Analysis of antigenic proteins of grouper iridovirus and the application of antibody
Date Issued
2007
Date
2007
Author(s)
Lin, Yang-Yan
DOI
zh-TW
Abstract
The genome of grouper iridovirus (GIV) consists 140 Kb and encodes 120 open reading frames. The results of Western blotting showed that two proteins with 60 kDa and 45 kDa, respectively, were recognized by antibodies produced with GIV viral particles. We propose the 45-kDa protein, a deduced major capsid protein (MCP), is encoded by the gene, named GIV045R, and the 60-kDa protein might be translated from the gene, named GIV042L or GIV010R. These three genes were subcloned into pET expression vector, and their recombinant proteins were produced by E. coli expression system. Because of the difficulty to effectively express MCP, we decided to divide the MCP into four segments, named MCP1, MCP2, MCP3 and MCP4 to express. The results of Western blotting showed that all the recombinant proteins have antigenicity, but compared to the others recombinant proteins, that MCP3 and GIV042L were with strong signal. These two recombinant proteins will be the target in the future experiment.
The grouper larvae were injected intraperitoneally with 2 μg of the recombinant proteins alone or combined. 21 days post-vaccination, challenged by intraperitoneally injection with 50 μl of 106 TCID50 ml-1 of GIV solution. Blood was collected and tested for ELISA after 38 days. Results of ELISA showed that all the recombinant proteins had raised titer against GIV, the group of MCP mix and GIV042L even higher than inactivated GIV-injected control group. The results showed some similarity between ELISA and Western blotting. Our results show that vaccination with the recombinant proteins may trigger the grouper larvae immune response, thus leading to protection upon live viral challeng.
In this study, two polyclonal antibody against GIV from rabbit and goat were purified and used to develop an indirect sandwich ELISA for detection of GIV. The indirect sandwich ELISA could detect the purified GIV at the concentration of 36 ng and the GIV RV at the dose 3.125x106 TCID50 ml-1. Our results show that the indirect sandwich ELISA has speciality and sensitivity. These results could further be applied to immunoassay strip for the rapid diagnosis of GIV infection in grouper.
Subjects
石斑魚虹彩病毒
重組蛋白
多株抗體
間接三明治型ELISA
grouper iridovirus
recombinant proteins
polyclonal antibody
indirect sandwich ELISA
SDGs
Type
thesis
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