Production of Polyclonal Antibodies Against Calpastatin Gene Domain I from Skeletal Muscle of the Kamphaeng Saen Beef Breed

Abstract

Calpastatin is an endogenous inhibitor of the calpain (EC.3.4.22.17, Ca⁺-dependent cysteine proteinase) proteolytic enzyme system, which plays a major role in the postmortem tenderization. Complementary strand DNA of calpastatin gene derived from domain I of Longissimus dorsi muscle
from Kamphaeng Saen beef breed was amplified using the 1AF_1DR primer. Analysis of PCR results indicated the presence of product of approximately 402 bp. Nucleotide sequencing of the cDNA for bovine calpastatin corresponded with published domain I of calpastatin nucleotide sequences from bovine skeletal muscle and bovine heart calpastatin in Genbank (accession no. L14450 and NM_174003, respectively). PCR products were cloned into the plasmid pDrive cloning vector and subcloned into the plasmid pET28a expression vector. Recombinant calpastatin corresponding to domain I had the size approximately 21 kDa. The recombinant calpastatin used as immunogens applied to an immunized rabbit. Polyclonal antibodies, were raised in the rabbit against recombinant calpastatin, specifically recognized the calpastatin protein of approximately 130 kDa as determined by the Western immunoblot analysis. These results indicated that calpastatin domain I was presented within the recombinant
calpastatin protein and direct subcutaneous of immunization of recombinant calpastatin into rabbit was effective in polyclonal antibodies production against calpastatin.