Parvalbumin Content and Expression Analysis
The relative content of fish parvalbumin was analyzed through both
protein- and transcriptome-based approaches. For the protein approach,
fresh fishes including tuna, salmon, halibut, cod, grouper, catfish,
grass carp, and tilapia were purchased from local wet market.
Genus/species of the fishes were confirmed by DNA barcoding upon DNA
extraction with QIAamp DNA mini kit (Qiagen) and PCR with specific
primers: Fish-F1: 5′-TCTCAACCAACCATAAAGACATTGG-3′ and Fish-R1:
5′-TATACTTCTGGGTGCCCAAAGAATCA-3′; Fish-F2: 5′-CATCCTACCTGTGGCAATCAC-3′20 . One milligram of raw fish meat was manually
homogenized in 10mL ice-cold phosphate-buffered saline (PBS) until a
smooth paste was achieved 21 . Protein was then
extracted overnight at 4°C with constant stirring. The protein extract
was centrifuged and supernatant was filter-sterilized through a 0.22 μm
polyethersulfone membrane. The fish protein extracts analyzed fresh upon
extraction. Ten microliters of fish extracts were then separated on a
13.5% SDS-PAGE gel according to their molecular weights using a Mini
PROTEAN SDS-PAGE system (Bio-Rad). Protein bands were stained with
SimplyBlue SafeStain (Thermo Fisher). Densitometry analysis was then
performed to determine the relative band intensity of parvalbumin on
SDS-PAGE by Image Lab (Bio-Rad) using salmon parvalbumin as the
reference band (i.e. band intensity = 1). Four pieces of fish meat were
randomly picked for protein extraction and resolved on separate SDS-PAGE
to ensure reproducibility of parvalbumin level analysis. While in
transcriptome-based approach, we processed publicly available RNA-seq
data of fish meat, ensuring quality, and de novo assembly with Trinity22. Aligning transcripts with known allergens
from AllergenOnline 23 via blast method
identified homologous parvalbumin transcripts.