Prokaryotic expression of variable region protein of adeno-associated virus 9 capsid and preparation of polyclonal antibody
Objective To express and purify the protein of variable region of adeno-associated virus 9(AAV9)capsid in prokaryotic cells,and prepare rabbit polyclonal antibody against it.Methods DNA sequence encoding variable region of AAV9 capsid protein was designed,synthesized and inserted into prokaryotic expression vector pET-30a.The obtained plasmid pET-30a-AAV-VR was transformed to E.coli BL21(DE3),induced by IPTG to express the multivalent antigenic peptide and purified by Ni-NTA resin under denaturation conditions.Male Japanese large-eared white rabbits were immu-nized with the AAV9 variable region protein after dialysis and renaturation to prepare polyclonal antibody,which was determined for the antibody potency by indirect ELISA,and for the specificity by Western blot and cellular immunofluo-rescence.Results The recombinant prokaryotic expression plasmid pET-30a-AAV-VR expressing the variable region of AAV9 capsid was constructed correctly as identified by Xho I and Bgl Ⅱ digestion.The expressed protein was recognized by His tag antibody after purification with a relative molecular mass of about 20 000.The potency of rabbit polyclonal antibody was 1:10 240 000,which specifically recognize AAV9 capsid protein.Conclusion The capsid variable region protein of AAV9 was successfully expressed and rabbit polyclonal antibody with high potency was prepared,which laid a foundation of the subsequent development of AAV vector and the research of AAV biological function.
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