Isolation And Identification Of CIAV,Cloning And Expression Of Its VP1 Gene And Preparing Monoclonal Antibody Against VP1

Chicken infectious anemia(CIA)caused by chicken anemia virus(CAV)is a serious immunosuppressive disease of poultry,which has caused huge economic losses to the poultry industry globally.Little is known about the molecular mechanism of CIAV infection and its immunosuppression.Among the three proteins VP1,VP2 and VP3 encoded by the CIAV genome,the capsid protein VP1 is considered to play an important role in mediating CIAV infection and inducing the production of neutralizing antibodies.VP2 encodes bifunctional phosphatase activity and VP3 encodes apoptin.How the VP1 of CIAV binds to its receptors on the surface of susceptible cells to initiate virus infection and its molecular basis need to be further explored.In this study,a CIAV isolate was successfully isolated and identified,the whole genome was sequenced and analyzed.The VP1 gene of the isolated CIAV was cloned and expressed,and the stable cell lines expressing VP1 were generated.Five monoclonal antibodies specific to VP1 of CIAV were prepared by using the synthesized peptide epitopes as immnogenes.All these data provide materials and lay a foundation for further investgating the molecular epidemiology of CIAV,developing diagnostic techniques of CIAV,identifying cell receptors for CIAV infection and elucidating the molecular mechanism for VP 1-mediated CIAV infection1.Isolation,identification and genetic evolution analysis of CIAV-JS-CZ isolateIn order to detect and identify the pathogen for chickens suspected with infectious anemia in a certain area of Jiangsu province,PCR detection and viral isolation using MDCC-MSB1 cells with blind passages were carried out.PCR results showed that the specific bands for CIAV could be amplified in tissues from these diseased chickens and from the MDCC-MSB1 cell cultures.The isolated CIAV was named as CIAV-JS-CZ.Three pairs of primers were designed for sequencing the whole genome of CIAV-JS-CZ isolate,and the genome sequence and bioinformatics were analyzed with biological software.The results showed that the whole genome size of CIAV-JS-CZ was 2298bp.The nucleotide homology between the CIAV-JS-CZ isolate and 23 CIAV isolates from Genbank was between 96.1%and 99%.The homology of CIAV-JS-CZ with the Japanese isolate(AH9410)was as high as 99%,and that with the Guangdong isolate(GD-K-12)from China was the lowest,which was 96.1%.Further analysis revealed that the amino acid at position 394 of CIAV-JS-CZ isolate was glutamine(Q),which indicating that CIAV-JS-CZ might be highly pathogenic.The isolation and identification of CIAV-JS-CZ enriches the database of CIAV isolates and lays a foundation for further CIAV research.2.Cloning of VP1 gene and construction of its expression cell line.In order to explore the role of VP1 in CIAV infection and identify its potential host interaction proteins,the genome of CIAV-JS-CZ was used as a template,and the whole VP1 gene was amplified by PCR and ligated and cloned into eukaryotic expression vector pcDNA3.1-Hygromycin.The recombinant plasmid was named as pcDNA3.1-Hygromycin-VP 1 after sequencing.Indirect immunofluorescence assay using the specific polyclonal antibody against VP1 demonstrated that 293T cells transfected with pcDNA3.1-Hygromycin-VP1 showed specific bright green fluorescence,while 293T cells transfected with control vector pcDNA3.1-Hygromycin did not show specific bright green fluorescence.Since we observed that LMH cells could be used as potential target cells for CIAV infection,LMH cells were transfected with the recombinant pcDNA3.1-Hygromycin-VP1 plasmid for cell lines selection.Through hygromycin B screening and limited dilution cloning,the LMH cell lines expressing VP1 were developed.The acquisition of eukaryotic expression vector for VP1 and the preliminary construction of its expression cell line laid a foundation for the identification of molecular basis and cellular receptor for VP1-mediated CIAV infection.3.Preparation of monoclonal antibody against VP1 of CIAVIn order to prepare monoclonal antibody against VP1,two peptides Peptide-1 and Peptides-2 were designed and synthesized on the basis of the hydrophilicity and antigenicity of VP1,and were used as immunogens to immunize Balb/c mice.Synthesized peptides and 293T cells transfected with pcDNA3.1-Hygromycin-VP1 were used as screening antigens.Mouse spleen cells and SP2/0 cells were fused by routine methods,and the hybridoma cells were screened and identified by ELISA and IFA.Finally,five hybridoma cell lines secreting monoclonal antibody against VP1 were obtained.One hybridoma cell line was named as CIAV-VP1-1C5 for epitope Peptide-1,and the other four hybridoma cell lines were named as CIAV-VP1-2E3,CIAV-VP1-2B3,CIAV-VP1-3F4 and CIAV-VP1-3C10 respectively for epitope Peptide-2.The five monoclonal antibodies against VP1 not only had good ELISA reactivity with the synthesized epitope peptide Peptide-1 or Peptides-2,but also could recognize VP1 protein stably expressed in LMH cells by IFA.The development of monoclonal antibody against VP1 of CAV not only provides a reagent for the further development of CIAV diagnostic technology based on VP1 protein,but also lays a foundation for the identification of cell receptors for VP1-mediated CIAV infection.