Production Of Nanobodies Against Porcine Parvovirus VP2 Protein And Its Application In Virus Detection

Sows infected with Porcine Parvovirus(PPV)in pregnancy early can cause reproductive failure,leading to stillbirth,miscarriage,and mummified fetuses.This disease has spread widely around the world,causing serious economic losses to the world’s pig industry.However,the lack of PPV detection methods which have high sensitivity and specificity,simple operation and low production cost seriously hinder the development of comprehensive prevention and control technology for the disease.The VP2 protein is the main component capsid protein for the PPV,which contains the major antigenic epitopes of the virus and has good immunogenicity,it is the main target antigen for developing PPV detection technology.Nanobody is a special IgG that deletion light chain and first constant region(CH1)of heavy chain in camelids.It has a small molecular weight and is known to the smallest antibody fragment that can bind intact antigen.Compared with traditional IgG in mammals,it has the advantages of structural stability,easy genetic engineering and low production cost.At present,nanobodies have been widely used for diagnosing different diseases based on above advantages,and it shows the characteristics of high sensitivity,specificity and low production cost,and has a good market application prospect,but the method of detecting PPV using nanobodies has not been reported.Therefore,based on the characteristics of VP2 protein and Nanobody,this study firstly expressed PPV VP2 protein prokaryotically,and used phage display technology to screen and prepare Nanobody against the protein,and then used the nanobody to establish a method for detecting PPV,this method laid the foundation for the development of new diagnostic technology for PPV.The main contents and results of this study are as follows:1.The recombinant plasmid pET-28a-VP2 was successfully constructed.Then VP2 protein were co-expressed with the chaperone protein Tf16 in the host strain BL21(DE3).SDS-PAGE analysis indicated that VP2 recombinant protein was successfully expressed(70kDa)and soluble expression;finally,VP2 recombinant proteins were purified with Ni-NTA column and obtain a higher purity protein,the yield is about 20 mg/L.2.The purified VP2 recombinant proteins were used to immunize 6-week-old Bactrian camel as an immunogen.After 5 times immunization,the titer of specific antibodis against VP2 protein reached to 1:512,000 in the camel serum;the Bactrian camel peripheral blood was isolated after 5 days the last immunization.A Bactrian camel heavy chain antibody variable region phage display library with a capacity of 3.15×109 were constructed by nested RT-PCR,restriction enzyme digestion,ligation and transformation.The VP2 recombinant protein was used as a screening antigen.After three rounds screening,five kinds of nanobodies against VP2 protein were obtained.Finally,it was found that Nb5,Nb12,Nb18,Nb19 and Nb56 could bind with natural PPV virus by immunofluorescence.3.The screened nanobodies were expressed using eukaryotic expression and prokaryotic HRP fusion expression,respectively,and then detecting PPV virus by paired assay,the results showed that Nb19 as a capture antibody with prokaryotic expression and eukaryotic expression of Nb56-HRP as a detection antibody was best.And then the "Nb I-Antigen-HRP(?)Nb Ⅱ" sandwich ELISA method for detecting PPV virus was successfully established by optimizing the reaction conditions.The minimum amount of PPV detected by this method was 100 TCID50.In summary,this study for the first time to prepare specific nanobodies against PPV VP2 protein,and successfully expressed Nanobody-HRP fusion protein anti-VP2 protein,and successfully established a double-nanobody sandwich ELISA for detecting PPV virus.This method shortens the detection time,simplifies the operation,and saves the use of HRP-labeled secondary antibodies.This study laid the foundation for the establishment of rapid detection method for PPV virus and the study of VP2 protein function,and provided materials for the development of the virus infection prevention and control technology.