Prokaryotic Expression Of The Main Antigenic Region Of PPV NS1 Protein And Its Application In Dot Immunobloting

Porcine parvovirus is a major pathogens which could lead to reproductive failure of sows. In additional, it can also to increased hazard with other pathogenic.The recently reports suggest that there may be have relation in leptospirosis, PPV, PCV2, PRRSV together to result pig chronic interstitial nephritis.PPV have ability to tolerate heat and acid, It is difficult to kill PPV with usual method of disinfection . So PPV cause a tremendous economic losses to livestock production. Because porcine parvovirus infection often showed continuous low-dose, long-term carriers of the virus infected pigsand the clinical symptoms was not obvious, it has brought to the clinical detection difficult. At present, the conventional PPV detection technologies are virus isolation and identification, neutralization test, ELISA and PCR methods, these methods have advantages and disadvantages, but they can not meet the clinical rapid, accurate diagnosis of the disease demands, these diagnostic methods has affected the lag time for rapid detection of the epidemic. Therefore, the establishment PPV specific, sensitive, rapid and accurate diagnostic method has important practical significance.PPV genome-encoded structural protein VP2 and non-structural protein NS1 may stimulate the pig to produce antibodies, with good immunogenicity, which is a conserved sequence of NS1 sequences. VP2 protein is used for vaccine research, while the NS1 proteins were mostly used in the detection of antibodies against PPV. Antigen detection method can be used to detect PPV.Its advantage is direct and accurate, but the PPV elimination virus in only 14-day, not detoxification period not detected PPV. The detection of antibodies is not required to consider the issue of detoxification period, additional test PPV NS1 antibodies can be use to distinguish infected with wild virus or immunization by peptide vaccine and inactivated vaccine in pigs.In view of the above research background, this study chosen porcine parvovirus NS1 protein as the object of the following experiments. First, in order to NCBI published in Porcine Parvovirus NS1 protein gene sequence as the reference to design a pair of specific primers for PPV NS1 protein major antigenic domain gene cloning, sequencing results showed that the cloned sequence of length 1236bp, using BLAST for comparison, The results show that pNS1 sequence with PPV NADL-2 strain, SR-1 strain, Tai'an strain, NJ strains, Kresse strain, China strain, ZJ strain, VRI-1 strain similarities were 98%, with Nanjing200802 strain, HN -Z1 strains, Nanjing200801 strain, S-1 strain, HN-Z3 strains, NJ-2 strain, nanjin-1 strain, vaccine virus IDT strain, 27a isolates strain the similarity of 97%.We can see pNS1 sequence have closer affinity to the S-1 strain in evolutionary tree analysis by using MEGA4.0.2. Main antigenic region of PPV NS1 protein gene was subcloned into prokaryoutic high-expressing vector pET-28a, pGEX-6P-1 and pQE30. By screening the final selection of PET-28a-pNS1 for the final expression vector . The result showed that the sequenced pNS1 gene was identical with the protosequence and the target gene which was inserted into expressing vector had uniformity, and had correct direction and site. BL21(DE3) competent cell was transformed by positive recombinant plasmid and was induced to express by IPTG. Expession product being analysized through SDS-PAGE, target protein and vector multimetric histidine protein were confluently expressed, and a target approximative band of 47.8kD. The expressing quantity of recombinant protein is influenced by the concentration of IPTG In the condition of 0.6M IPTG being induced 7 hours, the extrinsic protein of bacteria is more than in any other conditions. Western-blot confirmed that the fusion protein was detected with good immunogenicity. Finally, a large number of target protein expression, after preliminary purification used in the dot immunoblot, established a dot immunoblot technology-based diagnosis of PPV antibody method.We use this method to test a small number of samples .The testing results compared to results by PPV LAT kit confirmed that this method has a certain specificity and sensitivity,beside easy to operate and fast. The results of this experiment lay foundation for the development a specific, sensitive, rapid and accurate diagnostic test strip of porcine parvovirus.