Molecular Cloning and Expression Profile of Putative Antilipopolysaccharide factor in Chinese Shrimp(Fenneropenaeus chinensis)

Abstract

A new antimicrobial protein gene of the anti-lipopolysaccharide factor family (tentatively named as ALFFc) has been cloned from hemocytes of the Chinese shrimp Fenneropenaeus chinensis by rapid amplification of 3' and 5' complementary DNA ends with polymerase chain reaction. The full-length complementary DNA of ALFFc consists of 600 bp with a 369-bp open reading frame, encoding 123 amino acids. The deduced peptide contains a putative signal peptide of 25 amino acids and mature peptide of 98 amino acids. The molecular mass of the deduced mature peptide is 13799.16 Da. It is highly cationic, with a theoretical pI of 10.3. The deduced amino acid sequence of ALFFc showed 56% homology with sequences of Tachypleus tridentatus and L. polyhemus. The tissue expression profile of this gene was studied by Northern blot, and ALFFc transcripts were mainly detected in hemocytes, gill, and intestine. RNA in situ hybridization showed that ALFFc was constitutively expressed in hemocytes. Capillary electrophoresis reverse transcriptase PCR was used to quantify the variation of messenger RNA transcription level during the artificial infection process with Vibrio anguillarum. Significant enhancement of ALFFc transcription appeared during the first 24 hours in response to Vibrio infection. These results provide useful information for understanding the function of ALFFc in shrimp.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

“…There were two conserved cysteine residues and a disulphide array in EsALF, which were identical to that of other ALFs [14]. More importantly, out of the 11 positively charged amino acids, 5 residues were clustered mainly within the disulphide loop consisting of 22 residues, which was the representative characteristic of ALFs from horseshoe crabs and shrimps [9,14,16,[18][19][20]. Another typical characteristic was the highly hydrophobic NH 2 -terminal region, including Figure 8 Bactericidal activity of the expression product, the negative control and blank.…”

“…The identification and characterization of immune effectors are believed to be helpful for elucidation of immune defence mechanisms and for the diseases control in crab aquaculture because of their potential use as therapeutic agents and genetic improvement biomarkers on disease-resistant strain selection. In crabs, many AMPs has been identified and characterized up to now, such as 6.5 kDa proline-rich cationic protein [33], Callinectin [34], Carcinin [35], Scygonadin [36] and ALF [9,[14][15][16][17][18][19][20]. However, molecular features and functional studies on AMP genes were still deficient in Chinese mitten crab.…”

“…Because one of the seriously infectious bacterial pathogens in Chinese mitten crab is Gram-negative bacteria in the genus of Vibrio [9], in which lipopolysaccharide (LPS) is believed to play an important role in the process of pathophysiology, identifying and characterizing AMPs with LPS binding or neutralizing activity is particularly attractive to control E. sinensis disease. In the last decades, several LPS-binding proteins or peptides had been characterized in vertebrates and invertebrates, such as bactericidal increasing protein (BPI) [10], lipopolysaccharides-binding protein (LBP) [10], lipopolysaccharide and b-1,3-glucan-binding protein (LGBP) [11,12], C-reactive protein [13] and anti-lipopolysaccharide factor (ALF) [9,[14][15][16][17][18][19][20].…”

“…Subsequent studies indicated that ALF from L. polyphemus exhibited strong antibacterial activity against Gram-negative R-type bacteria through neutralizing the lipid A, a common toxic moiety of LPS [21,22], and also the inhibitory effect on the growth of Gram-positive bacteria. Afterward, similar proteins were identified from the haemocytes of Litopenaeus vannamei [15], T. tridentatus [20], Penaeus monodon [17,18], Fenneropenaeus chinensis [9], Marsupenaeus japonicus [16], Pacifastacus leniusculus [23], and Scylla paramamosain [24], etc. The expression profile of ALFs indicated that the mRNA transcripts for the protein could be induced by Vibrio infection [9,18] or LPS administration [16], suggesting their crucial roles in crustacean innate immunity.…”

“…Afterward, similar proteins were identified from the haemocytes of Litopenaeus vannamei [15], T. tridentatus [20], Penaeus monodon [17,18], Fenneropenaeus chinensis [9], Marsupenaeus japonicus [16], Pacifastacus leniusculus [23], and Scylla paramamosain [24], etc. The expression profile of ALFs indicated that the mRNA transcripts for the protein could be induced by Vibrio infection [9,18] or LPS administration [16], suggesting their crucial roles in crustacean innate immunity. More importantly, the discovery of various ALFs and their derivatives had attracted great interest in mammalian health as candidate therapeutic or prophylactic agents for the management of septic shocks [22,25,26].…”