Preparation Of Nanobody Against Porcine Circovirus Type ? Cap Protein And Establishment Of A Competition ELISA Method

Porcine circovirus associated disease(PCVAD)is the related diseases caused by porcine circovirus infection.5-18 weeks old piglets are susceptible,and the infected pigs often have clinical symptoms such as progressive weight loss,developmental delay,jaundice,and swollen lymph nodes.Because immunosuppression can be caused by circovirus infected,the infected piglets were mainly manifested as postweaning multisystemic wasting syndrome(PMWS).It also causes various diseases such as porcine dermatitis and nephropathy syndrome(PDNS),porcine respiratory diseases,congenital tremors,reproductive disorders syndrome,fetal myocarditis,and reproductive failure.Since its discovery in 1974,the disease has spread widely around the world,causing serious economic losses to the world's pig industry.Cap protein,the major structural protein of PCV2,contains the major antigenic epitope of the virus,and has good immunogenicity to stimulate neutralizing antibodies.Nanobody is a special Ig G in camelids naturally missing light chain and the first constant region(CH1)of heavy chain.It has a small molecular weight and is known to the smallest antibody fragment that can bind intact antigen.Compared with traditional Ig G in mammals,it has the advantages of stability structure,easy genetic engineering and low production cost.At present,nanobodies have been widely used for diagnosing different diseases based on its advantages,and it shows the characteristics of high sensitivity,specificity and low production cost,and has a good market application prospect.In this study,specific nanobodies against PCV2 Cap protein were screened by phage display technology,and a competitive ELISA method based on the screened nanobody for rapid detection PCV2antibody was established.The main contents and results of this study are as follows:1.Camel serum Ig G was purified,and rabbit anti-camel Ig G antiserum was obtained by immunization adult rabbits with purified camel serum Ig G,which provided materials for titer detection in the preparation of nanobodies.2.Adult male Bactrian camels were immunized with purified Cap protein.After 4immunizations,the titer of the serum against Cap protein reached 1:1,024,000;One week after the last immunization,the peripheral blood lymphocytes of the camels were isolated and the VHH phage display library with 5×10~6 capacity and abundant diversity was constructed by nested RT-PCR,restriction enzyme digestion,ligation and transformation.Then,the recombinant Cap protein was used as a screening antigen,and 19 Cap protein-specific nanobodies were obtained after 3 rounds of screening.3.The screened nanobody fusion with HRP was expressed using eukaryotic expression system to provide materials for the establishment of a rapid competition ELISA assay based on nanobody.The optimal antigen coating concentration was 1?g/m L,the optimal dilution of the expressed nanobody was 1:400,the dilution ratio of swine sera was 1:10,and the reaction condition was at 37°C for 30 min.The positive and negative sera were used to evaluate the method.Sensitivity and specificity were 99.1%and 100%,respectively.The repeatability was evaluated by intra-plate and inter-plate repeated test,and the coefficient of variation of intra-plate PI value was 1.3%-7.4%,and the coefficient of variation of inter-plate PI value was 3%-7.23%,which means the repeatability of the method is good.By comparing the test results with commercial kits,the coincidence rate can reach 99.2%.The results of cross reaction test with other positive sera showed that no cross reaction occurred with other positive serums.In summary,specific nanobodies against PCV2 Cap protein were screened and prepared,and anti-Cap protein nanobody fusion with HRP was expressed using eukaryotic expression system.A competitive ELISA detection method based on eukaryotic expressed nanobody for detection PCV2 antibody was established.In this method,the use of HRP-labeled secondary antibodies is eliminated,the detection time is shortened,and the operation is simplified.The established method can be used for rapid detection of PCV2 antibody levels.