Study On Rapid Detection Method Of Avian Influenza Based On Excessively Tilted Fiber Grating Graphene Biosensor

Avian influenza(AI)is a systemic or respiratory organ-borne disease caused by Influenza A virus.The World Organization for Animal Health(OIE)has defined it as a category A infectious disease,which seriously harms the poultry industry.Moreover,it poses a huge threat to public health,and the state has taken AI prevention and control as an important task.At present,the main detection technologies for avian influenza include hemagglutination-hemagglutination inhibition(HA-HI)test,enzyme-linked immunosorbent assay(ELISA),polymerase chain reaction(PCR),and colloidal gold.The first method is sensitive,but it is greatly affected by external factors which reduces the practical value of this method.ELISA is suitable for large-scale serological investigation,but it also has disadvantages including long time consumption,low specificity,complex process,and inconvenient on-site operation.although PCR can directly detect the virus gene,but the equipment required is expensive and has been greatly restricted in clinical testing.The colloidal gold method has been available at home and abroad,but its detection sensitivity is not high,it can only be qualitative and cannot be quantified.This method can only be used for aided detection for the diagnosis of disease.Therefore,exploring efficient,specific and accurate detection methods is of great significance for the prevention and control of avian influenza.The excessively tilted fiber grating(Ex-TFG)immunosensor has the advantages of high detection sensitivity,good specificity,label-free,portable device and low cost,and has potential for clinical detection.Previously,our laboratory has successfully applied Ex-TFG immunosensors to porcine circovirus type 2(PCV2),Newcastle disease virus(NDV),heart failure biomarker N-terminal pro-nasal natriuretic peptide(NT-proBNP),calcium reduction The detection of primordial(PCT)and programmed death ligand 1(PD-L1)combines the specificity of antigen-antibody reaction with the sensitivity of FBG sensor detection,and has made breakthroughs in detection sensitivity and published High-level research papers.In this paper,we prepared avian influenza virus HA antigen by prokaryotic expression system,and prepared monoclonal antibodies against HA by PEG1500 cell fusion technology.After modifying the macroscopic angle fiber,the GO dispersion was coated to make the GO uniformly fixed on the surface of the fiber to form a coating.Layer,using EDC and NHS to activate the coating carboxyl group(-COOH),blocking the prepared blocking solution to block unbound sites,detecting experimental samples such as different titer avian influenza virus standard solution,and repeating the specificity of the sensor,clinical sample testing,etc.for detailed verification.Therefore,an avian influenza virus immunosensor based on Ex-TFG was initially established.The sensor has the advantages of high specificity,low detection cost and short time consumption,and provides a novel method for clinical detection of avian influenza virus.The content of this paper is as follows:1.In order to realize H5N1 hemagglutinin(HA)protein high expression in Ecoli,this paper aimed to construct the recombinant plasmid with H5N1 HA gene and prepare the HA protein by prokaryotic expression system.According to the H5N1-HA(ID=DQ023145.1)gene sequence published by GenBank,without changing the amino acid sequence of the HA protein,DNAWorks2.4 software was used for codon optimization,and the whole HA gene was synthesized by chemical method.The synthesis was carried out by Shanghai Biotech and sequenced.The enzymes were designed in the 5' and 3' segments of the HA gene.Cut sites BamHI and EcoRI.The synthesized HA gene was digested with BamHI and EcoRI,and the digested product was recovered and inserted into the pET28a(+)vector which was also digested with BamHI and EcoRI,and the HA gene was cloned into pET28 a using the DNA Ligation Kit.In the vector,the recombinant plasmid pET28a(+)/HA was constructed.The recombinant plasmid was transformed into E.coli BL21(DE3)sensory cells,and various expression factors(temperature,inducer concentration,time)were optimized,and the optimal induction conditions were screened for large-scale expression.The recombinant protein was purified by His-tag nickel column,and the purified recombinant protein was analyzed by SDS-PAGE purity analysis,bisquinolinecarboxylic acid(BCA)concentration determination and Western-blot specific identification.2.The H5N1-HA protein prepared above was used as immunogen,and BALB/c mice were immunized by long-term immunization method.The spleen of the immunized mouse spleen was prepared into a spleen cell suspension,and then mixed with myeloma cells,and PEG1500 was used as a fusion.The agent is subjected to cell fusion;the antibodies in the cell supernatant after the multi-cloning and the subcloning are screened by indirect ELISA,and the