Construction Of Phage Display Nanobody Library For Selection The Nanobodies Against Porcine Circovirus Type 2 (PCV2) Cap Protein

Porcine circovirus type 2(PCV2) is a major causative agent for PCV2-associated diseases(PCVAD). PCVAD are widespread in pig-producing countries and cause large economic losses for the swine industry in the world annually. PCV2, a covalently closed circular chain of negative single DNA strands virus, is known one of the smallest animal viruses. Capsid protein of Cap is the major structural protein of PCV2, which is also the target for PCVAD diagnosis and prevention.Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In order to establish a fast and effective method for PCV2 detection, a Camelus Bactrianus was immunized with commercial PCVAD subunit vaccine to construct the immunized phage display VHH library. Subsequently, Cap VLPs antigen was coated in the immune tube to screen the specific VHHs to Cap by biopanning and ELISA. As a result, the screened VHHs will provide high quality antibodies for PCV2 detection. This study is divided into three parts as below:1. Preparation of Cap VLPsFirst,the pET32a-SUMO-Cap vector was constructed and transformed into E.coli BL21(DE3) for recombinant Cap expression. The purified recombinant Cap was digested with SUMO protease Ulp and flowed through the Ni2+ affinity resin to collect the native Cap protein. The result of transmission electron microscopy(TEM) indicated that the native Cap protein could self-assemble into virus-like particles(VLPs) in vitro.2. Modification of phagemid vector pCANTAB5EFirst, a linker with two different Sfi I restriction enzyme sites was inserted into the multiple cloning sites of commercial vector pCANTAB5 E between Sfi I and Not I sites. Then a positive and negative double selection cassette CAT/Ccdb was further inserted between two Sfi I restriction enzymes sites to obtain the result phagemid vector p CANTAB5E-Ccdb-cm for VHH library construction. Comparing with the commercial pCANTAB5 E phagemid vector, our modified phagemid vector pCANTAB5E-Ccdb-cm could dramatically increase the VHH cloning efficiency.3. A Camelus Bactrianus was immunized with commercial PCV2 submit vaccines five times with 2 weeks intervals. The humoral immune response was monitored in serially diluted serum by enzyme-linked immunosorbent assay(ELISA). Peripheral lymphocytes were isolated from whole blood, then total RNA was extracted for the amplification of VHH fragments by RT-PCR. The VHH fragments and pCANTAB5E-Ccdb-cm vector were Sfi I enzyme-digested, ligated and electroporated into E. coli TG1 competent cells to obtain the primary phage display VHH library. The size of the generated library was 6×106, the recombinant rate was 100% and the insertions were diverse. The results indicated that the VHH library was successfully constructed. Cap VLPs antigen was coated in the immune tube to select Cap specific VHHs through 4 rounds biopanning and phage ELISA analysis. And four positive recombinant phage clones was screened with high and specific binding affinity against Cap.In this study, a Camelus Bactrianus-derived phage display VHH library was generated and identified. In addition, four VHHs with good reactivity against Cap were obtained. These VHHs could provide the high quality antibodies for PCV2 detection and PCV2 pathogenesis study.