| Double-sandwich enzyme linked immunosorbent assay(DS-ELISA)is a sensitive detection method for different antigens.The establishment of highly sensitive DS-ELISA requires two specific antibodies respectively recognizing different epitope.The way to obtain the monoclonal antibody conventionally is that full-length protein was used as the immune antigen as well as screen antigen.However,this method exists some problems.Firstly,the obtained antibodies may not be successfully used for antibody pairing of DS-ELISA.Moreover,the epitope recognized by obtained antibody is not clear,which requires a lot of subsequent epitope identification work.To solve the above problems,we take the establishment of the proGRP DS-ELISA as an example and design a new epitope immune strategy to obtain monoclonal antibodies that can be applied for DS-ELISA.First of all,the full length protein of proGRP is analyzed by bioinformatics software.According to the three-dimensional structure of the protein,the immunogenicity scores of proGRP epitope and the existence of glycosylation sites,we choose three sequential amino acid which without interference in spatial structure as the targeted epitope of antibody used in DS-ELISA.Theoretically,antibodies against these epitopes can suitable paired for DS-ELISA.But epitope as a hapten,has only immunoreactivity,and can’t effectively induce immune response.The previous studies have shown that using virus-like particles(VLPs)and virus vector as carriers to present antigens can effectively increase the copy number of antigens and enhance the immunogenicity of antigens.So we choose the core protein of Hepatitis B virus(HBc)and type 5 Adenovirus(Ad5)as the carrier to express proGRP epitope,and use cross immunization strategy to further enhance the immune effect.In order to increase the probability of obtaining targeted monoclonal antibodies,we adopt the strategy of cross screening in the process of antibody screening by using the E fragment of progranulin(PGRN-E)fusion protein.In order to prove the feasibility of this epitope immun strategy,as well as acquire suitable matched antibodies for DS-ELISA,this topic mainly includes the following three parts:1.Prediction of proGRP B-cell epitope and generation and purification ofimmunogens carrying B-cell epitope.1)Prediction of proGRP B-cell epitopes:We used different bioinfonnatics software,such as COBEpro database and Bepipred database to predict proGRP B-cell epitope.Based on proGRP 3D structure,glycosylate modification,and immunogenicity scores,three candidates ofproGRP B-cell epitope,EP1:KSTGESSSVSER ranging from 30 to 41aa,EP2:ENRNHQPPQPKA ranging from 69 to 80aa,and EP3:NQQPSWDSEDSS ranging from 83 to 94aa,were selected.2)Preparation of the recombinant adenovirus vector carrying proGRP epitope modification in hexon and expression cassette secreting HBc-proGRP epitope in El region.By means of molecular cloning,the proGRP epitope gene fragment was inserted into HVR5 region of adenovirus backbone vector pAd5-Hexon-HRV5-BamH l-LacZ-Sful/E3-CMV-luciferase-T2A-eGFP.The obtained vector was called pAd5-Hexon-proGRP EP1-3/E3-CMV-luciferase-T2A-eGFP.At the same time,the proGRP epitope was cloned into El shuttle vectorpAd5-E 1/H1H2-HBc-MCS-Flag.The obtained vector was called pAd5-E 1/H1 H2-HBc-proGRP EP1-3-Flag.Then ScaI linearized pAd5-E 1/H1 H2-HBc-proGRP EP1-3-Flag and ClaI linearized pAd5-Hexon-proGRP EP1-3/E3-CMV-luciferase-T2A-eGFP were co-transformed into E.coli BJ5183.Then,the adenovirus vector pAd5-E1/H1H2-HBc-proGRP EP1-3-Flag/Hexon-proGRP EP1-3/E3-CMV-luciferase-T2A-eGFP were obtained by homologous recombination,which expressed the secretory HBc-proGRP fusion protein in El region and the small peptide modified in Hexon region.The three adenovirus vector constructed above was linearized by PacI,transfected into HEK293 cells,and amplified after packaging.Finally,the corresponding adenovirus particles were purified by CsCl density gradient centrifugation.3)Construction of prokaryotic expression vector carrying HBc-proGRP epitope fusion protein and expression and purification of HBc-proGRP epitope fusion protein.proGRP epitopes were