Cloning, Expression Characteristics And Functional Analysis Of Several Genes In Grass Carp As Perforin

Perforin (also known as pore-forming protein, PFP) is a glycoprotein which is produced by the activated cytotoxic T lymphocytes (CTL) and natural killer cells(NK). The cytolytic effect of perforin is a mechanism of anti-virus, killing microbial-infecte-d cells and tumor cells. Perforin is a very important non-specific immune factor. In order to understand the function of perforin, we amplified the cDNA of grass carp perforin C-terminal peptide. It contains a protein kinase C conserved region 2 (C2). Meanwhile, we use PCR site-directed mutagenesis to construct point mutations (D429A, D435A, D483A, D485A). These cDNA were connected with pET32a, then transformed to expression bacteria DE3. PFP-C and the four mutations were expressed by a prokaryotic expression system and then purified by affinity chromatography. It showed a significant haemolytic activity when PFP-C tested with rabbit red cells, the optimal pH for haemolytic activity was 7.5, and its haemolytic function dependents on Ca2+ rather than Fe3+ and Mg2+. Even in the presence of Ca2+, we can't see the haemolysis when four point mutant proteins tested with rabbit red cells. It means that the four mutant amino acids may be the key amino acids binding to Ca2+. So after mutant, it can't bind to Ca2+, and the haemolytic activity was lost.Glucose regulated protein 78 (GRP78), as a cellular stress-related protein, could regulate endoplasmic reticulum (ER) homeostasis by strongly responding to intracellular or extracellular stressors. The full length sequence of GRP78 was 2 490 bp, contained a 5'untranslated region of 155 bp with seven heat shock elements and a 3'untranslated region of 373 bp. The open reading frame was 1 962 bp which could code a 653 amino acid peptide. The deduced amino acid sequence of grass carp GRP78 contained three signature sequences of HSP70s in the N-terminus, and an endoplasmic reticulum retention sequence of KDEL in its C-terminus. The grass carp GRP78 shared the high homology with that of human and other animals. The analysis of adaptive evolution demonstrated that GRP78 was highly conservative and undergoing strong functional constraint, which illustrated it was very important in the course of evolution. The RT-PCR analysis showed that the expression of grass carp GRP78 was significantly enhanced in liver, kidney and other tissues after heat shock at 34℃and challenged by Poly I:C compared with the normal level.The full sequence of Annexin A4 was 1 307 bp, contained a 5'untranslated region of 104 bp and a 3'untranslated region of 237 bp. The open reading frame was 966 bp which could code a 321 amino acids peptide. The grass crap Annexin A4 had a tail (N-terminal region) and a core domain (C-terminal region). The N-terminus was composed of 15 residues. The core domain was made up of four similar repeats, each covered a type II calcium binding site. And there was a KGD motif in the fourth site. The grass crap Annexin A4 had high expression level in liver, kidney, spleen, heart, gill and intestine, but it was not detected in muscle and brain. Additionally, down-regulated expression of Annexin A4 was detected in all of the six tissues after grass carp was challenged by Poly I:C. Phylogenetic tree analysis revealed grass carp Annexin A4 shared the highest homology with zebra fish(Danio rerio). It has not been detected any positive selection sites by adaptive evolutional analysis, therefore Annexin A4 are highly conserved and undergoing strong functional constraint.