The Prokaryotic Expression Of Nonstructural Protein Nsp10 Of PRRSV And Its Indirect ELISA Method

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectio us disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized by sow reproductive disorder and piglet breathing disorder.Also known as "porcine blue-ear disease".PRRS is persistence in Chinasince the first report,.The outbreak of HP-PRRSV in 2006 brought great economic losses to China.In this study,samples were collected from a pig farm suspected of PRRSV infection in Guangdong province.RT-PCR was used to identify PRRSV positive samples.After filtration,the positive samples were inoculated into monolayer of Marc-145 cellsand blindly passaged on Marc-145 until the observation of cytopathic effects(CPE)After being further proved by immunofluorescence(IFA)staining,the PRRSV strain was successfully isolated and named as ZSBS-GD.The whole genome of the virus was sequenced.The full length nucleotide sequence of the genome was compared with JXA1,CH-1a,HB-1(sh)/ 2002,VR-2332,and the homology were 91.3%,88.1%,89.3% and 84.3%,respectively.A 29 + 1 amino acid deletion was observed in Nsp2,indicating that the strain was JXA1-like strain.phylogenetic evolution analysis shown that the strain was located on a new independent branch,and it was presumed that this is a new divergent strain related to the highly pathogenic strain in China.,which is also indicating it belongs to the North American type.Based on the sequence of ZSBS-GD,a pair of specific primers targeting Nsp10 gene were designed.Then the amplified Nsp 10 fragment was inserted into PET-28 a prokaryotic expression vector,the successfully constructed plasmid was named as PET-28a-Nsp10.The recombinant plasmids were transformed into BL21(DE3)competent cells and induced by IPTG.The expressed protein was detected by SDS-PAGE and the size of the protein was around 55 KD.Western-blot showed that the recombinant protein had good immunogenicity.The temperature,concentration of IPTG and time for induction were optimized and the following conditions were used :1mmol/L IPTG was used for induction at 37?for 5h to induce the expression of target protein.The purified recombinant protein was used as the coating antigen and diluted at a certain dilution ratio.The PRRSV positive and negative sera were diluted at a certain dilution ratio to optimize the reaction conditions of each step.The indirect ELISA detection me thod was successful established.The optimized conditions were as follows: 4.3? g/mL of recombinant protein was used for coating at,37? for 2h or 4? overnight;the blocking solution was 1% BSA,and the serum was diluted 1:200 and incubated at37? for 1.5h;The dilution of Enzyme-labeled secondary antibody was 1:5000,incubated at 37?for 1h;Substrate(TMB)was incubated at room temperature for 20min;The criteria for positive/negative are as follows:OD450>0.317 was treated as positive,OD450<0.281,was treated as negative.No significant difference between this method and the commercial IDEXX PRRSV antibody detection kit was found and the coincidence rate was 92.4%,the relative sensitivity and specificity was 93.1% and 90.9%,respectively.It meets the basic requirements for clinical diagnosis,which laid the foundation for the future establishment of commercial test kit.