21029750
not annotated - annotated - LINNAEUS only
Detection of infectious myonecrosis virus using monoclonal antibody specific to N and C fragments of the capsid protein expressed heterologously.
The gene encoding the capsid protein in ORF1 of the genome of infectious myonecrosis virus (IMNV) (GenBank AY570982) was amplified into three parts named CP-N (nucleotides 2248-3045), CP-I (nucleotides 3046-3954) and CP-C (nucleotides 3955-4953). The CP-N fragment was inserted into expression vector pTYB1 while CP-I and CP-C were each inserted into expression vector pGEX-6P-1 for transformation of BL21 E. coli strain. After induction, intein-CP-N (84 kDa), glutathione-S-transferase (GST)-CP-I (60 kDa) and GST-CP-C (62 kDa) fusion proteins were produced. They were separated by SDS-PAGE and electroeluted before immunization of Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to CP-N and one MAb specific to CP-C were selected for use for detection of natural IMNV infections in Penaeus vannamei by dot blotting, Western blotting and immunohistochemistry. There was no cross-reaction with shrimp tissues or common shrimp viruses including white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV), Penaeus monodon nucleopolyhedrovirus (PemoNPV), Penaeus stylirostris densovirus (PstDNV) and Penaeus monodon densovirus (PmDNV). The detection sensitivities of the MAbs were approximately 6 fmol/spot of purified recombinant intein-CP-N protein and 8 fmol/spot of GST-CP-C as determined by dot blotting. A combination of all three MAbs resulted in a twofold increase in sensitivity over use of any single MAb. However, this sensitivity was approximately 10 times lower than that of one-step RT-PCR using the same sample. Immunohistochemical analysis using MAbs specific to CP-N and CP-C in IMNV-infected shrimp revealed intense staining patterns in muscles, the lymphoid organ, gills, the heart, hemocytes and connective tissue.
Ann file
T1 Species 13 41 infectious myonecrosis virus
N1 Reference T1 Taxonomy:282786
T2 Species 210 238 infectious myonecrosis virus
N2 Reference T2 Taxonomy:282786
T3 Species 240 244 IMNV
N3 Reference T3 Taxonomy:282786
T4 Species 549 561 BL21 E. coli
N4 Reference T4 Taxonomy:562
T5 Species 936 940 IMNV
N5 Reference T5 Taxonomy:282786
T6 Species 1115 1140 white spot syndrome virus
N6 Reference T6 Taxonomy:342409
T7 Species 1142 1146 WSSV
N7 Reference T7 Taxonomy:342409
T8 Species 1149 1166 yellow head virus
N8 Reference T8 Taxonomy:96491
T9 Species 1168 1171 YHV
N9 Reference T9 Taxonomy:96491
T10 Species 1174 1194 Taura syndrome virus
N10 Reference T10 Taxonomy:142102
T11 Species 1196 1199 TSV
N11 Reference T11 Taxonomy:142102
T12 Species 1202 1238 Penaeus monodon nucleopolyhedrovirus
N12 Reference T12 Taxonomy:259389
T13 Species 1240 1247 PemoNPV
N13 Reference T13 Taxonomy:259389
T14 Species 1791 1795 IMNV
N14 Reference T14 Taxonomy:282786
T15 Species 1250 1281 Penaeus stylirostris densovirus
T16 Species 1283 1289 PstDNV
T17 Species 955 971 Penaeus vannamei
T18 Species 1295 1321 Penaeus monodon densovirus
T19 Species 1323 1328 PmDNV
T20 Species 781 791 Swiss mice
N15 Reference T17 Taxonomy:6689 Penaeus vannamei
N16 Reference T15 Taxonomy:1593240 Penaeus stylirostris densovirus
N17 Reference T16 Taxonomy:1593240 Penaeus stylirostris densovirus
#1 AnnotatorNotes T20 TODO check
#2 AnnotatorNotes T1 TODO check specificity
#3 AnnotatorNotes T2 TODO check specificity
#4 AnnotatorNotes T3 TODO check specificity