Screening And Identification Of Bovine Coronavirus Single-chain Variable Fragment

Bovine coronavirus disease is an important bovine disease in the world.Its pathogens can cause newborn calf diarrhea,adult cattle winterdysentery and respiratory diseases,and often mixed with other pathogens,which has brought huge economic losses to the development of the global cattle industry.In order to prevent the widespread of bovine coronavirus disease,we need to diagnose of BCoV infection at the early stage and take corresponding control measures.At present,the main methods that can be used to detect the pathogen of bovine coronavirus are reverse transcription PCR technology and ELISA.Among them,the ELISA kit mainly relies on import,which is high in cost and inconvenient to operate,while reverse transcription PCR is only suitable for detection of a small amount of samples.In order to establish a more convenient method,lower detection cost,and more suitable for large-scale detection of new methods for detecting pathogens,we intend to express and purify BCoV N protein and construct a BCoV N protein ScFvphage display library in order to obtain specific ScFv with high binding activity,which is expected to be used in the development of new rapid detection reagents for BCoV.The contents and results of this study include:1.Prokaryotic expression of BCoV N protein and mouse immunityThe constructed recombinant expression vector pET28a-N was transformed into E.coli BL21 cells and successfully expressed.After that,we purified and identified the target protein.The results showed that the recombinant protein was about 54 kDa,which was soluble expression,and it could be specifically recognized by His monoc-lonal antibody.The purified N protein was identified by SDS-PAGE with high purity and the protein concentration was 1.5 mg/mL.We used recombinant N protein to im-munize BALB/C mice.After three immunizations,we separated the mouse serum and detected the antibody titer by ELISA.The results showed that the antibody titer could reach 1/10~5,indicating that the recombinant N protein which was prepared in this exp-eriment has a good solubility and immunogenicity.2.Construction of BCoV N phage single chain antibody library and screening of Scfv against N proteinThe spleen of the BALB/C mice immunized with N proteinthe was removed under sterile conditions,after that we extracted the total RNA from the spleen lymphocytes and transcribed into cDNA by RT-PCR.The cDNA and designed mice antibody specific primers were used to amplify the VH and VL of antibodies by PCR and assembled into ScFv with a short peptide(linker)by Overlap-PCR.The purified ScFv gene was ligated with the enzyme-digested vector and then transferred into supercompetent cells.After expansion and purification,we constructed a ScFv phage display library with 8×10~8 individual colonies.Based on the established ELISA single-chain antibody detection technology for N protein,we screened of 32 higher positive ScFv.After analyzing the sequences,19 positive ScFv genes were found to be correct.Five positive ScFv with higher positive values were selected to test their phage antibody binding activity.Among them,the positive ScFv-154 showed high reactivity to N protein,which has replaced the previous foundation for the further exploration of the application of ScFv in the diagnosis and prevention of bovine coronavirus disease.