| Goose plague (GP) was an acute and sub-acute septicemic infection disease in 4-20 day old gosling by caused goose parvovirus (GPV). Infected goslings have 90-100% of mortality rate. It was one of the important infectious diseases for gosling. This disease was described first in China by Fang Ding-Yig at Yang Zhou district in 1956, followed by reports in various countries in the world. GPV has been classified as a member of the parvovirus genus of the parvoviride. GPV contains a single DNA molecule of about 5.1kilobases in length. The genome of GPV contains two major open reading frames (ORF),left one encoding for the non-structural proteins NS1 and NS2 and the right one encoding for the VP1, VP2 and VP3 structural proteins. VP3 was the major structural protein, as well as it was a major protective antigen and enable to induce the body to produce neutralizing antibodies. So the genes of VP3 were the important candidate genes for study on genetic engineering vaccine and detecting antigen. GPV inactivated and attenuated whole virion vaccines were be used to prevent GPVI, but these vaccines have some side-effects to spread virus and to cause sub-clinical infection. So this disease has not been effectively prevented. It was the main cause that lacking of fast and effective diagnostic and identifing methods of GPVI has not been effectively controlled. The routine diagnostic methods for GPVI were the Agargel precipitin assays (AP),Serum-neutralization test (NT),Immunofluorescent antibody assay (IFA) have been established. These methods have played important role in control GP, but which have lower sensitivity and use longer time. These methods have not suitable for the needs of efficiently control GP. A PCR method was established to identify GPV and GPMV by the digested fragments, but have not found the following reports about using this method. This study aimed at established effective and fast molecular biological and immunological identifying methods of efficiently control GPVI. PCR and DIG-labeled nucleic acid probes detecting methods for quantification and half-quantification were developed by two pairs of primers of VP1-VP3, NS1 nucleotide and by it's amplified products. To detect method of Indirect-ELISA and Dot-ELISA were developed by the expressed products of some nucleic acid fragment for quantify and determine GPV antibody. The results of this study showed that: 1. Cloning VP1-VP3 non-repeated nucleic acid sequences and obtaining products of PCR about 0.6kb in length. The sequencing result showed that the product of PCR contains 534 bp, which encodes 178 amino acid residues. The nucleotide and amino acid sequences have 100% homology with same region nucleic acid of GPV B reference strain. 2. The PCR detection methods were developed by two pairs of primers to amplify VP1-VP3 and NS1 nucleic acid. The specificity assay showed that two pairs of primers disable to amplify the control nucleotide of GPMV and AIV. Sensitivity assay showed that the pair of primers of VP1-VP3 nucleotide can detect 4.4fg (0.01LD50 GPV)of GPV nucleic acid, and the other pair can detect 44pg((0.01LD50 GPV) of GPV nucleic acid. The result of detecting 7 groups organs from GPV infected goose showed that two pairs of primers can fast and efficiently detect GPV DNA, and the positive rate was 100%. But the detecting sensitivity of NS1 nucleic acid primers was less than VP1-VP3 primers of 1000 times, so that the pairs of primers to amplify VP1-VP3 nucleotide fragment were selected to develop PCR detecting method. PCR have a strongly amplifying function, which could directly to detect GPV DNA from the extracted DNA of GPV infected gooses, so PCR was an effective and fast method to detect GPV. 3. The amplified product of VP1-VP3 nucleotide and NS1 nucleotide were labeled with Digoxigenin and developed the DIG-labeled nucleic acid probes to detect GPV DNA. The specificity assay showed that two labeled probes all enable to hybridize with target nucleotide products of PCR, DNA recombinant plasmid and nucleic acid from different GPV strain, and disable to hybridize with the control nucleic acid of GPMV and AIV. The sensitivity assay showed that NS1 and VP1-VP3 DIG labeled nucleic acid probes were able to detect 6.6pg and 9.9pg of GPV DNA respectively. The result of using two labeled probes to detect 2 groups organs from GPV infected goose showed that positive rate was 100%, which indicate that DIG-labeled probe was a fast detecting, suitable detecting large number samples one time, un-use specific equipment and suitable popularized method to diagnose GP. 4. The sensitivity of DIG-labeled probe method was less than PCR method. The sensitivity (4.4fg) of PCR method by VP1-VP3 primers was higher of 2250 times than the sensitivity (9.9pg) of DIG-labeled probe method. The sensitivity (44pg) of PCR method by NS1 primers was lowerr of 6.7 times than the sensitivity (6.6pg) of DIG-labeled probe method. 5. pGEX-(VP1-VP3) recombinant expressive plasmid was constructed and over expressed in GST fusion proteins. The suitable expression and purification conditions were confirmed. The provided recombinant plasmid pGEX-VP1, pGEX-VP2, pGEX-VP3, pGEX-VS1 and pGEX-NS2 by this laboratory were also expressed and purified successfully by the Prepared Glutathion Sepharos 4B after renaturing the expressed products. According to the result of Western blot and Dot-ELISA, selecting VP3 protein as the antigen to develop the Indirect-ELISA and Dot-ELISA method for quantifying and determining GPV antibody, selecting VP1-VP3 protein as the antigen to develop the Indirect-ELISA and Dot-ELISA method for identifying GPV antibody and VP3 sub-unit antibody. The result was satisfactory after detecting antibody titer of 90 serum samples from immunized with whole GPV vaccine. The result was relative to the neutralizing test antibody titer. All the above, these detecting method have been developed, which indicated that the high technological products of the cloned nucleic acid and expressed products of GPV structural proteins and non-structural proteins gene were have been applied to practical and were have beenused to produce economic returns. The PCR and labeled probe detecting methods were suitable for determining the GPV nucleic acid before antibody produce, and the ELISA methods were suitable for detecting infected antibody and determining immunized antibody. These methods have important practical significance and use value for fast diagnosis, control epidemic situation, epidemiological investigation, detecting immunizing antibody titer, identifying natural infection, and dispelled the trouble about identifying infection antibody when using VP3 gene engineering vaccine, which lay the academic and technical foundation for further development and assemble detecting reagent boxes. |
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