Epitope Identification Of Hsamp32 And Hph20 By Phage Display

Background and PurposeEpitope,or antigenic determinant,was an antigen domain with affinity to antibody in Ag-Ab complex.Epitopes play a very important role in the structure and function of protein antigens.Precise epitope mapping may help design more reasonable vaccines and diagnostic reagents,epitope-based vaccines can be easily distinguished from wild-typeviruses,which facilitate the eradication of virial diseases. Phage-displayed peptide libraries technology provides a new approach to disclose protein structure and function,and has been used to map largenumber of epitopes.Anti-sperm antibodies(anti-sperm antibodies,ASAB) was a male body autoantibodies,belonging to allogeneic antibody for women,9%～36%of infertile couples' bodies contain ASAB(male 8%～21%,female 6%～23%).ASAB has been proved to be the immune cause of infertility,at the same time,ASAB human antibody can also be used for contraceptive vaccine research,the ideal CV sperm antigen-specific shows on the sperm surface,involving sperm zona pellucida binding, and is related to human immune infertility,such as fertilization antigen(fertilization antigen,FA-1),hyaluronidase PH20,sperm protein 10(sperm protein,SP-10),human sperm acrosome membrane-associated protein 32(Human Sperm Acrosomal Membrane-Associated Protein32,hSAMP32) and so on.PH20 and SAMP32 were GPI-anchored sperm membrane protein-specific.Although hSAMP32's and hPH20's roles in immune infertility have ever been reported,so far,the hPH20 and hSAMP32 epitopes have not been reported yet.To understand the antigen structure of hSAMP32 and hPH20 better,we have carried out hPH20 and hSAMP32 epitope identification, which will facilitate to design scientific and rational vaccines and diagnostics,Materials and Methods1,This study will extract plasmid DNA with the molecular biology methods from strains of the group kept hPH20 and hSAMP32 gene,BamHⅠ/ XhoⅠdouble-enzyme digestion and DNA sequencing,the purified plasmid DNA as a template to preserve.Using Primer premier 5.0 software biology software designed hSAMP32,hPH20 sub-gene primers,divided into Sa,Sb,Sc;Pa,Pb,Pc,Pd.All primers are restriction sites upstream EcoRI,downstream of the HindⅢvector used for T7select10-3b,all fragments amplified by PCR and T vector,transformed and sequenced.2,after purification of each fragment digested vector,they associated with the T7 phage,display in the T7 phage packaged in vitro,which constructed the hSAMP32 and hPH20 gene-targeted peptide library.3 with ASAB positive serum we had a five-time screening,panning 5 gene fragment, combined with the results of the analysis TMHMM online.4 In the areas that Pa,Sa epitope located,by protein epitope analysis software design and synthesis biology epitope peptide.5 ELISA identification of peptide epitopes of the biological response,SPSS statistical analysis.Results1 Sa,Sb,Sc,Pa,Pb,Pc,Pd,were the targeted sequencing genes,respectively,343bp, 343bp,352bp,400bp,403bp,397bp,367bp.2 hPH20 and hSAMP32 gene-specific peptide library was constructed successfully, the capacity for 2x105pfu/mL.3 Pa,Sa were the area where epitope locates.4 epitope peptideSAMP32:6 AVQDAGL A H E G E G E E E T E; 37ETEDVSNRNVVKEVEFGM;76ESKCVVRVEECRGPTDC;95GKPISESLESVRLACIHTS;PH20:82 KISLQDHLDK AKDITFY.5.Identification results of ELISA show that the 6th peptide,peptide 37,peptide 76, peptide 82,the above 4 epitope peptides of the biological response well.ConclusionIn this study,the epitope of hPH20and hSAMP32 has been preliminary identificated, and it provides some theoretical basis for contraceptive vaccines and the development of peptide drugs.