| Specific immunoglobulin yolk (IgY), regarded as a promising alternative to antibiotics, has attracted considerable attention of the researchers, because that it cannot cause drug residues in animal products or bring the resistance of the bacterials. It is known that IgY can be easily decomposed under the action of the mucus in digestive tracts, however, the studies show that oral administration of specific IgY, without directly contacting with pathogens, can prevent the animals from the non-gastrointesinal diseases caused by pathogens or viruses. Whereby, researchers guess the mechanism of the IgY inhibiting the colonization and reproduction of pathogens in the lesions, and treating diseases of non-digestive tracts may be combining with the pathogens in the form of the activated fragments. The objective of this study was to prepare the bioactive fragments of the specific IgY by the methods of the enzymolysis in vitro and the genetic engineering, and investigate the trend and distribution of the fragments including the fluorescent recognization site.The bioactive fragments of the IgY was prepared by the enzymolysis in vitro. Laying hens were immunized with formalin-killed A. hydrophila, further the IgY was purified from the water-soluble fraction by the two-step salt precipitations and ultrafiltration. The maximum titer of IgY tested by ELISA reached up to1:51200; SDS-PAGE showed the purity of IgY finally purified was from30.5%to62.2%. Immunofluorescence and the inhibition of the growth in vitro showed that the specific IgY could bind specifically to A. hydrophila. SDS-PAGE of the IgY treated with the pepsin documented IgY was broken down into Fab’ and pFc" fragments incubating with the pepsin for24h; Western blot, ELISA and the inhibition of the growth in vitro showed Fab" of the IgY significantly inhibited of growing of A. hydrophila.The bioactive fragments of the IgY was prepared by the genetic engineers.The primers of the variable regions (VH and VL of the IgYwere designed according to the sequences from the variable-framework regions of the chicken IgY reported in the Genebank.In order to extract the total RNA, the blood was collected from the laying hens immunized with killed S. aureus. The oligo (dT)18primers and the mRNA were used to synthesize the cDNA. The primers of the VH and VL were added into the cDNA template to amplify the sequences of VH and VL by PCR. The gene sequences connected pMDl8-T vectors were DNA sequenced and the results showed that the size of the fragments VH and VL were about390bp and330bp, respectively. The sequences were compared with the gene sequences reported to analysize the sequence homology, and screened the specific sequences expressing the proteins binding to S. aureus. VH2, VH7, VL1and VL4genes were digested with HindⅢ and Nhe I, and cloned into the similarly digested vectors pET28a. The recombinant coli BL21containing the recombinant vectors were induced with IPTG and treated with the ultrasonication. SDS-P AGE showed that the expressions of VL1and VL4failed; VH2and VH7expressed in the E. coli BL21were intracellular soluble. Compared with the control group, the VH2and VH7had significant binding capacity of S. aureus by the indirect ELISA (P<0.05). This research supported using the technology of the genetic engineers to prepare the bioactive fragments of the IgY, because the specific component prepared by the enzymolysis included nonspecific fragments, which could affect the subsequent experiments.In conclusion, the bioactive fragments of the enzymatic components from the specific IgY treated with the pepsin was Fab’, on which was the antigen binding site; the genetic engineering technology successfully expressed the two bioactive fragments, VH2and VH7. |
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