Preparation Of Monoclonal Antibody Against Duck Circovirus Cap Protein And Immunocolloidal Gold Test Strip

Duck Circovirus(DuCV)is a single stranded circular DNA virus.DuCV was found in Germany for the first time,and DuCV was found in Ducks in China in 2005.DuCV can destroy the immune system of ducks,cause immune suppression of ducks,and lead to reduced immunity of the body,thus causing secondary infection of other pathogens,increasing the probability of double and multiple infections or aggravating other diseases,resulting in the death of ducks.In recent years,the infection rate of DuCV in duck flocks is on the rise,and the harm to large-scale breeding farms is more and more serious.Therefore,DuCV has become one of the important viruses that harm duck farms.However,there is still a lack of a rapid and effective detection method for DuCV.Colloidal gold immunochromatographic strip has the advantages of rapid diagnosis,high specificity,simple operation,and so on.It is very suitable for the use of rapid detection method of clinical sample.DuCV Cap protein is known to be an important target protein for DuCV antigen and antibody detection.Therefore,in this study,Cap protein was used as the target protein to prepare monoclonal antibody and develop colloidal gold immunochromatographic strip for rapid detection of DuCV,providing important technical support for rapid diagnosis and prevention of DuCV.In this study,Nuclear localization signals(NLS)at the N-terminal of DuCV Cap were removed,that is,the 5-terminal 1-108 bp region of Cap gene was removed and the remaining109-774 bp region was retained.His tag was introduced at the N-terminal to facilitate antigen purification.Finally,the nucleic acid sequence was cloned into the E.coli expression plasmid p ET-28 a.The successfully constructed plasmid was named p ET-28a-Cap and transformed into E.coli strain BL21.The high expression of recombinant Cap protein was obtained.By optimizing the expression conditions,the recombinant protein was finally induced at 37? for 3h in IPTG with the final concentration of 0.1 m M,which was the best induced expression condition.The recombinant protein was mainly expressed in E.coli in the form of inclusion body.Two Balb/c female mice were immunized with recombinant DuCV Cap protein emulsified with Fredrin's adjuvant.After three times of immunization and one enhanced immunization,serum antibody titer of the two mice was determined to be1:20,480 by ELISA,which met the requirements before cell fusion.Spleen cells of the mice were fused with mouse myeloma cells.ELISA method was used to screen positive pores that could secrete specific antibodies against DuCV Cap protein.A hybridoma cell line 6A1 that could secrete stably anti-Duc V Cap protein was finally obtained.Western Bolt results showed that monoclonal antibody 6A1 strain could specifically bind to both recombinant DuCV Cap protein and natural Cap protein.The Ig G type of monoclonal antibody 6A1 was identified as Ig G1 subclass and Kappa light chain.Then the hybridoma cells were intraperitoneously injected into female Balb/c mice to produce ascites,and monoclonal antibodies were prepared in large quantities.ELISA results showed that the antibody titer of ascites in mice reached 1:102400.At the same time,two healthy New Zealand rabbits were immunized with recombinant DuCV Cap protein prepared with Freund's adjuvant to prepare polyclonal antibodies against DuCV Cap protein.After three times of immunization,the titers of ELISA antibodies were all 1:16,3840.Western blot analysis showed that,the rabbit polyclonal antibody can specifically bind to both recombinant DuCV Cap protein and natural Cap protein.High purity murine monoclonal antibody and rabbit polyclonal antibody against DuCV Cap protein were obtained by purifying mouse ascites and rabbit serum with Protein A+G.Purified rabbit anti-Ducv Cap polyclonal antibody was used as the test line(2 mg/m L),purified mouse 6A1 monoclonal antibody(64 ?g/m L,p H 8.0)was used as the gold standard antibody,goat anti-mouse Ig G(1 mg/m L)was used as the quality control line,and colloidal gold test strip was assembled.The DuCV antigen colloidal gold immunochromatographic strip did not cross react in the detection of MDPV,GPV,DPV,NDV,AIV,DHV,FAd V and DRV,the specificity of the strip was good,and the sensitivity of antigen detection was up to15.6 ?g/m L.In the detection of 39 suspected sick duck samples,the coincidence rate of the colloidal gold strip and PCR two methods was 94.9%,the detection results of high accuracy,at the same time,the strip has good repeatability and stability,can complete the sample rapid detection within 10 min,the results show that,The colloidal gold immunochromatographic antigen detection strip based on DuCV Cap protein monoclonal antibody can detect DuCV in clinical samples quickly and specifically,and can be used for primary diagnosis and screening of DuCV.