Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests

Figures & data

Figure 1. Genome organization of BFDV focusing on the two major ORFs encoding the Rep protein (ORF V1) and the capsid protein (ORF C1). ORFs are labelled according to their localization on the virus (V) or complementary (C) strand. The positions of the primers are indicated (small arrows), with nucleotide (nt) numbering according to Niagro et al. (1998).

Figure 2. Expression of C1 protein variants in E. coli. 2a: Schematic presentation of the amino acid sequences deduced from the nucleotide sequences of the C1 expression plasmids. 2b: Analysis of protein expression by SDS-PAGE followed by Coomassie brilliant blue staining. Bacterial lysates (lysates) were obtained at 5 h after induction with IPTG using a buffer containing 8 M urea. His-tailed proteins were purified (purification) from the lysates using Ni-NTA-agarose affinity chomatography. neg., negative control derived from bacteria transformed with the vector alone; M, molecular weight markers: 94.0 kDa, 67.0 kDa, 43.0 kDa, 31.0 kDa, 20.1 kDa, 14.4 kDa. 2c: Purification of C1₃₉₋₂₄₄-His using Ni-NTA-agarose affinity chomatography, analysed by SDS-PAGE followed by Coomassie brilliant blue staining. The bacterial lysate (L) was obtained by sonication at 5 h after induction of the culture with IPTG. F, flow-through fraction; W, flow-through fraction after washing; 50 to 500, fractions after elution with 50 to 500 mM imidazole solution; M, molecular weight markers (kDa). The fractions after elution with 120, 200 and 300 mM imidazole were pooled and used in further experiments (used fractions). The arrows indicate the positions of C1_39–244 or C1_39–244-His.

Figure 3. Immunoblot analysis of sera collected from chickens immunized with C1_39–244-His. 3a: Analysis of reactivity with C1_39–244-His using sera obtained at 0, 7, 14, 28, 35, 100, 114 and 135 days after the first inoculation of the protein. An additional booster immunization was performed at day 100. 3b: Analysis of reactivity with supernatant of an organ suspension prepared from a BFDV-infected Rueppels parrot (P. rueppelli) and concentrated by ultracentrifugation (lane 1), crude organ suspensions of two BFDV-infected budgerigars (lanes 2 and 3), or C1_39–244-His (lane 4). Immunoblot analysis was performed using the chicken sera obtained before immunization (left) or 114 days after immunization with C1_39–244-His (right). Lane M, molecular weight markers: 97.4 kDa, 67.2 kDa, 45.0 kDa, 31.0 kDa, 21.5 kDa, 14.4 kDa. Arrows indicate the positions of C1 and C1_39–244-His. Additional faint bands in lanes 2 and 3, observed after using the chicken sera obtained 114 days after immunization, are indicated by white arrows.

Figure 4. Analysis of psittacine sera. 4a: Results of a HI assay using 4 haemagglutinating units of an organ homogenate prepared from a BFDV-infected budgerigar. 4b: ELISA using purified C1_39–244-His as antigen. A serum dilution of 1:160 is shown. 4c: Immunoblot analysis using purified C1_39–244-His as antigen. The arrow indicates the position of C1_39–244-His.

Table 1. Comparison of HI titres, ELISA titres and immunoblot reactivity obtained for 11 randomly selected psittacine sera

Serum (bird number)	HI titre (scoring^a)	ELISA titre (scoring^b)	Immunoblot reactivity (scoring^c)
1	<1:2 (−)	1:40 (−)	+/− (?)
2	1:8 (+)	1:160 (+)	+ (+)
3	1:2 (−)	1:80 (−)	− (−)
4	1:4 (−)	1:80 (−)	− (−)
5	1:4 (−)	1:40 (−)	− (−)
6	1:8 (+)	1:40 (−)	+ (+)
7	1:8 (+)	1:160 (+)	+ (+)
8	1:4 (−)	1:80 (−)	+/− (?)
9	1:4 (−)	1:80 (−)	− (−)
10	<1:2 (−)	1:40 (−)	− (−)
11	1:8 (+)	1:640 (+)	+ (+)
^a HI titres ≥1:8 are scored positive (+), lower titres are scored negative (−).
^b ELISA titres ≥1:160 are scored positive (+), lower titres are scored negative (−).
^c A clearly visible band (+) is scored positive (+), a faint band (+/−) is scored questionable (?), no band is scored negative (−).

Niagro, FD , Forsthoefel, AN , Lawther, RP , Kamalanathan, L , Ritchie, BW , Latimer, KS , and Lukert, PD , 1998. Beak and feather disease virus and porcine circovirus genomes: intermediates between the geminiviruses and plant circoviruses , Archives of Virology 143 (1998), pp. 1723–1744.

 PubMed Web of Science ®Google Scholar