Preparation Of Nanobodies Against Transmissible Gastroenteritis Virus N Protein And Development Of The Nanobody-based Competitive ELISA

Porcine transmissible gastroenteritis((Transmissible gastroenteritis of pigs,TGE))is an acute gastrointestinal disease caused by porcine transmissible gastroenteritis virus(TGEV).The main clinical symptoms of piglets are watery diarrhea,feces contain blood or milk clots and foul smell,severe dehydration occurs,death occurs within a week,and the fatality rate is almost as high as 100%.The clinical symptoms of adult pigs are depression,loss of appetite,vomiting,watery diarrhea,dehydration and other clinical symptoms,the disease has brought huge economic losses to the pig industry.N protein is one of the important structural proteins of porcine transmissible gastroenteritis virus and plays an important role in the life cycle of the virus.The amino acid sequence of virus is highly conserved;functionally,N protein plays an important role in the synthesis,replication,translation and pathogenicity of viral RNA,so N protein is of great significance for the development of clinical detection technology and therapeutic drugs.Nanobodies can not only maintain the binding force,affinity and specificity of traditional antibodies,but also have the advantages of structural stability and preparation in vitro.Compared with the traditional Ig G,the longer protruding part formed by the three CDR regions of the nano-antibody gives it the ability to bind to the antigen concealed epitope.Therefore,nanoantibodies play a vital role as research tools,diagnostic tools,and therapeutic drugs.In this study,the prokaryotic expression system was used to express TGEV N protein with excellent characteristics.After immunizing Bactrian camel,8 strains of specific nanobodies against N protein were screened by phage library display technique,one of which(Nb43)could compete with natural antibodies in clinical serum.The nanobody sequence was constructed into p EGFP-N1-RANbodies eukaryotic expression vector,and the recombinant plasmid was expressed by HEK 293 T eukaryotic expression system.The nanobody Nb43,expressed with HRP was used as competitive antibody,and a competitive ELISA method for specific detection of porcine transmissible gastroenteritis antibody was established.Main results:1.Expression and purification of TGEV N protein.The recombinant plasmid p ET-21b-N was transformed into Trans BL21DE3 expression competent cells,and the soluble N protein was expressed after induction by IPTG inducer.The soluble N protein was purified by Ni column affinity chromatography.The purified N protein was mixed with aluminum adjuvant and used to immunize Bactrian camel.2.Screening and identification of specific nanobodies against TGEV N protein.Blood samples were collected from Bactrian camels immunized for 5 times and peripheral blood lymphocytes were isolated.The RNA in lymphocytes was inversely converted to DNA.The c DNA was used as a template to obtain the VHH gene sequence by nested PCR,and the sequence was inserted into the phage p CANTAB 5E vector.The specific nanobodies against TGEV N protein were screened by phage library display technology.A total of 8 needle-specific nanobodies were obtained after 4 rounds of screening,one of which could compete with the natural antibodies in clinical serum.3.Development of the nanobody-based competitive ELISA.By chessboard titration of ELISA,the optimal dilution ratio of antigen coated was 1?g/m L,the optimal dilution ratio of nanobody was 1:100,the optimal dilution ratio of serum was 1:10,the optimal incubation time was 40 min,the optimal color time was 10 min.The Cut-off value of this method was 22.4% by detected 110 negative clinical serum.According to the evaluation,the accuracy of this method is high,and the coincidence rate with the detection result of indirect ELISA is 97.8%;the sensitivity is strong,and the clinical serum dilution can reach 1:40;the repeatability is good,the coefficient of variation of PI value between plates is between 5.32% and 8.81%,and the coefficient of variation of PI value in plates is between 2.64% and 5.96%.The specificity is good,and there is no cross reaction with other virus antibody positive clinical sera.Finally,it is determined that this method has good clinical application ability.In summary,the nanobody against TGEV N protein was generated which is used in a competitive ELISA for detecting TGEV antibody.This method can be used to detect antibodies against TGEV and to evaluate the level of antibodies after vaccination.