Generation And Characterization Of Anti-Idiotypic Nanobodies To Antibody Specific To PCV2 Cap Protein

Porcine circovirus type 2（PCV2）is the major pathogen of porcine circovirus associated disease（PCVAD）and this virus is widely present in pigs and wild boars,which is a problem for the pig industry all over the world.Capsid protein（Cap）is the only structural protein of PCV2 and contains the major antigen epitopes,so this protein is a key target for developing multiple vaccines.Anti-idiotypic antibody（AId or Ab2）is a specific antibody against the idiotype on the variable region of the antibody（Ab1）.According to the different positions on the Ab1 they combined,AId can be divided into 4 types:α-AId、β-AId、γ-AId andδ-AId.Among them,β-AId can recognize the antigen-binding site on Ab1 and has the same antigenic determinant as the antigen.Therefore,β-AId is regard as an antigen substitute to develop new anti-idiotypic vaccines.Camelidae serum contains conventional antibody and heavy chain-only antibodies which the light chains and the first constant region（CH1）of heavy chain was deleted.Because Hc Ab has only one type of heavy chain variable region（VHH）,they can be screened by phage display technology.Due to its small molecular weight（about 15k Da）,the separate VHH region is also called nanobody.Compared with traditional antibodies,nanobodies have many advantages such as smaller size,higher specificity,affinity and ability to bind antigens,low cost and easy to have mass production.They have been widely used in the detection and diagnosis of many diseases.Based on the above background,this study successfully obtainedβ-anti-idiotypic nanobodies which can mimic the key epitope of PCV2 Cap protein.The main results are as follows:1.Using Protein G to purify ascites containing anti-PCV2 Cap protein m Ab 1E7 with a yield of about 1.6 mg/m L.The recombinant p ET28a-PCV2 Cap protein was successfully expressed in E.coli BL21（DE3）and successfully purified by nickel column with a yield of about 20 mg/L.Western blot and indirect ELISA was used to verify the m Ab 1E7 can combine with PCV2 Cap protein.2.Camel was immunized with purified m Ab 1E7 6 times,2 mg each time.Two weeks after the fifth immunization,the titer of the antibody specific to m Ab 1E7 in camel’s serum is 1:10⁶.One week after the sixth immunizations,the peripheral lymphatic blood was collected and lymphocytes were isolated.RNA was extracted from lymphocyte cells and amplify the VHH gene by nested PCR.A 7×10⁹ phage library was constructed.Finally,10recombinant nanobodies were screened from the recombinant antibody library which bind the variable region of the m Ab 1E7 heavy chain specifically.3.Ten yeast recombinant plasmids VHH-p PICZαA were constructed and transformed into competent cells X-33 and positive clones were selected and induced by methanol with a production of 70～80 mg/L.Ten nanobodies can bind with m Ab 1E7 specifically was confirmed by Indirect ELISA.Blocking ELISA confirmed that ten nanobodies could block the combination between PCV2 Cap protein and m Ab 1E7 effectively,with a blocking rate of 96%.4.Peptides of PCV2 Cap protein and Nb61 were synthesized,then determined the position and key amino acids of the PCV2 Cap protein(⁷⁵NINDFL⁸⁰)and Nb61(¹⁰¹NYNDFL¹⁰⁶)recognized by m Ab 1E7 with Pep Scan technology and the epitopes of both Nb61 and PCV2 Cap included 6 key amino acids to combined with m Ab 1E7 and the epitope on CDR3 region(¹⁰¹NYNDFL¹⁰⁶)of Nb61 is consistent with the 5 key amino acids of PCV2 Cap protein epitope.Using software UCSF Chimera and Pymol to make a 3dimensional picture of PCV2 Cap protein and the target anti-idiotypic nanobody（Nb61）.BALB/c mice were immunized with Nb61 and Nb39 and Ab3 in the serum of the mice against the initial antigen PCV2 Cap protein was detected.In summary,this study screened PCV2 Cap protein anti-idiotypic nanobody by phage display technology firstly,successfully expressed it with Pichia yeast expression system and purified with nickel column.Verified the anti-idiotypic nanobody isβ-AId by blocking ELISA successfully.The preparation of PCV2 Cap protein anti-idiotypic nanobody can provide the foundation for searching PCV2 susceptible cell surface receptors,the development of PCV2 diagnostic and therapeutic reagents and the development of new anti-idiotypic vaccine.