[Immunoadjuvant effect of the extracellular domain of canine CTLA-4 on the VP2 protein of canine parvovirus].
OBJECTIVE
Investigation into the adjuvant effect of the extracellular domain of canine cytotoxic T lymphocyte-associated antigen 4 (CTLA-4).


METHODS
We first amplified the cDNA for the extracellular domain from blood lymphocytes using RT-PCR and then amplified the VP2S gene fragment for major antigenic epitopes of the VP2 protein of canine parvovirus (CPV) using PCR. After sequence analysis, we inserted the VP2S fragment into expression vector pQE-31 with or without the coding sequence for the extracellular domain of canine CTLA-4. After transformation of the two recombinant vectors into E. col, we studied recombinant protein expression by isopropyl beta-D-1-thiogalactopyranoside (IPTG) induction. Finally, we immunized BALB/c mice with the same dose of the purified protein VP2S or CTLA-4-VP2S and compared their antibody responses by enzyme-linked immunosorbant assay (ELISA) and haemagglutination inhibition (HI) assay.


RESULTS
After 30 cycles of amplification, agarose gel electrophoresis revealed expected PCR products for both gene fragments. Sequence analysis showed that the amplified extracellular domain was 99.2% identical to previously published sequence without mutation in the hexapeptide motif (MYPPPY) for B7 molecule binding. After IPTG induction, the vector-transformed E. coli expressed expected 29-kDa VP2S protein and 42-kDa CTLA-4-VP2S protein. Western blotting showed that both VP2S and CTLA-4-VP2S proteins were recognized by CPV-specific antiserum. After two times of immunizations, the VP2S-specific antibody appeared from week 2 and reached the highest level at week 4 in CTLA-4-VP2S-immunized mice. In VP2S-immunized mice, however, the specific antibody appeared from week 4 and reached the highest level at week 5. The CTLA-4-VP2S-immunized mice had a 100-fold higher ELISA antibody and 10-fold higher HI antibody compared to VP2S-immunized mice.


CONCLUSIONS
The extracellular domain of canine CTLA-4 had strong immunoadjuvant effect on its fused protein.
Wu
Zhi
Z
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China. yzwuzhi@163.com
Sun
Huaichang
H
Zhang
Xinyu
X
D000276:Adjuvants, Immunologic / Q000737:chemistry* / Q000235:genetics; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000917:Antibody Formation; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D060908:CTLA-4 Antigen; D036022:Capsid Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification; D004274:DNA, Recombinant / Q000235:genetics; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D007114:Immunization; D051379:Mice; D017993:Parvovirus, Canine / Q000737:chemistry* / Q000276:immunology*; D017434:Protein Structure, Tertiary; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000276:Adjuvants, Immunologic; D000906:Antibodies; D015703:Antigens, CD; D060908:CTLA-4 Antigen; D036022:Capsid Proteins; C556707:Ctla4 protein, mouse; D004274:DNA, Recombinant