ligated into the prokaryotic expression vector of pET-28a/HBc-MCS-His constructed by our laboratory.The obtained vector was called pET-28a/HBc-proGRP EP1-3-His.Then,the vector was transformed into BL21(DE3)competent cells.The HBc-proGRP epitope fusion protein was expressed and purified,then detected by SDS-PAGE and Western blot.2.Preparation of hybridoma secreting MAbs against proGRP different epitopes.1)Construction of prokaryotic expression vector carrying PGRN-E-proGRP epitope and expression and purification PGRN-E-proGRP epitope fusion protein.proGRP epitope were cloned into the prokaryotic expression vector pRSET-B/PGRN-E-MCS-His constructed by our laboratory.The obtained vector was called pRSET-B/PGRN-E-proGRP EP1-3-His.Then,the vector was transformed into BL21(DE3)competent cells.PGRN-E-proGRP epitope fusion protein were expressed and purified,then detected by SDS-PAGE and Western blot.2)Preparation of MAbs against proGRP epitope.With conventional monoclonal antibody preparation processes,through three-round cloning,two strains hybridomas secreting MAbs against hproGRP epitope 1 were screened,which were named anti-proGRP-EP1-1 and anti-proGRP-EP1-2 respectively.Four hybridomas secreting McAbs against anti-hproGRP epitope 2 were obtained,which were named anti-proGRP-EP2-1,anti-proGRP-EP2-2,anti-proGRP-EP2-3 and anti-proGRP-EP2-4 respectively.One hybridoma secreting MAb against proGRP epitope 3 was obtained,named anti-proGRP-EP3-1.The specificity of the obtained monoclonal antibodies was detected by Western blot,immunofluorescence staining and immunoprecipitation.The results showed that 7 strains MAb can recognize the liner structure of proGRP,and all 6 strains MAb can recognize proGRP spacial structure except foranti-proGRP-EP2-1.3.Establishment and application of proGRP DS-ELISAfor detecting proGRP.1)Construction prokaryotic and eukaryotic expression vector carrying full-length proGRP and their expression and purification.To produce eukaryotic and prokaryotic proGRP used for testing sandwich ELISA system,full-length proGRP isoform3 fused with His tag at the 3’-terminal was amplified and subcloned into commercialized vector pcDNA3.1 and pET28a respectively,the obtained plasmids were named pcDNA3.1/kozac-SP-proGRP-6His and pET28a/proGRP-His.pCDN A3.1/kozac-SP-proGRP-His was transfected into HEK293 cell.The supernatant was collected 72 hours post-transfection,and the eukaryotic proGRP protein was purified through Capturem His-Tagged Purification Miniprep Kit.The prokaryotic proGRP protein was induced by IPTG in the cells of BL21(DE3)and purified by Ni-NTA.The results of SDS-PAGE and Western blot analysis showed that eukaryotic or prokaryotic proGRP proteins have been obtained successfully.2)Establishment and application of proGRP DS-ELISA.The obtained MAbs against the three epitopes were used for antibody pairing experiment by ELISA.Then proGRP DS-ELISA is established by optimizing the titer of capture antibodies,the concentration of detecting antibodies and Avidin-HRP.The DS-ELISA established in this study was used to detect the serum of SCLC patients and the supernatant of SCLC cell line.The results showed that the DS-ELISA established in this study can sensitively detect the level of proGRP in different samples.In summary,the results obtained through the above research are as follows:1)Three linear epitope that can be used to prepare antibodies for DS-ELISA were predicted by using bioinformatics software;2)Using epitope immunization strategy,seven monoclonal antibodies specific to proGRP have been successfully prepared;3)The best matching antibodies used in the proGRP DS-ELISA was successfully obtained;4)the proGRP DS-ELISA established can sensitively detect the proGRP levels in different samples.To sum up,the novel peptide-based immunization strategy adopted in this study laid a foundation for the preparation of monoclonal antibodies for the other epitopes,which has an important application prospect in the preparation of neutralizing antibodies against checkpoint molecules used in tumor immunotherapy and antibodies used in sandwich ELISA for detecting other tumor markers. |
