by 0.1%BSA-PBST and added to plate to incubate for 40min at 37℃. Substrate (TMB) was added to incubate for15min at 37℃. Stop solution (1M H2OS4) were added to measure the OD₄₅₀ value by ELISA reader, tocalculate S/P value of each sample and to judge the result.467 Sheep and goat sera were collected from Shandong province. The sera were detected by developedELISA test. Samples's S/P value (sample's OD₄₅₀/reference positive serum's OD₄₅₀) were calculated andthreshold was acquired by S/P's average adding 3 times standard deviation and finally it is 0.27. Infectedarea's 67 sera were detected by developed i-ELISA and C-ELISA. The C-ELISA which is from AHI PPRreference laboratory of OIE was based on H-protein MAb and its antigen is sonicated virus. The coincidentpercentage of I-ELISA with C-ELISA is 79.7%(((8+47)/69). The relative specificity and sensitivity of I-ELISAwith C-ELISA were 94%(47 of 50 serum samples) and 42% (8 of 19 serum samples), respectively.3 Transcription of PPRV N gene and developing of RT-PCR E. coli JM109 were cultured and PUC-PPRV-N vector were extracted. NP1 were used as forward primer and NP2 were used as reverse primer.PPRV N gene were amplified by NP1 and NP2. Then, N gene were purified, and sequenced to pGEM-Tplasmid to construct pGEM-T-PPRV-N. The production was transformed to E. coli DH5αcompetence cells.Recombined vector which was selected by blue-white selection were identified, then extrcted by alkaline lysistest. The vector was digested by Nde I and lineared. Linearing production were purified by PhenolChloroform Test. DNA concentration were measured and then to perform transcription test in vitro by using linering plasmid as template. The production were purified and measured, then diluted to its finalconcentration is 6×10¹⁰ copies/2μL. and it will be stord at -80℃in 0.1mL amount as positive control.Using production of transcript as template and using NP3 and NP4 as primers, the ideal procedure of RT-PCRwere determined. In the tube, 2.5μL 10×Taq buffer, 2.0μL dNTP, 1.0μL MgCl₂, 1.0μL NP3, 1.0μL NP4, 1.0μL template, 1.0μL AMV, 1.0μL RNase, 0.25μL Taq polymerase and 14.25μL DEPC H₂Owere added. The amplification was camed out according to the following programme: reverse transcript at 42℃for 40min; heating step at 95℃for 5 min, followed by 30 cycles of denaturation at 95℃for 30s,annealing at 52℃for 30s and the extension at 72℃for 30s for primers, and finally extension at 72℃for7min. RNA of CDV and NDV were extracted, using them as sample and using production of reversetransecription as positive control to perform RT-PCR test. The specifity of RT-PCR were identified becauseonly the control was amplified. Production of transcription in vitro were diluted 1/10 continuously, and usingthem to perform RT-PCR. The sensitivity of RT-PCR PPRV is measured and it is 40copies per reaction.Those results in above indicated that: PPRV N gene were synthesized, PPRV N protein were expressedand purified, I-ELISA of PPRV N protein were developed. The coincident percentage of I-ELISA with C-ELISAis 79.7%. PPRV N gene was transcripted and it was used as positive control in RT-PCR. Theprocedure of PPRV RT-PCR was determined by NP3 and NP4. Serological test and pathogeny test of PPRVwere developed in this study, it was indicated that we have the ability to survey PPRV in China.