Investigation Of B-cell Linear Epitopes Existed In Nsp2 Of A Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) whichis the member of arteritis virus families is the main cause for reproductive failure of pregnant sows and piglet respiratory symptoms. PRRSV is also the important pathogen to cause serious economic losses for pig industry, and has formed a huge threat to the world's pig industry. The non-structural protein 2 (Nsp2) is considered to be one of the immunogenic proteins of PRRSV. In this study, the B-cell linear epitopes of the Nsp2 of a North American type Chinese strain PRRSV TaiPing (TP, deriveing from the Institute of Guangdong Wen's Group)were predicted by Bio-software and further identified by ELISA using sera of 8 experimentally infected pigs. The results presented in this report indicated that the Nsp2 contains the highest amount of B cell linear epitopes(BLE), that provided valuable information for developing the PRRSV marker vaccine and other vaccines using the Nsp2 epitopes.In detail, the nucleotide sequence (2850bp)of Nsp2 was amplified by RT-PCR, encoding 950 amino acids. A total of 27 BLE in Nsp2 were predicted based on the antigenicity, hydrophilicity, surface accessibility and flexibility by bio-software. Then the predicted BLE and positive peptide were all sent to biology company to synthesize.Using positive peptide, PRRSV positive and negative serum, ELISA was established by optimizing dilution of antigen and serum, coated time of antigen, concentration of HRP, incubation time of serum, type and concentration of sealing fluid, blocked time and incubation time of HRP. The sequential blood samples were collected from all the 8 experimental pig which inoculated with PRRSV TP60 by intranasal and intramuscular routes at days 0 (zero), 15, 22, 30, 45, 60, 75 and 90 dpi and tested for PRRSV-specific antibodies by using a commercially available ELISA kit (Idexx Labs, Inc). The result showed that the serum of experimental group were all positive in 7dpi except one, and all of the serum were also positive from the 15dpi~90dpi. The control group were negative all the time. By the serum and optimised ELISA, eleven of these 27 Nsp2 BLE were reactive with 80~100% of the examined sera. Antibodies reacted with these BLE appeared in the 15~22dpi, and can sustained for at least 45d, but the immunogenicity of these BLE were different, that may be due to the individual differences in the immune system of pigs.The result of conservation analysis show that 3-KV10 ,12-PD15 had the best conservation(95%). By comparing other BLE detived from the American-type strains which have been reported, two new BLE: 3-KV10 (K243TNRATPEEV252) and 20-ST14 (S518GSAPVPAPRRTVT531)were found. Other BLE have different degrees of overlap with the ones have been reported. In all of the overlap department, 12-PD15 (P76ERVRPSDDWATDED90) ,7-TV15 (T490SSEPMTPMSEPVLV504) ,22-SQ15(S546SQTE YEAFPLAPSQ560) and 9-VD16 (V781GSFTDLPSSDGVDVD796) were relatively low in variation. Considering all the factors, the BLE,12-PD15 (P76ERVRPSDDWATDED90) and 3-KV10 (K243TNRATPEEV252), are considered ideal potential serological markers for PRRSV marker vaccine, which were immunogenic, immunodominant, immunoreactive and conservative during PRRSV infection in pigs.