Preparation Of Yolk Antibody About Anti-S1 Protein Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea is a highly contagious acute intestinal infectious disease caused by porcine epidemic diarrhea virus,which has a rapid onset,high transmission rate and high mortality rate of piglet leading to huge economic losses to the pig industry in our country.There are existing large variation between the current epidemic PEDV strains and vaccine strains resulting in low protection rate in existing vaccine,so the development of anti-PEDV vaccine and preparation is of great practical significance.According to the Genbank PEDV S gene sequence(accession No.JN601050.1),a pair of primers were designed.The 520 bp gene was amplified from diseased piglets tissues by RT-PCR technology,which was connected to the pET-32 a expression vector to obtain the recombinant expression plasmid pET-32a-S1 to soluble the size of 33 kDa recombinant fusion protein through inducing 6 h by 0.4 mmoL/L IPTG.The recombinant protein was purified by NTA agarose gel and the concentration was measured by nucleic acid protein analyzer reaching to 3.2 mg/mL.In addition,the recombinant protein had good specificity by Western Blot.Using recombinant protein diluted as antigen coated ELISA plate,chicken serum as samples and enzyme-labeled Rabbit-anti-chicken IgY as second antibody,we established a high specificity and sensitivity,good repeatability and stability of the indirect ELISA for the detection of chicken serum and yolk antibody against PEDV.At the same time,50 primiparous laying hens were divided averagely into tissue inactivated vaccine group,virus inactivated vaccine group,S protein vaccine group,combined immunization group and control group for immunity test and specific antibody titers in serum was detected by self-established indirect ELISA.The results showed that 4 immune groups after the third immunization could all produce high levels of antibodies and S protein immunization group’ specific antibody titers in serum was higher than that in other groups.In order to select the best production process of yolk-antibody powder and yolk-antibody frozen powder,we collected the high-immunity egg of S proteinimmunization group after 7 days in third immunization to develop yolk antibody by spray drying technology and freeze drying technology and the antibody titer was detected by indirect ELISA.The results showed that liquid egg yolk antibody titer had the average loss of 1.71 titers using spray drying technology after adding protective agent by controlling temperature and this process is suitable for mass production.The treatment of sick piglets was tested by applying high-titer yolk antibody.In the control group,mortality rate was 82.50%.In the egg-yolk antibody powder group,the mortality rate was25.33%.In the egg-yolk antibody liquid group,the mortality rate was 21.74%.In the egg-yolk antibody powder group,the mortality rate was 24.07%.It reveals that yolk antibody has certain clinical efficacy.