| Porcine epidemic diarrhea(PED) is a highly contagious intestinal disease of pigs caused by porcine epidemic diarrhea virus(PEDV). The main characteristic of PEDV was vomiting,diarrhea and loss of appetite in a variety of age pigs, especially piglets. PEDV can infect pigs of any age, from neonates to sows or boars. In recent years,the outbreaks of porcine epidemic diarrhea epidemic caused heavy economic losses for pig industry in world. To investigate the genetic variation of porcine epidemic diarrhea virus and prevent and treat the disease timely and effective, this study successfully isolated a strain of PEDV(PEDV-HN/01)and establish the good specificity and sensitivity of the indirect ELISA method by S1 protein and has a good therapeutic effect of egg yolk antibody(Ig Y). The study is divided into three parts:1. The isolation and identification of the PEDVOur experiment successfully isolated the virus using Vero cells. In the isolation process,we added the pancreatin with concentration of 6ug/ml and found that the cells became lighter and turned into a ball. The cytopathic effect was first present at about 4th generation. The result of nucleotide homologies and phylogenetic trees show that PEDV-HN/01 strain was in the same branch with the other isolated strains after 2011 and remote to classical strain CV777 、 USA strain and Korea strain. Sequence analysis showed that the S1 gene of PEDV-HN/01 was 91.9﹪、91.8﹪ to that of USA strain and Korea strain. Titer of the virus was 10-4.3/0.1ml at 20 passage. The Vero cell cultures were detected by RT-PCR, IFA and animal regression test. At the last, the isolated virus was proved to be PEDV and was named PEDV-HN/01 strain.2. Prokaryotic expression of porcine epidemic diarrhea virus truncated S1 gene and development of an indirect ELISA based on expressed proteinPEDV truncated S1 gene was amplified and sequenced from PEDV-HN/01PMD-18T-S1. Furthermore, the truncated S1 gene was cloned into PET-32a(+)vector for expression in BL21( DE3),the recombinant protein S1 was expressed in soluble form after induction with IPTG. The recombinant protein S1 was about 45 ku and could specifically reacted with PEDV positive serum. Western-blotting showed that the purified recombinantprotein S1 retains better antigenicity and specificity.To establish the serum diagnosis method of PEDV infection, an indirect ELISA was developed for the detection of PEDV antibody with the N protein of PEDV as coating antigen expressed in E. coli. The optimized reaction conditions were as follows: antigen working concentration was 5.13μg /ml. Serum sample dilution was 1:100. It was coated at 4℃ for 12 h.The plates were blocked by 1.0% BSA incubated at 37 ℃ for 1h. The secondary antibody was diluted at 1:4 000,incubated at 37 ℃ for 1h.The TMB was incubated at 37 ℃ for 10 min. It was judged as positive when the cutoff value OD450nm≥0. 338,as negative when OD450nm<0.338. Under the optimized reaction conditions, the assay was able to specifically detect the positive serum of PEDV, but no cross-reactions were found for the positive sera of other virus,including TGEV、PRV、PCV-2、PRRSV、PPV、CSFV. The coefficients of variation for intra and inter-assay were 1.06% ~ 6.81%. Fifty pig serum samples from clinical suspected PEDV-infected pigs were detected for PEDV-specfic antibody. The results showed high coincidence with commercialize ELISA. Coincidence rate was 94.0%. Compared with virus neutralization test, both of them have good correlation.A total of 150 clinical serum samples obtained from pig farms in Henan Hebei Anhui Shandong province were detected. The positive rate was 42.00%.These results indicate that the indirect ELISA is a rapid, sensitive and specific for detecting antibodies against PEDV and has a potential to apply for diagnosis of PED and for serologic survey of PEDV infection.3. Preparation of PEDV yolk antibodyPEDV reproduced in Vero cells and the 3000 ml viruses were collected. The viruses were purified by concentration and chromatographic tec HN/01 iques. The SDS-PAGE showed that the tec HN/01 iques can remove all culture calf serum components. The hens were immunized with the viruses propolis vaccine. After the antibodies titer reached to 1:64 tested by double diffusion and eggs of hyperimmunity were collected. The hyperimmunized yolk antibodies were purified by chloroform extraction and salt precipitation with ammonium sulfate.SDS-PAGE showed that the viruses had better purification effect. Neutralizing antibody titer to PEDV with CV777 was PD50=10-2.1/0.1ml. The results proved the purified viruses had good immunogenicity and could provide a basis for the study hyperimmunized yolk antibody production. |
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