Publication: Improved immunodetection of Penaeus monodon densovirus with monoclonal antibodies raised against recombinant capsid protein
1
0
Issued Date
2011
Resource Type
File Type
application/pdf
ISSN
448486
Other identifier(s)
2-s2.0-78651480503
Rights Holder(s)
มหาวิทยาลัยศรีนครินทรวิโรฒ
Bibliographic Citation
Aquaculture. Vol 311, (2011), p.19-24
Suggested Citation
Srisuk C., Chaivisuthangkura P., Sukhumsirichart W., Sridulyakul P., Longyant S., Rukpratanporn S., Sithigorngul P. Improved immunodetection of Penaeus monodon densovirus with monoclonal antibodies raised against recombinant capsid protein. Aquaculture. Vol 311, (2011), p.19-24. doi:10.1016/j.aquaculture.2010.08.018 Retrieved from: https://hdl.handle.net/20.500.14740/7374
Abstract
Penaeus monodon densovirus (PmDNV), also called hepatopancreatic parvovirus (HPV), is a pathogen that causes stunt disease in the tiger shrimp, Penaeus monodon. In this study, the gene sequence encoding the capsid protein of PmDNV was cloned into two parts: PmDNV-N (837bp) and PmDNV-C (806bp). Both PCR products were cloned into the pGEX-6P-1 expression vector and transformed into the Escherichia coli BL21 strain. After induction, glutathione-S-transferase (GST)-tagged PmDNV-N and GST-tagged PmDNV-C proteins were obtained with molecular masses of 57.2 and 57.6kDa, respectively. The recombinant proteins were purified by SDS-PAGE and used to immunize mice for monoclonal antibody (MAb) production. Three hybridoma clones producing MAbs specific to PmDNV-N and two hybridoma clones producing MAbs specific to PmDNV-C were selected by dot blot, Western blot and immunohistochemistry. All five MAbs detect PmDNV infection in shrimp by dot blot and Western blot, but only PmDNV-specific MAbs can be used to detect PmDNV infection in the hepatopancreas by immunohistochemistry. The detection limit of PmDNV-N-specific MAbs obtained in this study is similar to the most sensitive MAb characterized in a previous study, approximately 50fmolμlμ1 of antigen as determined by dot blot; the sensitivity of PmDNV-C-specific MAbs is twofold less than the MAbs specific to PmDNV-N. Using three MAbs specific for PmDNV-N and PmDNV-C together is twofold more sensitive than using either MAb alone. Although the detection limit of combining MAbs was 25,000 times lower than one-step PCR-based methods, using these MAbs is still useful for confirmation of PmDNV infection which is more convenient than normal histopathology confirmation. © 2010 Elsevier B.V.