Development of Indirect Enzyme-Linked Immunosorbent Assay Based on the Dimeric Nucleocapsid Protein for Detecting Antibodies against Porcine Reproductive and Respiratory Syndrome Virus
In order to prepare N protein with good stability and immunoreactivity,N-dimer was designed and expressed,and an iELISA detection method for PRRSV antibody was established based on this protein.Firstly,the NADC30-like N protein gene sequence of the current dominant PRRSV strain in China was selected,and the N protein gene sequence was concatenated through a(GGGGS)3 linker.After codon optimization and synthesis,an N-dimer recombinant expression vector was constructed.The recombinant protein was expressed using prokaryotic expression system and purified by nickel ion chelation affinity chromatography.Its reactivity with PRRSV positive and negative sera was analyzed by Western-blot.Its stability after freezing and thawing and immunoreactivity were further compared with those of N-monomer.An iELISA was subsequently developed based on N-dimer as coating antigen,and evaluated for the specificity,sensitivity,and repeatability.The developed iELISA was finally applied to clinical serum sample detection.The results showed that under the conditions of 16 C and 0.2 mmol/L isopropylthiogalactoside,N-dimer was obtained with a large amount of soluble expression after 16 hours of induction.Western-blot results showed that N-dimer could specifically react with PRRSV positive serum;Compared to N-monomer,N-dimer coule be stably stored and had better immunoreactivity.We successfully established a PRRSV antibody iELISA detection method based on N-dimer,which had good specificity,sensitivity,and repeatability;Compared with the results of commercial test kits,the positive agreement rate was 96.88％,and the overall agreement rate was 96.86％,indicating a high agreement rate.In summary,this study successfully prepared N-dimer with good stability and excellent immunoreactivity;A PRRSV antibody iELISA detection method was established based on N-dimer.
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维普
