Prokaryotic Expression Of S And M Protein Of Pedv Variant And Develop Ment Of The Hybridoma Cell Line Secreting Monoclonal Antibody Against The S Protein

To obtain soluble expressed fusion protein M of PEDV in abundance, the intracelluar region(aa99~227)of gene M was cloned into the prokaryotic expression vector p ET-Ls La linked with a mushroom lectin tag. According to the recombinant plasmid of p ET32a-PEDV, which was constructed by our own lab and included gene S, M, N and ORF3 of PEDV ZMDZY-CH-11, one pair of specific primers was designed to amplify the gene M which included restriction sites, while without the signal peptide. And the PCR amplification product was inserted into the prokaryotic expression vector p Ls La-t M. Then the recombinant plasmid was transformed into the escherichia coli BL21(DE3), which was then induced by IPTG. SDS-PAGE analysis showed that the recombinant protein was expressed in a large amount, and the molecular weight of it was about thirty-six k D which was consistent with expectation. And the inclusion body could be easily purified by the affinity chormatography with lactose, which concentration was 2 mol.L-1.Western blot analysis showed that the purified protein could keep its reaction specificity.The epidemic PEDV strain(PEDV ZMDZY-CH-11) was propagated in newborn piglets, and the purified PEDV virion was achieved through differential centrifugation, and the whole protein concentration of it was about 19 mg/m L, determined by the light absorption method.The recombinant protein GST-SA of PEDV ZMDZY-CH-11 was obtained through the prokaryotic expression vector p GEX-SA which was also constructed by our own lab, and purified through the GST affinity chormatography. Western blot analysis indicted that the protein could react with anti-swine PEDV positive serum, specifically.The purified PEDV was used to immune BALB/c mice, the purified recombinant protein M of PEDV was used as detection antigen in respond. While, the recombinant protein S of PEDV was used as immunogen, the purified PEDV virion was used as detect the antibody against PEDV.When the purified recombinant PEDV M protein was used as detection antigen, the optimal conditions for the indirect ELISA were as follows, the concentration for coating was 10 μg/m L and the condition of coating was at 4 overnight, the blocking ℃material was 5% non-fat milk and the blocking time was ninety minutes, the dilution of the primary antibody was 1: 200, and the optimal condition for the primary antibody was at 37 ℃for ninety minutes. The appropriate dilution for the HRP-labeled goat-anti-mouse second antibody was 1:6000 and the chromogenic time was fifteen minutes.When the purifed PEDV was used for the detection, the suitable concentration for coating was 5.625 μg/m L and the coating condition was one hour at 37 and then at ℃4 overnight, the blocking material was 5% non℃-fat milk and the blocking time was sixty minutes, the dilution of the primary antibody was 1: 3200 and the working condition for the primary antibody was at 37 ℃for ninety minutes, the suitable dilution for the second antibody was 1: 8000 and the chromogenic time was also fifteen minutes.The cell fusion was conducted through traditional method, the screening was carried out by indirect ELISA, and three or four subclonings were practiced through limited dilutions. Finally, three hybridoma cell strains, which could stably secrete Mc Abs against protein S of the variant PEDV, were named 2D1, 1G6 and 2C10, respectively. Their subtypes were all Ig M. The ELISA titers of the MAbs in culture supernatant were 1: 1600, 1: 800, 1: 800 and in ascites were 1: 6400, 1: 3200, 1:3200, separately. Specific tests of them showed that they had no cross-reaction with porcine transmissible gastroenteritis virus(TGEV), porcine reproductive and respiratory(PRRSV) and porcine circovirus type 2 virus(PCV2).Western blot analysis of the MAb 2D1 showed that it could specifically recognize PEDV, while it couldn’t distinguish the variant PEDV strain from the vaccine PEDV CV777 strain. ELISA results showed that although the MAb could react with both of them, the OD values were obviously different, therefore ELISA test could be used as the first screening to identify them. The relative affinity(Ka) of the MAb 2D1 was 4.0 mol/L, determined by the thiocyanate elution method, and the value indicted that the MAb had a relatively high affinity. In indirect IF test, the MAb could recognize the protein S of the natural PEDV CV777 vaccine strain. The neutralization titer of it was 1: 20, which was calculated according to the Reed-Muennch method, using micro neutralization test, and the titer suggested that the MAb might not neutralize the infective activity of PEDV effectively.The double-antibody sandwich ELISA was tried to bulit with best efforts, using the Mc Ab in ascite of the hybridoma cell strain, but unfortunately failed, and the causes needed further researches.