Evolution Analysis Of Epidemic Canine Distemper Virus Strains And Construction And Screening Of Phage Library Of Anti-Canine Distemper Virus Vhh Antibody

Canine distemper(CD)is an acute and highly contagious infectious disease caused by canine distemper virus(CDV)with high morbidity and mortality up to 50%,which seriously affects the development of canine reproduction and fur animal reproduction.Therefore,in this study,clinical samples suspected of being infected with CD were collected in Jinan,and the CDV H gene was amplified and their genetic evolution was analyzed.At the same time,the purified CDV was used to immunize alpaca for constructing the CDV specific Nanobody phage library,then screened and expressed the CDV Nanobodies.Experiment 1: Cloning and Genetic Evolution Analysis of H Gene of Canine Distemper Virus in Jinan areaIn order to investigate the CDV infection status of pet dogs in Jinan area,56 clinical swab samples suspected of being infected with CDV were randomly collected from the pet hospitals in Jinan from January to June 2019.The full-length H genes were amplified by RT-PCR and sequenced,and their genetic evolution was analyzed.The results showed that 7 full-length H genes were amplified by RT-PCR from the positive samples.The nucleotide homology of H gene is 98.7%-99.9%,and the amino acid homology is 98.7%-100% among these strains.the homology is 90.0%-90.9%,and the amino acid homology is 88.8%-91.4%.when compared with the vaccine strains,the nucleotide homology is 90.0%-90.9%,and the amino acid homology is 88.8%-91.4%.Genetic evolution analysis showed that these 7 strains are all belong to current Asian type I strains of China,similar to Beijing strains(BJ17BC1)and Heilongjiang strains(HRB-E8 and HLJ-1-14),and formed a large scattered branch with Guangdong strains(GD1811and GD1810).The amino acid analysis of these seven H genes showed different degree of variation compared with the reference strains.Glycosylation site analysis shows that,six strains except SD-07 contained nine potential asparagine glycosylation sites,while SD-07 contains eight potential asparagine glycosylation sites.The g3(309)glycosylation site was different from the vaccine strains,but it was highly consistent with the Asia I wild strain.Predictive analysis of H gene antigens showed that seven wild virus strains were significantly different from vaccine strains.The study showed that the prevalent of CDV in Jinan area was mainly Asia type ?.These seven wild strains were with genetic differences with vaccine strains,and showed genetic diversity between these seven different strains.Experiment 2: Construction and identification of the CDV specific Nanobody phage libraryTo obtain a CDV specific nanobody phage library,ultra-purified CDV was used to immunize the alpaca,then amplified the Nanobodies(Nanobodies,Nbs)sequence by nested PCR,and constructed a CDVspecific nanobody phage library,and then selected 40 monoclonal phages and counted the number of positive clones,randomly selected 13 sequences for verification,and calculated storage capacity and analyzed library diversity.The results showed that after the fourth immunization,the serum antibody titer could reach 1:25,000,which met with the requirements of library construction.400 bp bands were amplified by nested PCR,which consistented with the expected Nbs size.The library capacity of these phage display library was determined to be 3.41×109 PFU,and the positive rate was 90%.The sequencing results showed that these 13 selected VHH fragments was different with each other,and the complementarity determined the best diversity of Region 3(CDR3).The above results indicated that this study successfully constructed a CDV-specific nanobody phage library.The library had large capacity,rich diversity,and high positive rate,which laid the foundation for the subsequent expression of Nanobody.Experiment 3: Screening and expression of canine distemper specific NanobodyThe purified CDV was coated on an ELISA plate,and the CDV-specific nanobody phage library was subjected to three rounds of panning to enrich phage that specifically bound to CDV.monoclonal phages were selected then phage-ELISA was used to identify and select phage with high binding capacity.These genes were expressed and purified by Pichia pastoris expression system,and their binding abilities were identified by ELISA and IFA.The results showed that after three rounds of panning,a total of 85 positive clones were enriched with a positive rate of 88.5%(85/96).Two Nanobodies were expressed by the yeast expression system.WB identification results showed that there were clear band at 15 kDa,which consistented with the expected Nbs size.Both Nanobodies could specifically binded to CDV in ELISA and IFA.The above results indicated two Nanobodies that specifically binded to CDV had successfully screened and expressed.In summary,this study has initially analyzed the genetic differences of current CDV epidemic strains in Jinan area,which will provide the targeted and effective CD control measures.the CDV specific Nanobody phage library was successfully constructed,and two Nanobodies specifically binding to CDV were expressed and purified,which provided an experimental basis for the later establishment of canine distemper diagnostic methods and screening of Nanobodies with neutralizing activity.