Genetic Modification Of M13 Phage And Its Application In Ultrasensitive Detection Of Avian Influenza Virus

Avian influenza virus(AIV)belongs to the Orthomyxoviridae family of influenza A virus.According to the difference of the glycoprotein hemagglutinin(HA)and neuraminidase(NA)on its surface,it can be divided into 16 HA subtypes and 9 NA subtypes.In recent decades,highly pathogenic AIV H5N1 and H7N9,as well as low pathogenic AIV H9N2,have spread widely in China,causing huge economic losses to the poultry industry and posing a potential threat to public health.Although H9N2 is a low pathogenic AIV,its host range is becoming wider,virulent,and even the possibility of infecting humans,thus it is crucial for the ultrasensitive detection of H9N2 AIV.Common methods used for AIV detection include antigen hemagglutination titer determination,polymerase chain reaction(PCR),quantitative polymerase chain reaction(PCR),test strips,enzyme-linked immunosorbent assay(ELISA),etc.These methods have advantages,however none of them have the three advantages such as easy-to-operate,time-saving,and highly sensitive.Surface plasmon resonance(SPR)biosensor is an emerging detection instrument developed in the past three decades.It has the advantages of simple operation,fast analysis and high sensitivity.However,SPR is not sensitive enough to analyze samples from early infection with pathogens.Therefore,current research on SPR technology focuses on improving its sensitivity.M13 phage is a filamentous virus particle with 880 nm in length and 6 nm in diameter.The capsid protein of M13 phage have the ability to display exogenous protein molecules,therefore,it is widely used in the construction of phage display libraries for screening polypeptides or antibodies with specific affinity to target molecules.M13 phage particle is stable at a temperature below 80℃ or pH 3.0-11.0 for a long time.In addition,the M13 phage can be genetically engineered to bind to metal materials.These advantages enable M13 phage the potential as an excellent biological probe for detection of cells,pathogens and biomarker molecules.In this study,a phage composite probe with surface-modified Au nanoparticles(AuNPs)was obtained by genetically engineering of M13 phage,and the N-terminal of p Ⅲ protein displayed the target molecule affinity peptides.This probe was used for SPR biosensor,and an enhanced surface plasmon resonance(ESPR)method based on M13 phage probe for ultrasensitive detection of H9N2 AIV.1.Genetic modification and characterization of M13 phageM13 phage is composed of five capsid proteins:p Ⅲ,p Ⅵ,p Ⅶ,p Ⅷ and p Ⅸ,among which the major capsid p Ⅷ contains about 2700 copies,and the rest is about 5 copies of the minor capsid protein.p Ⅷ and p Ⅲ are located in the body and tip of M13 phage,respectively.According to the receptivity of different proteins to foreign genes,8 peptides of Au binding peptide(AuBP)and 7 peptides of H9N2 binding peptide(H9N2BP)were selected to be displayed on the recombinant M13 phage.Using the M13 phage as a vector,AuBP was displayed on the N-terminus of p Ⅷ and H9N2BP on the N-terminus of p Ⅲ by homologous recombination technology to construct a dual-affinity recombinant phage M13@H9N2BP@AuBP that can bind to both AuNPs and H9N2 AIV.The ELISA results showed that the recombinant phage had a high specific affinity for H9N2 AIV.At the same time,a qPCR method was also established to accurately quantify M13 phage.2.Characterization of recombinant M13 phage specifically binding to AuNPs and H9N2 AIVWith genetic modification,the recombinant phage M13@H9N2BP@AuBP has the ability to bind amphiphilic AuNPs and H9N2 AIV.In order to explore the effect of AuNPs with different diameters on the modification of M13 phage p Ⅷ protein,AuNPs with diameters of 2 nm,5 nm and 15 nm were screened for binding.Results by macroscopic characterization,UV-Vis,TEM,etc.shows that 5 nm AuNPs are more suitable for M13@H9N2BP@AuBP modification.TEM results showed that the modified AuNPs probe M13@H9N2BP@AuNPs still had good H9N2 AIV binding properties.In addition,the probe can be stored stably at pH range of 4.0-10.0,and for 15 d at room temperature and at least 21 d at 4℃.Therefore,the M13@H9N2BP@AuNPs probe is stable and specific and can be used as a good probe for the detection of H9N2 AIV.3.Establishment of M3 phage-based ESPR ultrasensitive detection of H9N2 AIVSPR is one of the commonly used techniques for studying protein-protein interactions and quantitative studies,as well as determining their equilibrium and kinetic parameters.In recent years,it has also been employed for detection of pathogens.Traditional SPR can achieve label-free detection,but its sensitivity is relatively low.Combining specific probes with the traditional SPR can significantly improve the detection sensitivity.In this study,the probe M13@H9N2BP@AuNPs was used to establish an ESPR method with good correlation.The standard curve equation is y=70.4691n(x)+25.066,where y refers to the resonance unit(RU)value,x refers to the concentration of H9N2 AIV,the correlation coefficient of the established standard curve was R2=0.9907.When ESPR is used to detect H9N2 AIV,it will cause a larger polarization phenomenon,resulting in a larger RU change.This probe method can detect H9N2 AIV with a limit of detection(LOD)as low as 6.245 copies/mL,and the detection time of a single sample is less than 10 min.The LOD of traditional SPR is about 116.134 copies/mL,and the sensitivity of ESPR method is about 18.5 times more sensitive than that of the traditional SPR one.When comparing our ESPR with qPCR,the detection of high concentration samples,the results showed that the sensitivity of two methods is comparable;for the detection of samples at very low concentration,the result by qPCR were negative,while the result by ESPR were positive.4.M13 bacteriophage ESPR probe for detection of early infected samples with AIVThe established ESPR method based on M13 phage was further validated by clinical detection.Mainly by inoculating SPF chicken embryos with H9N2 AIV at a gradient concentration of 101-104 copies/μL and setting an infection time of 4-48 h as a detection variable,the collected chicken allantoic fluid samples were assayed with common laboratory methods.Objectively compare ESPR method with antigen hemagglutination titer determination,PCR,qPCR,test strip and other methods in terms of sensitivity,detection time,operation steps,etc.The results show that the test strip and ESPR test take the shortest time,and it only takes about 10 minutes to obtain the test result;the detection steps from simple to complex are:test strip test,ESPR,chicken embryo hemagglutination titer determination,qPCR and qPCR;the order of sensitivity of these techniques from low to high is:chicken embryo hemagglutination titer determination<test strip detection<PCR<qPCR<ESPR.ESPR can detect H9N2 AIV low-concentration infection and early infection,which is expected to be applied to rapid screening of clinical samples.