| Monkey B virus is must be negative in the common experimental monkey clones. It can infect a variety of animals and humans, and human infection has the mortality up to 80%, and survivors have neurological sequelae, while monkey infection only shows slight symptoms or no symptoms, but the virus will hide in ganglion continually and reactive periodically. This characteristic is most possible to lead the false-negative test results. MBV just has one serotype, and has stable antigenicity, and its nucleotide sequence and amino acid sequence have been determined. The envelope glycoprotein gD has been shown to have a high diagnostic potentiality.Simian type D retrovirus and Simian immunodeficiency virus are must be negative in the SPF clones. SRV and SIV are the pathogens of simian AIDS, and they both can cause immune deficiency symptom. However no symptoms because of SIV in its natural hosts, only causes SAIDS when cross-species infection occurring. SRV has 7 serotypes, of which 1-3 have been seauenced. and the amino acid seauence of the nucleocapsid protein P27 is highlv conserved. The capsid protein P27 of SIV contains the major antigenic determinant, and has group antigen, are considered to be the sign of SIV detection. This study contains three main aspects:1. The detection of MBV, SRV, SIV serum antibodies of experimental monkeys from Shanghai. There were 475 serum samples totally, the positive of MBV serum antibodies was 34, and the positive rate was 7.158%; the positive of SRV serum antibodies was 20, and the positive rate was4.211%; the positive of SIV serum antibodies was 1, and the positive rate was 0.2105%. The positive rate has no correlation with year or the total number of samples. 2. The expression, purification and identification of proteins.The pET28a-gD (MBV) recombinant plasmid was transformed into the BL21 (DE3) strain, then expressed gD protein, and molecular weight was 47.5kD approximately. After NI-agarose affinity chromatography purification and identificated with Western blot assay, It showed that the protein had antigenicity, then measured the concentration was 3.726mg/mL.The pET28b-p27 (SRV) recombinant plasmid was transformed into the Rosetta (DE3) strain, then expressed P27 protein, and molecular weight was 26.2kD approximately. After NI-agarose affinity chromatography purification and identificated with Western blot assay, It showed that the protein had antigenicity, then measured the concentration was 2.043mg/mLThe pET28a-p27 (SIV) recombinant plasmid was transformed into the Rosetta (DE3) strain, then expressed P27 protein, and molecular weight was 24.6kD approximately. After NI-agarose affinity chromatography purification and identificated with Western blot assay, It showed that the protein had antigenicity, then measured the concentration was 1.604mg/mL. 3. The establishment and evaluation of the indirect ELISA method.Using MBV gD as antigen to establish an indirect ELISA method for detecting serum antibody against MBV. we have determined the optimum conditions:antigen 1:1280 dilution (concentration was 2.913μg/hole) and sera 1:100 dilution, worked for 60min; HRP secondary antibody diluted 1:20,000, worked for 60min, substrate worked for 15min. OD> 0.326 judged as positive, OD< 0.255 judged as negative. Experiments showed that gD protein did not have cross reaction with SRV or SIV serum antibodies. The coincidence rate between this established method and full virus ELISA kits detecting results was 96.74%. The coefficient of variation was less than 14% in the repeating tests. Then we applicated this method to detect 37 serum samples from Shanghai in 2016,1 positive was detected, and the positive rate was 2.703%.Using SRV P27 as antigen to establish an indirect ELISA method for detecting serum antibody against SRV, we have determined the optimum conditions:antigen 1:640 dilution (concentration was 3.193μg/hole) and sera 1:100 dilution, worked for 60min; HRP secondary antibody diluted 1:20,000, worked for 60min, substrate worked for 15min. OD≥ 0.297 judged as positive, OD≤ 0.244 judged as negative. Experiments proved that P27 protein did not have cross reactions with MBV or SIV serum antibodies. The coincidence rate between this established method and full virus ELISA kits detecting results was 97.87%. The coefficient of variation was less than 9% in the repeating tests. Then we applicated this method to detect 37 serum samples from Shanghai in 2016,2 positive was detected, and the positive rate was 5.405%.Using SIV P27 as antigen to establish an indirect ELISA method for detecting serum antibody against SIV, we have determined the optimum conditions:antigen 1:640 dilution (concentration was 2.508ug/hole) and sera 1:100 dilution, worked for 60min; HRP secondary antibody diluted 1:10,000, worked for 60min, substrate worked for 15min. OD≥ 0.269 judged as positive, OD< 0.256 judged as negative. Experiments proved that P27 protein did not have cross reactions with MBV or SRV serum antibodies. The coincidence rate between this established method and full virus ELISA kits detecting results was 98.91%. The coefficient of variation was less than 3% in the repeating tests. Then we applicated this method to detect 37 serum samples from Shanghai in 2016,0 positive was detected, and the positive rate was 0. |
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