| Avian influenza(Avian influenza,AI)of H9N2 subtype not only causes about 30%mortality of broilers,but also causes a serious drop in egg production rate of laying hens and breeders,causing serious economic losses to China’s poultry industry.In addition to infecting poultry,H9N2 subtype avian influenza virus(Avian influenza virus,AIV)can also infect humans and other mammals.Therefore,the prevention and control of the disease also plays an important role in public health.Virus infection monitoring is one of the important means of disease prevention and control.At present,the monitoring methods of influenza virus infection in humans or different animals mainly include RT-PCR,hemagglutination and hemagglutination inhibition tests,ELISA,etc.Hemagglutination and hemagglutination inhibition tests are widely used in clinical monitoring of H9N2 AIV infection and evaluation of antibodies after vaccine immunization in chickens.However,on the one hand,this method requires the preparation of red blood cells and the results need to be subjectively determined;on the other hand,it is easy to produce false positives when monitoring other animals for H9N2 AIV infection.The ELISA method has the advantages of simple operation and high throughput,and has been widely used for monitoring H9N2 AIV infection.However,relying on the ELISA method established by traditional antibodies,the production process is complex,and usually requires the use of secondary antibodies and secondary antibody labels,resulting in higher prices,which seriously hinders its commercialization.Nanobodies are the variable regions of heavy chain antibodies in camels,and have the advantages of small molecular weight,high stability,and easy genetic modification.Especially the fusion of Nanobodies with different reporter genes has been widely used in the diagnosis of different human and animal diseases.Such methods are often simple to operate and low in production cost.However,H9N2 AIV has no reports on the screening of Nanobodies and the use of Nanobodies to establish diagnostic methods.In this project,prokaryotic expression of H9N2-NP recombinant protein was used to immunize Bactrian camel to construct phage library;then,phage display technology was used to screen Nanobodies against the protein;We performed fusion expression and used it as a competitive antibody to establish a competitive ELISA for detecting anti-H9N2 antibody in animal serum.1:Prokaryotic expression and purification of H9N2 AIV nucleocapsid protein(H9N2-NP)Using the synthesized gene sequence encoding H9N2 NP protein as a template,the prokaryotic expression recombinant plasmid p ET-28a-H9N2-NP of H9N2-NP protein was constructed by PCR amplification,enzyme digestion,ligation and transformation.The positive recombinant plasmid was transformed into E.coli expression competent cell Transetta(DE3).After induced expression by IPTG,the recombinant recombinant protein H9N2-NP with expected size of 56 k Da was successfully expressed by SDS-PAGE analysis.Western blot analysis showed that H9N2-NP can react specifically with positive chicken serum.After the recombinant protein was purified by a nickel column(Ni),the recombinant protein H9N2-NP with a high purity of 3 mg/m L was obtained.2:Screening and preparation of H9N2-NP protein specific NanobodyThe purified H9N2-NP protein was used to immunize Alxa Bactrian camel.After 5immunizations,the peripheral blood of the immunized camel was collected,and the indirect ELISA was used to detect the antibody titer of H9N2-NP protein in the serum of the immunized camel reached 1:128000;Lymphocytes were isolated,total RNA was extracted,nested PCR amplification,double enzyme digestion,ligation and electrotransformation into TG1 competent cells to construct a phage library with a storage capacity of 4.9×10~8transformants.Subsequently,through phage display technology,after 3 rounds of panning,5strains of specific Nanobodies against H9N2-NP protein were successfully screened.The results of blocking ELISA showed that Nanobody H9N2-NP-Nb5 could be obviously blocked by positive chicken serum from binding to antigen H9N2-NP protein.3:Establishment and evaluation of ELISA method for detecting H9N2 antibody in serum based on NanobodyUsing the established rapid expression platform of Nanobody and HRP fusion protein,construct recombinant eukaryotic expression vector p CMV-N1-H9N2-NP-Nb5-HRP.Then,the positive recombinant plasmid was transfected into HEK293T cells,verified by IFA and ELISA,the Nanobody H9N2-NP-Nb5 and HRP fusion protein was successfully expressed and secreted into the cell supernatant,and still remained reactive with the H9N2-NP protein.Subsequently,the optimal antigen coating volume of the competitive ELISA method was determined by chessboard titration to be 100 ng/well,and the optimal dilution ratios of Nb5-HRP and the chicken serum to be tested were 1:320 and 1:10,respectively.The time was 20min and the best color development time was 10 min.The cut-off value of the competitive ELISA was 14%.It has been verified that the sensitivity of the method is 99.4%,the specificity is 100%,and the agreement rate with the existing commercial methods is 91.4%.The established competitive ELISA is suitable for monitoring H9N2 antibody in animal serum.In summary,this subject successfully screened and prepared five nanobodies against H9N2-NP protein for the first time.Then,express and prepare the fusion protein of anti-H9N2-NP protein nanobody and HRP,and use the fusion protein to establish a competitive ELISA method for detecting anti-H9N2 antibody in animals,the method is simple,time-consuming,and has good sensitivity and specificity.It can be widely used in serological detection of H9N2 AIV infection,which lays a foundation for the prevention and control of H9N2. |
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