Study On Antibody Spectrum Of Influenza Virus Based On Phage Display Technology

As an important zoonotic disease,avian influenza virus(AIV)has attracted widespread attention.Moreover,AIV can spread widely and uncontrollably through migratory activities of migratory birds,making the epidemic rage around the world and causing huge losses.At the same time,highly pathogenic avian influenza virus subtypes such as H7N9 and H5N1can also infect humans,threatening the safety of human life.Avian influenza virus has many subtypes and high variability,which makes surveillance,detection,vaccine development and disease prevention and treatment more difficult.At present,the conventional detection methods of avian influenza are not only limited to the sampling site,but also can only detect one known virus at a time.It can only realize one-to-one detection of antigen and antibody.It is difficult to achieve a wide range of detection.So it is urgent to establish a simple,high throughput and high specificity method for detection of avian influenza virus.An antibody spectrum scanning technique was established to detect avian influenza virus in this paper.This method is an organic combination of phage display technology,immunoprecipitation and high-throughput sequencing analysis.Using the characteristics of T7 bacteriophage,we constructed a T7 bacteriophage library which displays antigenic variability gene fragment of avian influenza virus.The bacteriophages that could bind to the antibodies in the samples were isolated by immunoprecipitation.After DNA of the captured bacteriophages was extracted,the sequence displayed by T7 bacteriophage was obtained by high-throughput sequencing,and then the genus of the viral fragments displayed by T7bacteriophage was analyzed,so as we can achieve the purpose of detecting avian influenza virus.This method has high sensitivity,strong specificity and wide coverageand.It not only can detect virus although the virus is very low or cleared in the body,but also can detect all kinds of avian influenza at once time.In this way,a large number of poultry and wild birds can be detected,early warning can be carried out according to their infection status,and timely preventive measures can be taken,so as to cut off the spread of avian influenza virus from the source of infection,reduce the risk of human disease and reduce economic losses.The results showed that by searching avian influenza virus antigen fragment in the Gene Bank,modifying and synthesizing,amplifying by PCR,fragment inserting DNA and vector DNA by EcoR I,Xho I double digestion,T4 ligase ligation and recombinant bacteriophage packaging in vitro,the T7 bacteriophage display avian influenza virus antigen variability gene library was successfully constructed.After identification,the library capacity of T7phage display library was 8×10~9 pfu,the titer of T7 phage display library was 4×10~8 pfu/mL.The 100 phage plaques selected randomly were recombined effectively,and the displayed protein also has the ability to bind with corresponding antibodies.Through the actual detection of serum samples of sentinel animal,it was found that the serums of Sentinel Mallard ducks in net Lake Hubei Province contained antibodies against H5N6 subtype avian influenza virus,which proved that the mallard duck in this area had been infected with avian influenza virus of H5N6 subtype.It is proved that this method can be used to detect avian influenza virus antibody in high throughput.