Expression of recombinant subgroup A avian leukosis virus gp85 and its application as a diagnostic antigen for antibody detection
Date Issued
2012
Date
2012
Author(s)
Hsu, Meng-Fang
Abstract
Avian leukosis viruses, ALVs are the pathogens of neoplastic diseases in chickens, the reproductive reduction and increased elimination may cause the economic loss of chicken flocks. The commercial available subgroup A avian leukosis virus (ALV-A) antibody (Ab) detection kit coated with whole virus may decrease the sensitivity and specificity because of the interfering of Ab from other subgroups. The purpose of the study is using baculovirus/ insect cells expression system to express ALV-A surface glycoprotein (gp85), and to evaluate the potential of the recombinant protein to be used as the antigen in enzyme linked immunosorbent assay (ELISA). Using the laboratory isolate TW-3577 as the cloning template, two recombinant baculoviruses, rgp85-baculovrius and rgp85sp-baculovirus were constructed and infect the Sf9 insect cells. The infected insect cells showed positive result of recombinant gp85 proteins in Western blotting, the target protein size were about 75 kDa. In the indirect immunofluorescence assay, recombinant baculovirus infected insect cell showed positive results reacted with anti-His mAb and ALV-A antiserum. The cell pellet which harvested at 72 hours post infection was lysed, sonicated, and directly used as the coated antigen. 72 field sera were collected to test the optimized ELISA reaction. Using virus neutralization test as the gold standard, the sensitivity and the sepecificity of the rgp85sp ELISA were 80% and 83.3%, respectively. Both values were not good enough for future test due to the impurity of the expressed protein. The purity of the expression protein should be enhanced for future development of this test.
Subjects
avian leukosis virus
subgroup A avian leukosis virus
exogenous viruses
env-gp85 glycoprotein
eukaryotic expression
baculovirus/insect cell expression system
enzyme linked immunosorbent assay (ELISA)
Type
thesis
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