Preparation Of Egg Yolk Antibody Against PCV2 And Study Of Its Protective Effect

In this study, specific IgY against porcine circovirus type 2 was prepared, and the effects of antibodies which inhibits virus wered investigated in vitro and in mice conditions. The main contents are as follows:1. Preparation of specific egg yolk antibody against PCV232 healthy laying hens of 22 weeks old were divided into 4 groups, with 8 replications per group. Control group injected with saline; Test group 1:inactivated PCV2 emulsified with Freund's adjuvant with increasing dose; Test group 2:inactivated PCV2 emulsified with Freund's adjuvant with maintain the same dose; Test group 3:inactivated PCV2, a total immunization for five times. The antibody level in serum and egg yolk was detected by both ELISA and immunity colloid gold; IgY was isolated from egg yolk by water dilution, salting-out, the second salting-out, dialysis and concentration; BCA method assay the protein concentration of each extraction; The purity of each extraction step was detected by SDS-PAGE. The results show that:The antibody level in IgY in all test groups was always higher than serum. Experimental group 1 received the highest antibody titers, it can reach 1:640 and maintain more than 8 weeks, Serum antibody level is always higher than the level of egg yolk antibody; Through water dilution, salting-out, the second salting-out, dialysis and concentration, we can finally get IgY 51.67 mg/10ml egg yolk; Detected by SDS-PAGE, IgY has high purity.2. Egg yolk antibody against PCV2 in vitro and in vivo neutralization testThe study of maximum non-toxic effect concentrations of IgY:IgY was diluted to different concentration in the 96-well cell culture plate, adding the cells and observing the CPE of cells by inverted microscope. The neutralization test in vitro of IgY:IgY was diluted to different concentration, and neutralize with 200 TCID50 of PCV2 in vitro for 1 h, then adding the cells. The cells were collected after 72 h, extracted viral DNA. The copy number of the virus were determined by fluorescence quantitative PCR. The neutralization test in vivo of IgY:Select 30 SPF 8 weeks BALB/c mice, divided into 3 groups. The negative control group:the SPF BALB/c mice were treated with normal saline,0.5 ml/per mice; The test group:IgY and PCV2 (107 TCID50/ml) neutralized for 1 h, the SPF BALB/c mice were treated with the mixture 0.5 ml/per mice; The positive control group:the SPF BALB/c mice were treated with PCV2 (107TCID50/ml) 0.5 ml/ per mice. The blood and tissue samples of the mice in three groups were collected on the 7 d and 14 d, and the virus replication situations and serum antibody levels in mice were detected. The result show that:IgY does not cause cells to produce CPE, however, IgY will promote cell growth, producing cells accumulation. And with the IgY concentration decreased, the phenomenon of accumulation decreased. When IgY concentration was 0.9 mg/ml, the accumulation disappears; The neutralization test in vitro of IgY results showed that:when the concentration of IgY was 225μg/ml, the copy number of PCV2 in cells were significant different between the negative control group and experiment group. experiment group IgY inhibited viral replication, and the virus copy number were declined with the increase of concentrations of IgY; the neutralization test in vivo of IgY results showed that:the IgY could inhibited the proliferation of PCV2 in mouse tissues and have a good protection for the mice when the concentration of IgY was 100 mg/ml.