Preliminary Identification Of Epitope Fragments Of Anti-i LRP Monoclonal Antibody

Objective:The preliminary identification of the epitope corresponding to the anti-iLRP monoclonal antibody lays the foundation for the further study of the importance of each amino acid in the antigenic epitope segment,providing a theoretical basis for the study of peptide vaccines targeting i LRP..Methods:1.According to the i LRP full-length gene sequence?GenBank No.NC₀00003.12?designed five pairs of primers,using pET30a-i LRP plasmid as a template,PCR amplified five fragments of interest,it was connected to the pET-30a plasmid vector,Five iLRP gene fragmented vectors were constructed.2.Double digestion and plasmid sequencing verified the successful construction of the five segmented vectors.3.The plasmids of pET30a-i LRP and five iLRP gene fragmentation vectors were transformed into BL21 competent cells respectively,and induced by the inducer IPTG to induce the protein expression,so that the pET30a-i LRP and the five recombinant vectors express a large number of target proteins,respectively.SDS-PAGE analysis of protein expression.4.The bacteria were collected,centrifuged after ultrasonication,and the supernatant and precipitate were collected,respectively,and analyzed by SDS-PAGE to determine the fraction of the target protein,and the protein was quantified.5.Using inclusion bodies as antigen and anti-iLRP monoclonal antibody as primary antibody,Western blot was used to identify proteins that specifically bind to mAbs,thereby inferring the truncated fragments where the epitopes are located.6.The target genes of the truncated body containing the i LRP epitope were identified and fragmented again.Four pairs of primers were designed and four recombinant expression vectors were constructed again to further narrow the range of the iLRP epitope.Results:1.Successfully amplified five target fragments of the expected size by PCR and connected the target fragment to pET-30a vector by T4 DNA ligase.Thus,five recombinant expression vectors were successfully constructed and named pET30a-iLRP-2186,pET30a-iLRP-2187,pET30a-iLRP-2188,pET30a-i LRP-2189,and pET30a-i LRP-2190,respectively.2.The plasmids of five recombinant expression vectors were verified by double enzyme digestion experiments to verify the successful construction of the vectors.The positive plasmids were then sent to Shanghai Shenggong for sequencing verification,and once again proved that the five recombinant expression vectors were successfuLly constructed.3.After induced by different concentrations of inducer IPTG,SDS-PAGE electrophoresis analysis showed that the five recombinant expression vectors were obtained at the positions of the expected protein size?28.3 KD,22.9 KD,17.5 KD,12.4KD and 6.7 KD?respectively.Expressed,and the optimal final concentrations of IPTG were 0.5 mM,0.5 mM,0.2 mM,0.8 mM,and 0.5 mM,respectively.4.By SDS-PAGE electrophoresis analysis,it was found that the target protein was expressed in the form of inclusion bodies.5.Western blot analysis showed that the proteins expressed by pET30a-i LRP-2186 and pET30a-i LRP-2187 can specifically bind to the anti-i LRP monoclonal antibody,so it is inferred that the epitope is on the target gene fragment of pET30a-i LRP-2187.6.The target gene fragment of pET30a-i LRP-2187 was again divided into four segments,and a recombinant vector was constructed by the same method.Four recombinant vectors,pET30a-iLRP-2187-30,pET30a-i LRP-2187-60,were verified by sequencing.Successful construction of p ET30a-iLRP-2187-90 and pET30a-i LRP-2187-120.After Western blot analysis,it was found that the proteins expressed by the four recombinant vectors can specifically bind to the anti-iLRP monoclonal antibody,and the antigen epitope is located on the target gene fragment of pET30a-i LRP-2187-120.Conclusion:In this experiment,we first constructed five i LRP gene fragmentation vectors.The SDS-PAGE analysis and Western Blot identification identified the epitopes of iLRP at 155aa-205aa,^[155]RYVDIAIPCNNKGAHSVGLMWWMLAREV LRMRGTISREHPWEVMPDLYFYR^[205],Subsequently,four segmented vectors were constructed again using the same method,and the i LRP epitope was further localized in 155aa-164aa,^[155]RYVDIAIPCN^[164],providing an important basis for the detailed study of the core sequence of the iLRP epitope.