selected positive wells are subcloned at least twice or more to obtain a stable antibody-secreting single.The positive hybridoma cells were cloned,the cultured cells were expanded,the monoclonal antibody ascites was prepared,the antibody was subtyped,and the corresponding purification method was selected according to the antibody subtype,and the purified antibody was subjected to biological characterization.3.After the outer membrane protective layer was scraped off on the surface of the extreme angle fiber grating(Ex-TFG),the fiber was cleaned with 5% diluted HNO3;after surface hydroxylation modification with 8 mg/mL NaOH solution,the surface of the fiber was made.With a hydroxyl group(-OH),the GO dispersion is uniformly coated,and the coating is observed by an electron microscope after drying and fixing treatment.The good water solubility of the graphene is extremely favorable for wrapping around the optical fiber to form a dense film layer.After well-coated,the avian influenza monoclonal antibody was immobilized and the superfluous site was blocked,and the avian influenza virus was detected,and the biosensitivity sensor's repeatability,specificity and clinical application were evaluated.Results:1.The recombinant strain pET28a(+)-HA/BL21(DE3)had the highest expression of HA protein when induced at 30 °C and IPTG concentration of 1 mmol/L for 8 h.SDS-PAGE electrophoresis showed that the expression of supernatant was obvious after bacterial cell lysis.Higher than the precipitate,indicating soluble expression,the purity of the HA protein purified by nickel column affinity chromatography was higher,reaching more than 90%.Western blot showed that there was a significant Western blot at 70 KD,which was consistent with the expectation,indicating success.High expression of HA protein in E.coli was achieved.2.HA monoclonal antibody was prepared,and a cell fusion was performed.Four plates were plated in a 96-well plate,and the number of clone growth was 380 wells,and the fusion rate was 98.95%.The number of positive wells was 323 wells,and the positive rate was 84.1%.After 3 subclones and screening,4 monoclonal antibody cell lines with anti-avian influenza HA were obtained,named 1H8-F12,4G1-G11,4G1-E12,4G1-B11;1H8-F12 after subtype identification,4G1-G11 is IgG2 b type antibody,4G1-E12 is IgG2 a type antibody,4G1-B11 is IgG1 type antibody;4 hybridoma cell lines are repeatedly frozen,Resuscitation and multiple passages can secrete high titer antibodies;Western-blot identification confirmed that the obtained monoclonal antibody is specific to avian influenza virus HA antigen;1H8-F12,4G1-G11 cells were intraperitoneally injected into BALB/c mice Ascites was collected and purified by ProteinA column.SDS-PAGE electrophoresis showed that the purity of the purified antibody reached more than 90%.The highest peak of the same batch was obtained,and the highest concentration was obtained by BCA method.2.6637 mg/mL.3.A series of experiments were conducted on macroscopic fiber hydroxylation modification,GO coating,coating activation,antibody immobilization,blocking,virus detection,reproducibility,specificity,clinical sample detection,etc.were carried out,and the test analysis was carried out.: The resonance wavelength shifts after surface hydroxylation modification,coating of graphene oxide,coating activation,and binding to avian influenza antibody were: 0.027 nm,0.065 nm,0.022 nm,and 0.096 nm,respectively,indicating that the modification was successful..The minimum detection limit(LOD)of the immunosensor for AIV is 0.1 ng/mL(1.43 pM),and the linear range of detection is 0.1-5000 ng/mL,which reaches the clinical detection level;the results of the repetitive experiment are basically the same,indicating that the sensor The repeatability was good;two different avian virus stocks and AIV blank allantoic fluid of NDV allantoic fluid and AIV allantoic fluid were tested and analyzed.The results showed that the sensor has high specificity for AIV and can meet the requirements of clinical testing.Conclusion:In this study,the recombinant pET28a(+)-HA/BL21(DE3)Ecoli was constructed,and the optimal expression conditions were screened to induce the expression of HA protein in large quantities.The H5N1-HA protein was highly expressed in E.coli,and the recombinant HA protein was prepared and purified.Four hybridoma cell lines stably secreting monoclonal antibodies were obtained.With the prepared antigen and antibody,the immunosensor based on GO integrated Ex-TFG was preliminarily established which was used to detect avian influenza virus.The recombinant HA antigen and monoclonal antibodies against HA prepared in this study will provide effective reagents for the diagnosis of avian influenza.The developed sensor has high detection sensitivity,good specificity and reproducibility,and can be used for real-time quantitative detection of clinical samples,which provides a novel detection method for the prevention and control of avian influenza.