Molecular Cloning And Functional Analysis Of Two C-type Lectins From The Shrimp Litopenaeus Vannamei

C-type lectins exist in almost all the animals. As a pattern recognition receptor(PRR), C-type lectin is believed to mediate pathogen recognition and play an important role in the clearance of pathogens in innate immunity. A number of C-type lectins in crustaceans have been isolated and characterized in recent years. In this study, two C-type lectin genes (named Lvlectin1 and Lvlectin2) cloned from shirmp Litopenaeus vannamei were studied by molecular biotechnologies including Real-time PCR,recombinant protein expression and RNA interference(RNAi).The 161, 075 expressed sequence tags of Litopenaeus vannamei published at the NCBI have been jointed, noted and cluster analyzed(data not published) in our lab. The contigs and singletons noted as lectin were analyzed and two C-type lectin fragments (named Lvlectin1 and Lvlectin2) were selected frrom the ESTs assembled library of L. vannamei. Lvlectin1 and Lvlectin2 share high identity with C-type lectin receptor from Portunus pelagicus and C-type lectin 3 from Fenneropenaeus chinensis respectively. The open reading frame of Lvlectin1 is 510 bp, encoding 169 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain (CRD) at the C-terminal. Lvlectin2 gene contains a ORF which is 474 bp. The deduced amino acid sequence of Lvlectin2 is 157 amino acids, containing a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain (CRD) at the C-terminal. There is a potential carbohydrate-bingding motif"EPS"(Glu120-Pro121-Ser122) presented in the CRD may support its ability to bind mannose-type sugars. In healthy shrimp L. vannamei, Lvlectin1 and Lvlectin2 were both mainly expressed in hepatopancreas. Real-time PCR analysis indicated that Lvlectin1 and Lvlectin2 transcripts level show significant change in hepatopancreas after the shrimp were artificially challenged with LPS, Micrococcus lysodeikticus and white spot syndrome virus(WSSV).According to the deduced amino acid sequence analysis of Lvlectin1, there was no key motif EPN or QPD which is predicted to bind to mannose or galactose in its CRD. The function of Lvlectin1 was investigated by recombinant expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21(DE3). The recombinant Lvlectin1 could agglutinate M. lysodeikticus and Vibrio anguillarum. The agglutinating activities were calcium-dependent and could be inhibited by D-galactose, D-Glucose, D-mannose and N-Acetyl-D-mannose.In order to study the antiviral function of Lvlectin1, RNAi-based silencing of Lvlectin1 gene resulted in significantly increasing mortality rate when the shirmp were challenged with WSSV, and the median lethal time was much earlier than the control.Four BAC clones containing Lvlectin1 gene were screened with special primers for Lvlectin1 from the Bacterial Artificial Chromosome (BAC) Library of Litopenaeus vannamei, which will be utilized to research genomic characters, transcriptional promoter and regulating element of Lvlectin1.These results suggested that Lvlectin1 was involved in the immune response against bacterial and WSSV infections and contributed to nonself recognition as a pattern recognition receptor in the innate immune system of shrimp L.vannamei.