In Vitro Selection Of DNA Aptamers To PRRSV Nucleocapsid Protein By SELEX

Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV), commonly known as "blue ear disease". The disease was reported in the United States in1987at the earliest and the virus was isolated in the Netherlands in1991, Harbin veterinary research institute in China was first reported in1996and isolated pathogens. At present, the disease is prevalent in many pig-breeding countries all over the world, resulting in a huge economic loss for global pig industry. PRRSV is positive single-stranded RNA viruses, belongs to the family Arteriviridae, order Nidovirales. The mature virus particle diameter about50-60nm, enveloped. Genome is about15kb and contains nine open reading frames (ORFs). ORF7encodes a15kDa nucleocapsid (N) protein which yields an icosahedral core structure. The N protein is the more abundant protein in the virus, highly immunogenic with4-5valuable epitopes, Therefore very suitable for disease diagnosis.Systematic evolution of ligands by exponential enrichment technology (SLEXE), a new type of combinatorial chemistry technology, is produced in the nineties of the last century. High affinity and specificity ligands of aptamer can be screened from a mass random oligonucleotide library by using the SELEX technology.In this study, we screen ssDNA aptamers specific for PRRSV N protein by SELEX technology, by measuring the affinity and specificity of aptamers with N protein to assess the application value of early diagnosis of PRRSV. The main contents include:1. Expression and purification of PRRSV proteinpET-30a (+)-PRRSV-N prokaryotic expression plasmid preserved in our laboratory, was transformed into E.coli BL21(DE3) and was induced by IPTG to express the6his-N protein of PRRSV WH3. PRRSV N protein were purified by Ni-NTA column and identified by PAGE. Result showed that high purity of fusion expression protein was got.2.The construction of the initial random oligonucleotide library and primersDesign and chemically synthesize an initial random DNA oligonucleotide library in vitro,which consisted of a central randomized sequence of40nucleotide flanked by two18nt primer hybridization sites. This library consists of a multitude of ssDNA fragments (-1015molecules). Primers were designed according to the fixed sequence and used in the SELEX procedure. 3.SELEX procedureIn the screening process, the first dsDNA library was generated by PCR with the initial ssDNA library as template, and then to generate ssDNA library by the asymmetric PCR. In the process of SELEX, ssDNA library firstly incubate with blank microliter, PRRSV GP5or PRRSV negative serum to minimize the enrichment of unspecifically binding oligonucleotides, and then incubate with N protein which fixed on the microliter plate, after a period of time, specific binding sequences are eluted. In the next round of screen, new dsDNA library is generated with the eluted ssDNA sequences as template and then ssDNA library is generated,screen and elution in turn, go round and begin again. In this study,15cycles were processed totally. Last screened specific sequence is cloned into pGEM(?)-T Vector, then transformed into DH5a, we picked the34Monoclonal colony. After identified by restriction enzyme digestion and PCR, sequenced, we obtained26affinity aptamer sequences.4. Aptamers feature analysisThe affinity and specificity between aptamers and PRRSV N protein were detected by ELISA using a biotin-labeled aptamer as primary antibody and HRP-streptavidin as secondary antibody. The results show that24affinity and specificity aptamers against the N protein was obtained and Some aptamers are affinity for PRRSV. Base composition analysis shows that26aptamer sequences are all rich in C or T, which may be associated with affinity of aptamer to PRRSV N protein. Sequence alignment analysis Suggests that7sequences appears2times and Some sequences has conserved motifs. Secondary structure analysis by DNAMAN indicates that these conserved motifs locate at the top of ring or in the sequence between two rings.This study established a SELEX method with microplate as medium, and using this method, we have successfully screened affinity aptamers for PRRSV N protein, making a preliminary study on application of the aptamer in PRRSV early detection.