Prokaryotic Expression And Functions Of A Neotype Micromolecule Antibody A?₄₂ScFv Against A?₄₂

Object:Design and prokaryotic express a neotype micromolecule antibody A?42ScFv which can specifically bind to A?42,verify that the antibody can bind to A?42 and inhibit its aggregation in vitro.Method:According to A?42 monoclonal antibody known sequence provided by GenBank,we select its heavy chain variable region CDR1,light chain variable region CDR3,added membrane peptide SEKDEL and cell-penetrating peptide TAT to design the sequence of micromolecule antibody A?42ScFv.Then we predicted the structure of A?42ScFv by Phyre2.The prokaryotic expression sequence was designed and constructed by genetic engineering,and the target fragment obtained by PCR reaction was verified by electrophoresis and sequenced after TA-cloning.The target fragment was linked to the pet-28 a plasmid vector through Xho? and Nco? restriction sites to construct the prokaryotic expression plasmid,and the target fragment was verified to be recombinant in the vector after enzyme digestion by electrophoresis.The prokaryotic expression plasmid was transformed into JM109,then the target protein was recovered.Prokaryotic expression plasmids were transferred to BL-21 expressing bacteria,and the expression products were tested in vitro by Native-PAGE protein electrophoresis,transmission electron microscope to verify the combing binding ability to A?42 and inhibit A?42 aggregation.Result:1.Electrophoresis was performed on the target fragment of the prokaryotic expression sequence obtained by PCR reaction,and a 150 bp fragment was observed,which was the size of the expected target fragment,and sequenced after TA-cloning confirm the above conclusion.2.Restriction enzyme Xho? and Nco? were used to ligate the product after TA-cloning and electrophoresis showed 150 bp target fragment band and 5200 bp carrier band after enzyme digestion,which was in line with the expectation.3.The target protein expressed by JM109 was recovered and sequenced,and the experimental results are in accordance with the expectation,which confirmed that the target gene was inserted correctly and A?42ScFv was successfully expressed.4.Since the isoelectric point of A?42ScFv was significantly different from that of A?42(the isoelectric point of A?42ScFv was 10.43,and that of A?42 was 5.31),After positive Native PAGE electrophoresis,only A?42 monomers,oligomers and fibrous bodies were observed normally,A?42ScFv expressed by BL-21 alone did not swim out of the sample hole due to the large positive charge.After co-incubated,no A?42 related band was observed.It was confirmed that A?42 and A?42ScFv combined in the reaction system without swimming out.5.Observed by transmission electron microscopy,compared with incubated with A?42ScFv at 37 ? for 1d,the aggregation degree of A?42ScFv was increase obviously.Conclusion:1.Gene of A?42ScFv which can be specifically bound to A?42 was successfully designed and cloned.2.PET28a-A?42ScFv recombinant plasmid carrying A?42ScFv was successfully constructed,and the recombinant plasmid was transformed into BL-21 bacteria,and can express functional A?42ScFv protein stably.3.A?42ScFv expressed by BL-21 can bind to A?42 and inhibit its aggregation in vitro.