Prokaryotic expression of chicken infectious anemia apoptin protein and characterization of its polyclonal antibodies.
In the present study recombinant VP3 (rVP3) was expressed in E. coli BL21 (DE3) (pLysS) and its polyclonal antibodies were characterized. SDS-PAGE analysis revealed that the expression of recombinant protein was maximum when induced with 1.5 mM IPTG for 6 h at 37 degrees C. The 6xHis-tagged fusion protein was purified on Ni-NTA and confirmed by Western blot using CAV specific antiserum. Rabbits were immunized with purified rVP3 to raise anti-VP3 polyclonal antibodies. Polyclonal serum was tested for specificity and used for confirming expression of VP3 in HeLa cells transfected with pcDNA.cav.vp3 by indirect fluorescent antibody test (IFAT), flow cytometry and Western blot. Available purified rVP3 and polyclonal antibodies against VP3 may be useful to understand its functions which may lead to application of VP3 in cancer therapeutics.
Molecular Biology Laboratory, Department of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243 122, India.
Shikha
S
Saxena
Gandham Ravi
GR
Kumar
Prafull
P
Singh
Uttara
U
Chaturvedi
Lovleen
L
Saxena
Rajiv
R
Kumar
Aditya Prasad
AP
Sahoo
Juwar
J
Doley
Rajmani
Amit
A
Kumar
Sudesh
S
Kumar
Ashok K
AK
Tiwari
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017727:Chicken anemia virus / Q000235:genetics* / Q000276:immunology; D002645:Chickens / Q000821:virology; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D050130:Oncolytic Virotherapy; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; C091717:VP3 protein, Chicken anemia virus