Establishment Of Nano-antibody-based ELISA Method For Detection Of Avian Influenza Virus H5 Subtype

Avian influenza is a zoonotic infectious disease caused by avian influenza virus infection.In recent more than a decade,H5 subtype highly pathogenic avian influenza has appeared in many countries around the world,which has caused serious harm to poultry production and human health.In this study,an ELISA detection method based on nano-antibody for H5 subtype of avian influenza virus was established,which provides an accurate and effective diagnostic method of the disease in clinic.In this study,the prokaryotic expression plasmid p ET-NB93 was constructed by cloning the nucleotide sequence of nano-antibody targeting H5 subtype haemagglutinin(HA)into p ET28a.The nano-antibody gene sequence and the alkaline phosphatase(AP)gene sequence were fused and cloned into the expression vector to construct p ET-NB93AP.The two plasmids were transformed into Escherichia coli BL21 for expression.After SDS-PAGE analysis,the optimal final concentration of IPTG induction of recombinant protein was 1.0m M,and the highest expression of IPTG was obtained at 37?.Then,the recombinant protein was purified by affinity chromatography for solubility or renaturation and the purification conditions were optimized.The results of Western blot showed that the recombinant protein could react with mouse anti-His label antibody,and there was no obvious non-specific reaction,which indicated that the purity of the recombinant protein was high.NB93 and fusion protein NB93AP were used for hemagglutination inhibition and chicken embryo neutralization tests with avian influenza virus H5 subtype strain r D8,strain ZQ105 and strain D7,respectively.The results showed that the hemagglutination inhibition titers of NB93 on r D8 and ZQ105(both of them are branch 2.3.4.4)were 8 log2 and 7 log2,respectively,but there was no significant hemagglutination inhibition effect on strain D7(branch 2.3.2).The hemagglutination inhibition titer of renatured recombinant protein NB93AP to r D8 strain was 2log2.The results of chicken embryo neutralization test showed that the neutralization titer of recombinant protein NB93 was 320 times for strain ZQ105 and0 for D7 strain,which was consistent with the results of hemagglutination inhibition test.It indicates that NB93 has a good binding effect on the H5 subtype influenza virus of branch2.3.4.4.The ELISA method for detecting H5 subtype influenza virus was established after the virus was coated.The optimum reaction procedure of ELISA was as follows:the concentration of coated virus was 10⁷ EID₅₀,coated for 1 hour at 37?,sealed at 3%BSA,sealed at 37?for 2.5 h,dilution of ELISA-1 was 50 times,incubated at 37?for 90 min,and colour developing time was 1.0 h.According to the statistical principle,the average value of OD(?)and standard deviation(S.D.)of negative sample OD value were used to determine that OD₆₂₀ of negative samples was positive when OD₆₂₀?(3+3S.D.The critical value of NB93-HRP is 0.461,that is,when OD₆₂₀?0.461,it is judged to be positive,and conversely,it is judged to be negative.The optimum reaction conditions of ELISA for NB93-AP were basically the same as that for NB93-HRP.The colour developing time of NB93-AP was 10h,and the critical value OD₄₁₀ was 0.432.The positive samples ZQ105 and Avian Influenza Virus H9,H7,subtype H6 and strain D7 of subtype H5,Gosling Plague Virus,Newcastle Disease Virus and Tambusu Virus were detected by ELISA.Except for the positive reaction of ZQ105,the results of other viruses were negative,which indicated that the established method had good specificity.Detection by NB93-HRP,when ZQ105 virus is 100 EID₅₀,the OD₆₂₀ is still greater than 0.461,while when NB93-AP is 800 EID₅₀,the OD₄₁₀ is still greater than 0.432,which indicates that the method has good sensitivity.