[Prokaryotic expression and antiserum preparation for major antigenic epitope region of major capsid protein of Chinese giant salamander (Andrias davidianus) iridovirus].
Objective To express the fusion protein of major antigenic epitope region of major capsid protein (MCP) of Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) and prepare the rabbit antiserum. Methods Using the genomic DNA of CGSIV Lueyang strain (CGSIV-LY) as a template, the gene fragment of major antigenic epitope region of MCP was amplified by PCR and cloned into the prokaryotic vector pET-21a(+) to construct the prokaryotic expression recombinant plasmid pET-21a-MCP. The recombinant plasmid was transformed into Escherichia coli BL21(DE3). His-tagged fusion protein was induced by IPTG. After identified by SDS-PAGE and Western blot analysis, the recombinant protein was purified by nitrilotriacetic acid (Ni-NTA) agarose resin. New Zealand rabbits were immunized with the purified recombinant protein to generate antiserum. Specificity and titer of the antiserum were determined by Western blotting and indirect ELISA, and then the antiserum was used to detect the CGSIV in the infected EPC cells by indirect immunofluorescence assay. Results The recombinant protein with the relative molecular mass of 29 000 was expressed. The prepared rabbit antiserum had a good specificity and a high titer. Indirect immunofluorescence assay showed that the antiserum could recognize CGSIV in the infected EPC cells. Conclusion The fusion protein of major antigenic epitope region of MCP of CGSIV is successfully expressed and the rabbit antiserum with a high titer and a good specificity been prepared.
Aquaculture and Fishery Engineering Laboratory, Yellow River Fisheries Institute, Chinese Academy of Fishery Sciences, Xi'an 710086, China.
Xiaoyuan
X
Zhou
Aquaculture and Fishery Engineering Laboratory, Yellow River Fisheries Institute, Chinese Academy of Fishery Sciences, Xi'an 710086, China.*Corresponding author, E-mail: zxlang029@sina.com.
Xinglang
X
Zhang
Aquaculture and Fishery Engineering Laboratory, Yellow River Fisheries Institute, Chinese Academy of Fishery Sciences, Xi'an 710086, China.
Qiuhong
Q
Jia
Aquaculture and Fishery Engineering Laboratory, Yellow River Fisheries Institute, Chinese Academy of Fishery Sciences, Xi'an 710086, China.
Yahui
Y
Han
Aquaculture and Fishery Engineering Laboratory, Yellow River Fisheries Institute, Chinese Academy of Fishery Sciences, Xi'an 710086, China.
Hongwei
H
Gao
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D000918:Antibody Specificity; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007106:Immune Sera / Q000276:immunology*; D017994:Iridovirus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014562:Caudata / Q000821:virology
D000914:Antibodies, Viral; D036022:Capsid Proteins; D007106:Immune Sera; D011994:Recombinant Proteins