Porcine reproductive and respiratory syndrome (PRRS) was first observed in America in 1987, and subsequently spread worldwide, becoming one of the main infectious diseases in the swine industry and the cause of substantial economical losses. PPRS is characterized by pregnant sow reproductive failure, highly mortality in piglets and respiratory symptoms in growing pigs . The PRRS virus (PRRSV), is a small enveloped virus with a single-stranded RNA genome containing eight ORFs. These are the viral replicase encoded by ORF1, which contains ORF1a and ORF1b; the virus envelope glycoproteins GP2, GP3, GP4 and GP5 encoded by ORFs 2-5 respectively ; the viral unglycosylated membrane protein M encoded by ORF6; and the highly conserved nucleocapsid protein N encoded by ORF7 .
GP5 protein is a major multifunctional glycoprotein of 25 kDa. It has six antigenic determinants that can induce the production of antibodies that neutralize viral infection in vitro . GP5 shows the greatest variability of the PRRSV proteins, and stimulates cellular and humoral immunity. Therefore, establishing methods for detection of mAbs using GP5 protein as the antigen is feasible.
Monoclonal antibodies (mAbs) play an important role in infectious disease research, diagnosis and therapy. The largest area of mAb application in virology has been in diagnosis. The use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal anti-sera and provides unlimited quantity and consistent quality of reagent supplies. In this study, we obtained a specific mAbs against PRRSV, which may serve as a useful tools in future studies of PRRSV and development of diagnostic kits for the detection of PRRSV.
The results showed a specific protein band with molecular weight of 42 kDa appeared after induction at 28 ℃ and 12h, which was consistent with the expected results. The analysis results suggested that the recombinant GP5 protein content accounted for 30% of the total thallus protein content. (Fig. 1).
mAbs were produced from mice immunized with PRRSV lzh/07, and the two specific clones described here were named 8C9 and 4B4. The purified monoclonal antibody by SDS-PAGE showed the molecular weights of the heavy chain and light chain were about 45.0 kDa and 25.0 kDa respectively (Fig. 2A), which was consistent with the predicted molecular weight.
Figure 2. SDS–PAGE and Western blot analysis of the purified mAbs 8C9 and 4B4. A: SDS–PAGE analysis. Lane 1 and 2, Purified mAb 8C9 and 4B4 respectively. Lane M is the protein marker. B: Western blot analysis of the purified mAb 8C9 and 4B4. Lane 1and 2, Purified mAb 8C9 and 4B4 respectively. Lane M is the protein marker.
Purified recombinant GP5 (0.5 μg/well) were separated on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad), incubated for 1 h in blocking solution (5% non-fat dry milk, 0.1% Tween-20 in phosphate buffered saline, PBS) and probed with a mAbs 8C9 and 4B4 diluted 1:5 000 in blocking solution. Specific anti-PRRSV antibodies were detected with protein A-horseradish peroxidase conjugate (Sigma) and the reaction developed with luminol. Affinity chromatography-purified GP5 proteins were stored at -70℃ for use as ELISA antigens (Fig. 2B).
The neutralization activities of prepared mAbs were shown in Table 1. In this test, the setting virus control, mAb control and cell control were normal until the time end, the result showed that the prepared 4B4 mAbs can protected 50% cell no CPE in the dilution reach to 1:512. But 8C9 can't neutralized ability to PRRSV. This indicated that 4B4 have neutralization activity, 8C9 have no this ability.
Table 1. Detection of mAbs neutralization titers by virus neutralization test
The two mAbs, 4B4 and 8C9, which showed the highest binding activity for PRRSV were selected and characterized, and found to specifically bind to PRRSV without cross-reactivity with SVDV and CSFV (Table 2). In an isotype test, 8C9 was found to be of type IgG1, whereas 4B4 belonged to type IgG2a. Their titers in cell culture supernatant were 1:104, and the titers of abdomen liquor were 1:4×105. In stability tests, the titers of prepared mAbs were maintained invariably when passaged to thirty generations (data not shown). All of these results showed that the selected mAbs possessed good specificity and high titers.
Table 2. The result of the cross-reaction test