Prokaryotic Expression Of Nucleocapsid Protein Of Bovine Parainfluenza Virus Type 3 And Establishment Of Indirect ELISA

Bovine parainfluenza virus type 3(BPIV3)is an non-segmented,negative-sense,single-stranded RNA virus.It is widely distributed in the world and can cause bovine respiratory syndrome(BRDC)with other pathogen.The symptoms of BPIV3 infection in cattle are different.Mild cases of cattle have cough,fever and other symptoms.When it was severe,there will be secondary bacterial infection,leading to bronchial cell necrosis and then cause pneumonia.The further development of cattle raising industry has been severely restricted,which has caused serious losses to the industry.Based on the current domestic research on BPIV3,and there are no particularly effective commercialization detection kits.Although there are testing kits abroad,the testing cost is high.Therefore,it is very important to establish a serological test method of BPIV3 with independent intellectual property rights.On the other hand,the study of BPIV3 infection is inseparable from the study of mechanism.The detection of BPIV3 mechanism and gene function requires quantitative detection of protein expression,however corresponding antibodies are lacking.It has been found that the N protein of BPIV3 is the main component of the nucleocapsid of the virus,which has good immunogenicity and can be used to prepare detection antibodies.The experiment mainly includes three aspects.First,the construction and purification of N protein prokaryotic expression vector were carried out.According to the literature report and bioinformatics analysis of N gene,the dominant antigen region of N protein 388 aa~500 aa was amplified to express N protein.The prokaryotic expression vector pET-32a-N was successfully constructed.It was found that N protein mainly existed in soluble form.It was high expression when the IPTG was 1 mM and the induction time was 8 h.Ni-NTA affinity chromatography was used for purification.The protein was purified well when NPI 70 be used,and the concentration of N protein was about 0.6mg/mL.Next,we prepare polyclonal antibody of N protein.Kunming mice were immunized with the mixture of the prepared N protein and Freund's adjuvant 1:1.After the first immunization,the mice were immunized twice,and then get the serum of mice.MDBK cells infected and uninfected with BPIV3 were detected with this serum.It was found that there was a band around70 kDa in the infected cells compared with uninfected cells,which consistent with the expected molecular weight of N protein.It proved that the polyclonal antibody can be used to detect the N protein of BPIV3 basic theory.Last,by using purified N protein as envelope antigen and optimizing the conditions,an ELISA method for detecting BPIV3 was established.The optimized conditions were as follows: Antigen concentration was 1.4 ?g/mL and should be coated at 37 ? for 2 hour;10%horse serum cultured at 37 ? for 1.5 hours;Serum dilution of 1:50,incubation at 37 ? for 1h;the second antibody was diluted with 1:2000,culture for 1 hour;TMB coloration for 15 min.The established ELISA method has good repeatability and specificity,and can be used for the preliminary detection of BPIV3.According to the established method,bovine serum was detected and compared with the imported kit,the total coincidence rate was 90.2%.The positive rate of bovine serum from Tianjin was up to 55.3%,the situation of BPIV3 prevention and control in this cattle farm is severe.In this study,polyclonal antibodies prepared with BPIV3 N protein can provide support for the basic theoretical research of BPIV3.The ELISA method established by N protein can reduce the detection cost to a certain extent and lay conditions for the development of detection kit.