Study On Antimicrobial Factors And A Pattern Recognition Protein Of Chinese Shrimp, Fenneropenaeus Chinensis

Research on innate immunity of the penaeid shrimp is motivated greatly by economical necessities. Indeed, the aquaculture of these organisms is now limited by the development of infectious diseases. Antibiotics have been widely used to control bacterial diseases, but long-term application of antibiotics can lead to bacterial resistance. Abuse of antibiotics can cause serious damage to the environment and human health. Anti-microbial peptides/proteins (AMPs) are a kind of important host immune factors, which are present in almost all phylum,and demonstrate a broad spectrum of antibacterial, antifungal and antivirual activity. Studying of AMPs is particularly attractive not only for progressing basic knowledge on immunity but also because they offer various possible applications for disease management in aquaculture. AMPs are promising substitutes of antibiotics. Pattern recognition proteins (PRPs) play an important role in innate immunity by recognizing common epitopes on pathogen surfaces, which usually are carbohydrate moieties. Three AMPs including Fenchin-Pen5, crustin and ALFFc, and a PRP,lipopolysaccharide- andβ-1, 3-glucan binding protein (LGBP) were cloned and characterized from hemocytes of Chinese shrimp, Fenneropenaeus chinensis in this paper. A new member of antimicrobial peptide genes of the penaeidin family, Fenchin-Pen5, has been cloned from the haemocytes of F. chinensis, by reverse transcription PCR (RT-PCR), 3' -RACE and 5' -RACE methods basing on the EST information. It was ranged into a new subgroup Pen5, which different from the three known subgroups of Pen2, Pen3 and Pen4. The Fenchin-Pen5 cDNA is 659 bp and the open reading frame of the cDNA encodes a 79 amino acid peptide. Fenchin-Pen5 contains a putative NH2-terminal signal sequence (1-19) followed by a mature peptide including 60 amino acid residues. The signal sequence of Fenchin-Pen5 is almost completely identical to that of other penaeidins. The mature peptide contains a proline-rich domain at the N terminus and 6 conserved cysteine residues at the C terminus, ant it shares 54% to 60% amino acid sequence identity with the mature penaeidins of Litopenaeus vannamei and L. setiferus. The mature peptide has a predicted molecular weight of 6446.53 Da, and a pI of 9.55. The expression and distribution of Fenchin-Pen5 in unchallenged shrimps were studied by RT-PCR. The results showed that the Fenchin-Pen5 transcripts were detected in hemocytes, gill, intestine and ovary of the shrimp, but little in hepatopancreas or muscle. The expressive profiles of penaeidin mRNA in hemocytes of Chinese shrimp challenged with killed bacteria (Vibrio anguillarum and Staphylococcus aureaus) were measured using semi-quantitative RT-PCR method. The results revealed that challenge with bacteria did not result in a significant increase of the mRNA level in hemocytes, but a brief decrease at 6h and 12 h after injection. We reported the molecular cloning and characterization of another AMP, crustin cDNA from the hemocytes of F. chinensis. The Chinese shrimp crustin cDNA is 489 bp in length with an open reading frame of 390 bp. The deduced amino acid sequence of this AMP consists of 113 amino acid residues of a mature peptide and a signal peptide of 17 amino acid residues. The Chinese shrimp crustin shares 40% and 66-76% amino acid sequence identity with that of Carcinus maenas and other penaeid shrimp. The mature peptide has a predicted molecular weight of 12324.5 Da, and a pI of 8.50. Like other animals' crustins, Chinese shrimp crustin possesses conserved sequence identities of proteins that have confirmed or putative proteinase inhibitory activity. All of these peptides have one four-disulfide core (4-DSC) domain, comprising approximately 50 amino acids and including eight cysteine residues in a conserved arrangement. Northern blotting showed that the cloned crustin genes were mainly expressed in haemocytes, gill, intestine, and RNA in situ hybridization (ISH) indicated that crustin gene was constitutively expressed exclusively in haemocytes. Capillary electro...