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Swine pseudorabies (Pseudorabies, PR) is caused by pseudorabies virus (Pseudorabies virus, PRV), a highly contagious, acute infectious disease. PRV is a herpes virus family (Herpesviridae) α herpes virus subfamily, China in 1948, the first time the pseudo-rabies reported in cats, according to the statistics of the summer of 2006, PR popular range has been expanded to 31 provinces, municipalities (districts), including Hong Kong and Taiwan, China's aquaculture industry has caused huge economic losses, therefore, to establish an effective diagnostic tests has important significance to the control and eradication of PR. The study screened hybridoma cell lines that secrete anti-PRV monoclonal antibody; prokaryotic expression system PRVgB gene antigen protein. The PRV hybridoma cell line screening and monoclonal antibody preparation: the Batha strain of pseudorabies virus vaccination in BHK-21 cells, the virus proliferation, cell cultures after repeated freezing and thawing three times, collected the virus, the use of differential centrifugation and sucrose density gradient centrifugation of purified virus using purified pseudorabies virus immunized BALB / c mice after three free, take qualified spleen cells were fused with NS0 myeloma cells using ELISA and immunohistochemistry screening hybridoma, be subcloned by limiting dilution method, the final two stable hybridoma cell lines secreting anti-PRV 2B2-C1 and 3F1-A1,. Specific experimental results show that: 2B2-C1 and 3F1-A1, does not react with porcine circovirus, swine fever virus, porcine reproductive and respiratory syndrome virus and normal BHK-21 cells, and good specificity. The two PRV monoclonal antibodies obtained to further establish specific, sensitive, accurate and rapid detection of PRV laid a foundation. PRV gB gene cloning and prokaryotic expression of antigenic region: PRV gB gene sequence published pursuant to Genbank, designed a pair of specific primers, PRV gB gene fragment (264bp) were amplified by PCR and cloned into pET- 28a expression vector was constructed expression plasmid was named pET-28a-gB, into pET-28a-gB BL21 engineered bacteria culture was induced by IPTG, and analyzed by SDS-PAGE and Western blotting results show that the gB gene fragments BL21 engineered bacteria highly expressed protein bands of the size of about 16 kDa, the expression of the protein can be PRV-positive sera. PRV gB gene expression of the major antigenic region laid the foundation for the gB monoclonal antibody screening and establish gB antibody detection.
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