Screening And Characterization Of Vhhs Against Newcastle Disease Virus Fusion Protein

Newcastle disease(ND), which caused by Newcastle disease virus(NDV), is thought to be one of the most seriously and highly contiguous poultry diseases. Although prophylactic vaccination is a major preventative measure to control ND, it cannot fully protect chicken from infection. Therefore; a novel method should be developed to effectively control the prevalence of ND. Fusion protein(F protein) is a major neutralizing and protective antigen of NDV and closely related to the viral virulence and pathogenicity. The antibodies target F protein could effectively block virus-cell fusion and protect chicken from NDV infection.Recently, the variable domain of heavy chain of heavy-chain antibody(VHH), which developed based on genetic engineering technology, is the smallest naturally occurring intact antigen binding unit with a molecular weight of approximately 15 kDa. Compared with conventional antibody, VHH has some unique properties: the molecular weight is small, easy to manufacture and expression; high solubility and stability; could recognize structures inaccessible for conventional antibodies and high affinity. Therefore, the potential application of VHH is recognized for many diagnostic and therapeutic purposes.Our study aimed to select VHH fragments that targeted to NDV F protein from a na?ve VHH library by yeast two-hybrid system, which provides new choice to control ND. The main results are as below:1. Construct a na?ve Camelus Bactrianus VHH Yeast Two-Hybrid library. The blood, bone marrow,spleen and Lymph node were collected from a healthy femal Camelus Bactrianus. And peripheral lymphocytes were isolated from blood samples using Ficoll Paque PLUS reagent. Then total RNA was extracted from peripheral lymphocytes. Reverse-transcription was performed to generate the first-stand cDNA. VHH fragments were amplified through two rounds PCR using two pairs of specific primers. NDV Camelus Bactrianus VHH yeast two-hybrid library was successfully constructed according to Clontech Mate&Plate library construction system user manual. The capacity and titer of the VHH Y2 H library were 2.07×107、7.6×108 cfu / mL, the percentage of library insertion diversity was more than 95%, which met the demand for Y2 H library screening. And this would lay the foundation for preparation VHHs against interested antigens.2. Screening VHHs against Newcastle diseases virus F protein. The neutralizing epitope of NDV F protein was synthesized and cloned into bait vector pGBKT7. After being verified by enzyme digestion and sequencing, the recombinant vector was transformed into yeast cells Y2 H Gold. Then self-activation and toxic action of the bait vector pGBKT7-F-neu were tested. Positive specific VHHs were obtained after screening the na?ve Camelus Bactrianus VHH Yeast Two-Hybrid library. And these positive clones were confirmed by PCR, sequencing alignment and re-transfromation.3. Expression and function identification of VHHs.Two VHH fragments were selected from the positive clones for expression by pHISE vector. The recombinant plasmids were transformed into E. Coli Rosetta and induced at 30℃ for 6 h by adding 0.4 mM IPTG. Among these VHH sequences, 2 selected clones could specificly react with NDV virions in indirect ELISA assay. And western blot result showed that the F protein could also be recognized by VHH. Additionally, the results of neutralizing assay demonstrated that both VHH1 and VHH2 were able to neutralize NDV F48E9 infection DF-1 cells.