Prokaryotic Expression Of PRRSV BJ3 Strain N And Nsp2 And Preparation Of Their Antibodies

PRRSV(Porcine reproductive and respiratory syndrome virus)mainly causes respiratory diseases of weaned piglets and fattening pigs,as well as reproductive disorders of sows.The pig industry caused great economic losses.PRRSV belongs to the arteritis virus family,arteritis virus genus,a single-stranded positive-strand singlestranded RNA virus,the genome length is about 15.4 kb,at least 11 open reading frames(ORF).The N protein is encoded by ORF7 and has a molecular weight of approximately 15 k Da.It is one of the most abundant,immunogenic,and most conserved proteins in PRRSV particles,and the host specific antibodies against this protein were first produced and exist for a long time With these characteristics of N protein,N protein has high application value in the detection of PRRSV.There are studies on monoclonal antibodies prepared against the N protein to distinguish between type I and type II strains;four antigen epitopes were identified using the anti-N protein monoclonal topic.Nsp2 is the largest and most variable protein in the PRRSV genome and is encoded by 21% to 23% of the genome.It is often used as a target for PRRSV monitoring;Nsp2 plays an important role in PRRSV replication,immunity,and host cell autophagy and apoptosis.Features.Currently,graduate students have used monoclonal antibodies prepared with Nsp2 protein to distinguish strains of different subtypes,and identified new epitopes using anti-Nsp2 protein.In this experiment,gene fragments of PRRSV BJ3 strain N and truncated Nsp2 were amplified by reverse transcription polymerase chain reaction(RT-PCR),and cloned into prokaryotic expression vector,and the recombinant protein was obtained using prokaryotic expression system;The purified recombinant protein was used to immunize BALB / c female mice and hybridoma technology to obtain anti-N protein and anti-Nsp2 protein high-immune serum and a hybridoma cell of anti-N protein monoclonal antibody.The main contents include:1.Prokaryotic expression of PRRSV BJ3 strain N and Nsp2To obtain N,Nsp2 protein of PRRSV BJ3 strain.First,the gene fragments of PRRSV BJ3 strain N(372bp)and Nsp2(1-831bp)were amplified by RT-PCR,the target gene was cloned into the cloning vector p MD-19 T,and then the N and Nsp2 genes were subcloned into prokaryotic The expression vectors p Cold I,p ET-32 a,and finally the prokaryotic expression vector was introduced into E.coli BL21(DE3)to optimize the induction temperature,induction time and final IPTG concentration,aiming to confirm the optimal expression conditions of the recombinant protein.Induction time and IPTG.Under the condition of constant final concentration,change the induction temperature,the induction temperature is set to 18 ?,25 ?,37 ?;under the condition of the induction temperature and the final concentration of IPTG,change the induction time,the induction time is set to 2h,3h 4h,5h;change the final IPTG concentration under the conditions of constant induction temperature and induction time.The final concentration of IPTG is set to 0.1 mmol / L,0.5 mmol / L,1 mmol / L,1.5 mmol / L.The results showed that the two recombinant prokaryotic expression vectors p Clod-N and p ET32a-Nsp2 were successfully constructed.After IPTG induced expression and condition optimization,it was determined that the recombinant protein expression was highest when the temperature was 37 ?,the induction time was 4 h,and the final IPTG concentration was 1 mmol / L.Purification and identification of recombinant proteins revealed that the two recombinant proteins mainly existed as inclusion bodies.2.Preparation of PRRSV BJ3 strain N and Nsp2 high-immune serumIn order to obtain high-immune serum against the recombinant proteins N,Nsp2 of PRRSV BJ3 strains,explore whether the obtained recombinant protein has good immunogenicity and reactive origin.First,the obtained N,Nsp2 recombinant protein was fully emulsified at a dose of 200 ?g each with an adjuvant 1: 1 to immunize BALB / c female mice,immunized three times,each time 15 d apart,after the third immunization,blood was collected from the eyeball To collect high-immune serum.The immunogenicity and reactogenicity of the recombinant protein were detected by ELISA and Western blotting.ELISA results showed that the high serum-free titers prepared with N recombinant protein and Nsp2 recombinant protein exceeded 3200,indicating that the N recombinant protein and Nsp2 recombinant protein had good immunogenicity;Western blotting test results showed that the specific recognition of N high-free serum was 16.7 The KDa band and the Nsp2 high-immune serum-specific recognition of the 31 KDa band are all in line with the expected size,indicating that the prepared N and Nsp2 proteins have good reactogenicity.The above test shows that the obtained N and Nsp2 proteins have good immunogenicity and reactogenicity,which can lay a good foundation for the next experimental study.3.Preparation of monoclonal antibodies against PRRSV BJ3 strain N and Nsp2 proteinIn order to obtain monoclonal antibodies against PRRSV BJ3 strain N,Nsp2 protein.Preparation of anti-N,Nsp2 by immunizing the obtained N,Nsp2 recombinant protein in BALB / c female mice,preparation of feeder cells,cell fusion,ELISA screening,limited dilution method,ELISA screening,expanded culture,Western blotting detection Protein monoclonal antibody.Western blotting results showed that the supernatant of anti-N protein monoclonal antibody hybridoma cells can specifically react with N protein expressed by His-tagged N recombinant protein and different type II PRRSV isolates,specifically identifying 16.7k Da,15 The k Da band was of the expected size and did not react with the His-tagged Nsp2 recombinant protein,indicating that the cell line secreted anti-N protein monoclonal antibodies and was able to recognize exogenous and endogenously expressed N protein.Western blotting results showed that the supernatant of anti-Nsp2 protein monoclonal antibody hybridoma cells can specifically react with His-tagged Nsp2 recombinant protein and Nsp2 protein of different type II PRRSV isolates,and specifically recognize 31 k Da,107 The k Da band,which was of the expected size,did not react with the His-tagged recombinant N protein,indicating that the cell line secreted monoclonal antibodies against the Nsp2 protein and was able to recognize the exogenous and endogenously expressed Nsp2 protein,but in subsequent A positive loss occurred in continuous subculture,which led to the failure of preparing hybridoma cells secreting anti-Nsp2 protein monoclonal antibodies in this experiment.IFA results showed that the cell line supernatant of anti-N protein monoclonal antibody can react specifically with the N protein of different type II PRRSV isolates.Then,the supernatant of the hybridoma cells with 13 consecutive antiN protein monoclonal antibodies was used as antibody,and the His-tagged recombinant N protein was used as the coating antigen,and the titer was measured by ELISA.400,indicating that the prepared anti-N protein hybridoma cells can stably secrete antibodies against PRRSV N protein.In summary,the PRRSV BJ3 strain N and Nsp2 prokaryotic expression vectors constructed in this experiment can well express recombinant proteins and recombinant proteins,and have good immunogenicity and reactogenicity.A hybridoma cell capable of stably secreting anti-N protein by hybridoma technology was obtained.The results of this experiment can lay a certain material foundation for the further study of the structure and function of PRRSV BJ3 strain N and Nsp2 proteins and the establishment of a rapid diagnosis of PRRSV.