Establishment And Application Of Indirect ELISA Methods For Detecting Antibodies Against PoAstV 2,PoAstV 3,PoAstV 5 By Using Recombinant Spike Antigen

Porcine Astrovirus?PoAstV?is a single-stranded positive-strand RNA virus that can cause viral diarrhea in pigs.There are five genotypes?PoAstV1-5?,and there are large differences among different genotypes.Its nucleotide sequence homology is only about 40%.At present,four genotypes of PoAstV have been found in different regions of China,which proves that the virus is widely distributed in China.Previously,the detection of PoAstV was mainly based on reverse transcription-polymerase chain reaction?RT-PCR?,although it was accurate but inefficient and required corresponding equipment,which was not conducive to popularization and application.Therefore,it is of great significance to construct a rapid and sensitive serological method for detecting PoAstV.At present,the research on capsid protein of astrovirus is focused on Human Astrovirus?HAstV?and the research on PoAstV antigen protein is very few.Therefore,this subject is based on the research of HAstV capsid protein,assuming that PoAstV capsid protein is also composed of three regions,namely Inner,Outer and Spike.According to the detected PoAstV 2,PoAstV 5 and published PoAstV 3-NC₀19494 strains as templates,the corresponding capsid protein expression primers were designed,and the three fragments encoding the capsid protein of PoAstV 2 were amplified to connect with pCold-TF prokaryotic expression vector.At the same time,three fragments of the amplified PoAstV 3 and PoAstV 5 capsid proteins are connected to a pHUE prokaryotic expression vector to express the corresponding recombinant proteins and purify the recombinant proteins.The corresponding recombinant proteins were successfully expressed and purified using prokaryotic expression system,and then identified by Western Blot to determine the expressed protein as the desired target protein.Subsequently,mice were immunized with the target proteins to obtain serum containing polyclonal antibodies.ELISA tests confirmed that Spike protein s had the strongest antigenicity.Therefore,the spike proteins were used as the antigen protein for indirect detection of PoAstV antibody.The optimum reaction conditions were determined through preliminary experiments.The optimal dilution concentration of coated antigen is 2.5 ng/ hole.The best concentration of serum dilution is 1: 100.The best dilution of goat anti-pig enzyme labeled secondary antibody is 1: 10000.The best substrate color development time is 12 min.The S/P thresholds of PoAstV 2,PoAstV 3 and PoAstV 5 were determined to be 42.9,31.07 and 29.85 respectively.Repeated experiments further verified that the coefficient of variation of Elisa Plate in the same batch or different batches was less than 15%,which confirmed that PoAstV-ELISA method had better stability.Then 272 serum samples from pig farms in six different counties and cities in Hunan and Jiangxi provinces were detected by the constructed ELISA serological detection method.It was found that the positive rate of PoAstV 2 was 35.66%,that of PoAstV 3 was 44.12%,and that of PoAstV 5 was only 2.94%,indicating that the epidemic trends of PoAstV of differ ent genotypes are quite different.The PoAstV 2-Spike,PoAstV 3-Spike and PoAstV 5-Spike proteins expressed in this study have good antigenicity,and the established ELISA method s have good stability and sensitivity,which provides a new method for the detection and prevention of PoAstV in the future,and is helpful for the research of PoAstV capsid protein in the future.