Generation Of Monoclonal Antibodies Against CPIV, Expression And Activity Analysis Of Single Chain Fv Protein

Canine parainfluenza virus(CPIV) is one of the main pathogens of canine infectious respiratory disease, and epidemic in almost all the countries raising dogs. CPIV infection mainly causes fever, runny nose and kennel cough in dogs, which is very similar to the symptoms of dogs infected with canine distemper virus. So, generation of monoclonal antibodies against CPIV is important for producing diagnosisand treatment reagents.Monoclonal antibodies(MAbs) generated by hybridoma technology are the most popular tools for diagnosis and treatment of diseases. In this study, Babl/c mouse were immunized with concentrated CPIV and then used for MAbs generation. Single chain Fv proteins have small molecular weight and strong penetration compared with MAbs,and play an important role in parsing the antigen-antibody complexes, designing cross protection antibodies, and detecting the change of virus genotypes.In conclusion, this research set up a system of antibody genes amplification and protein expression. The active proteins of 33B5 sc Fv can used as substitutes of nature antibody to some extent, and are important for further study of antigen-antibody complex and gene engineering vaccine.The concrete content includes:1. Preparation and activity analysis of monoclonal antibodiesCPIV was proliferated in Vero cells as references and concentrated by PEG6000. Titer of CPIV measured by TCID50 was increased from 2.6×105 PFU/ml to 7×106 PFU/ml after concentration. Haemagglutination litre of CPIV was measured as 23 by 1% of pig red blood cells agglutination assay.Immunized 6-week old Babl/c mouse with concentrated virus protein emulsified with Freund’s Adjuvant, and hybridoma fusion test was carried out when the antibody titer of mouse serum reached to 1.0 by ELISA assay. At last, we received 6 stable hybridoma lines after three subcloning and named as 13E8, 33B5, 51D8, 51E6, 51G12, and 54D10 separately. After assays of IFA and westernblot, we determined that antibodies secreted by13E8 and 51D8 were combined with P protein and antibodies from 33B5, 51E6, 51G12,and 54D10 were combined with antigen sites of N protein. To further determine the specific region of N protein these antibodies reacted with, recombinant plasmids were constructed, and then expressed different region of N protein(1-509, 40-509, 204-509, and40-375) in 293 T cells. Results of IFA assays showed that antibodies from 33B5 and 51G12 were reacted with antigen region in 375-509 aa of N protein, but antibodies secreted by54D10 and 51E5 were reacted with antigen region in 1-40 aa of N protein.2. Amplifying variable genes, expressing antibody proteins and analyzing activityWe successfully amplified antibody genes of 33B5, 51D8, and 51E6, and expressed by prokaryotic plasmid of p ET42 b. Results showed that all of the three proteins were expressed in inclusion bodies. In order to receive active protein, we chose variable genes of 33B5 for optimizing expression including: expressing different region of antibody,adding soluble tag, changing plasmids and strains for expression, and adjusting expression time, temperature and concentration of IPTG. Unfortunately, there was no use.All of the antibody proteins expressed in prokaryotic forms were inclusion bodies and in high quantity, but 33B5 sc Fv expressed in sf9 cells were enclosed in or on cell membrance and in low quantity.Finally we generated active protein by denaturation with urea and renaturation with gradient dialysis. Assays of ELISA, IFA, and Westernblot all confirmed the renatured proteins are specificity in antigen binding ability with nature MAbs.