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Construction,expression,purification And Potent Application Of Recombinant PCV2b Decoy Epitope Peptides

Posted on:2018-09-29Degree:MasterType:Thesis
Country:ChinaCandidate:J W LiuFull Text:PDF
GTID:2323330515982998Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Porcine circovirus(PCV)cause huge losses to the global aquaculture industry and are the causative agent of postweaning multisystemic wasting syndrome(PMWS).The main preventive approaches to Porcine circovirus stype 2(PCV2)are inavtivated vaccine and subunit vaccine.The quality detection of PCV inactivated vaccines is mainly the determination of potency value through the immune attack which evaluate the quality of vaccines according to the infection.Benefited from the development of technology,detecting the antigenic content in vaccines by fluorescent quantitative PCR and the serum titer by ELISA are gradually used to evaluate the quality of vaccines.Capsid protein is the structural protein of PCV with high immunogenicity.Decoy epitope is one of the six linear epitopes one the capsid protein inside the virus particles.However,the decoy epitope has more space advantage than other linear epitopes,it can produce generous anti-decoy epitope antibodies when exposed to outside which can suppress the production of neutralizing antibody,but can’t suppress the proliferation of virus.The quality detection method of the vaccine can not detect the existence of the null antibody.To evaluate the quality of vaccines more comprehensively,engineering the peptide of decoy epitope for detecting the null antibody is necessary.Firstly,We have transferred the gene sequence of the decoy epitope from PCV and then got the codon-optimized sequence primarily.We designed the primers of the epitope which is paialleled thrice,then engineered the recombinant p MD18-T-E plasmid including the PCR fragment and the p MD18-T plasmid vector.We engineered the recombinant p ET28a-E3 and p ET28a-E3-his plasmids which expressed in Escherichia coli later through subcloning.SDS-PAGE Gel recovery andNi-affinity chromatography were used to purify the E3 and E3-his proteins.We detected the anti-decoy epitope antibodies in Porcine serum immunized with inactivated vaccine and serum of mice immunized with D protein,Y protein and P protein by ELISA.These results indicates that E3 protein has the activity of detection and identification.The polyclonal antibody was prepared by immunized rabbits with purified E3 protein,then used to detect decoy epitope of the subunit vaccines(D protein,Y protein and P protein)before and after assembling by ELISA,providing a detection method for evaluating whether subunit vaccine assembled into VLPs correctly.Investigating the exposure status of inactivated PCV provide the reference for evaluating the quality and technology of inactivated vaccines.The results of ELISA showed that the polyclonal antibody had the activity of detection.The polyclonal antibody purified by DEAE ion exchange chromatography and affinity chromatography was detected by ELISA.The results showed that the antibody still haddetection activty providinghe basis for the preparation of Immune colloidal gold technique.This paper provided certain reference value for the quality detection of inactivated PCV vaccine and PCV subunit vaccine.Above all,the E3 protein constructed from the decoy epitope can be used to detect the anti-decoy epitope antibody in serum;The polyclonal antibody of E3 protein prepared by immunized rabbits and the specific antibody after purification can be used to detect the exposure of the decoy epitope,providing the platform technology for the quality detection of the PCV inactivated vaccine and subunit vaccine.
Keywords/Search Tags:Porcine circovirus, capsid proteins, decoy epitope, serum purification
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