Establishment Of A Colloidal Gold Test Strip For Detection Of Porcine Epidemic Diarrhea Virus And Study On New Subunit Vaccine

In recent years,the variant PEDV has become popular among pig farms in China.It is difficult to distinguish PED from TGE and PRV in clinical symptoms.Accurate detection is an important step to prevent porcine epidemic diarrhea.The colloidal gold test strip of porcine epidemic diarrhea virus based on immunochromatography is a rapid,sensitive and specific PEDV detection method.It is of wide application value for field diagnosis at the base level.At present,PEDV vaccines are mainly viral inactivated vaccine and attenuated vaccine.These vaccines have more or less shortcomings.Therefore,the development of a safe and efficient new PED vaccine is of great significance for the prevention and control of PEDV.The N gene of HB2015 strain was amplified by RT-PCR and sequenced.The results showed that the homology of the German strains?GER-L00721-2014?,CH/JXJA/2017,the French strain?KR011756-FR001-2014?and the Hebei strain?JF700126.1-HB-HS-2010?was 99.5%⁹9.9%.The homology homology between HB2015 isolates and CV777 and CH/S strains was 95.5%⁹5.6%,and there was a small variation.The HB2015 isolates were one of the evolutionary branches of the German strains?GER-L00721-2014?,CH/JXJA/2017,the French strain?KR011756-FR001-2014?and the Hebei strain?JF700126.1-HB-HS-2010?in 2014.Therefore,HB2015 strain belongs to the variant strain of PEDV G2 group.The recombinant N protein was expressed by pET-32a-N prokaryotic expression,then it was analyzed and identified by Western blotting.The PEDV N protein was purified and was used to immunize 6-week-old BALB/c female mice to prepare monoclonal antibodies,Western blotting,IFA and indirect ELISA analysis were used to identify the monoclonal antibodies specificity and titers.The results showed that the recombinant N protein was successfully expressed in E.coli and has good antigenicity.Two hybridoma cell lines stably secreting monoclonal antibodies against PEDV-N protein were screened and named as 3E7 and 5D3 respectively.The Indirect ELISA method could be used to detect the PEDV without cross reactions with those antigen of porcine reproductive and respiratory syndrome virus,classical swine fever virus,porcine transmissible gastroenteritis virus,type O foot and mouth disease virus and type A foot and mouth disease virus,The titers of ascites were 1:32000 and 1:64000,respectively.The two monoclonal antibodies had good specificity,stability and high titer.Colloidal gold was prepared by trisodium citrate reduction method,the colloidal gold conjugates with monoclonal antibodies 3E7.The T line is monoclonal antibody 5D3 and the C line is Goat anti mouse IgG coated on the NC membrane.The specificity,sensitivity and stability of colloidal gold diagnostic strip were performed.The results showed that the optimal McAb of immunogold gold was in the range of 20³0?g/mL,and the best pH value was 8.The 0.9mg/mL monoclonal antibody 5D3 was coated on the NC membrane as the detection line.The 1.0mg/mL Goat anti mouse IgG package was used as the quality control line on the NC membrane.The colloidal gold diagnostic strip could be used to detect the PEDV without cross reactions with those antigen of PRRSV,CSFV,TGEV,type O FMDV and type A FMDV.The minimum amount of virus that can detect 225 TCID₅₀,4 centigrade can be preserved for 180 days.The test strip has strong specificity,high sensitivity and good repeatability.Nucleotide and amino acid sequences of S gene of 31 strains of PEDV at home and abroad was analyzed.After optimizing the 390-789aa coding gene of S1,The expression of target proteins S1,S1-P28,S1-CD40L and S1-IgG1Fc in baculovirus expression system were emulsified with nano-adjuvant and 206 adjuvant respectively and evaluated in mice and pigs.The results showed that the neutralizing antibodies of nano S1-CD40L group were better than those of national standard?1:16?,The serum IFN-gamma and IL-4 levels of non-adjuvant group were higher than those of other groups,indicating that CD40L could significantly improve the immune response.