Recombinant Expression And Functional Analysis Of Two Proteins Related To Immunity Of Chinese Shrimp, Fenneropenaeus Chinensis

Based on the techniques of recombination, transmembrane protein FcTetraspanin-3and antibacterial protein FcCar related to the immunity of Chinese shrimp (Fenneropenaeus chinensis) were expressed, and the functional roles of these two proteins were detectedThe FcTetraspanin-3constitutive expression in all examined tissues and the expression of the gene were highly induced by white spot syndrome virus (WSSV) challenge. The large extracellular loop (LEL) of FcTetraspanin-3was cloned from the cDNA library of Chinese shrimp. We expressed and purified the recombinant peptide containing the LEL domain of FcTetraspanin-3, and produced the anti-LEL poly clone antibody. The expression of FcTetraspanin-3was observed by Real-time PCR and Western blot, the high expression appeared in hepatopancreas, ventral nerve cord and intestine. Also, the localization of FcTetraspanin-3-positive cells in intestine and hepatopancreas were revealed by immunofluorescence,FcTetraspanin-3was mainly localized at the membrane of columnar epithelial cells in intestine and glandular cells in hepatopancreas. The results of anti-LEL antibody blocking experiments shown that the antibody can significantly reduce the mortality of shrimp challenged by WSSV. Additionally, dsRNA interference was utilized to examine the functional role of FcTetraspanin-3in response to WSSV infection, and a sensible decrease of the viral copy number in the tetraspanin knockdown shrimp. These results suggested that FcTetraspanin-3might play an important role in response to WSSV infection, the LEL domain of FcTetraspanin-3might mediate the entry of WSSV. These works on the virus and host would be helpful for white spot syndrome virus disease control.In this investigation, a Crustin gene (named FcCar) was obtained from Chinese shrimp by3’RACE and5’RACE. The full-length cDNA of FcCar consists of465bp open reading frame (ORF) encoding a17.8kDa protein. A BLAST search revealed that FcCar is most similar in amino acid sequence to carcinin protein. This is the first carcinin gene obtained from penaeus shrimp. The transcripts of FcCar were primarily observed in the ovary. Expression analysis revealed the transcript levels of FcCar were up-regulated upon infection treatments of Vibrio or white spot syndrome virus (WSSV). The recombinant FcCar protein exhibited antimicrobial activity against some Gram-positive bacteria in vitro, but not any Gram-negative bacteria. The data suggest that FcCar might play an important role in the innate immunity of shrimp.Our study illustrates the expression of two kinds of Chinese shrimp immune-related proteins FcTetraspanin-3and FcCar in different tissues, the immune response of them after WSSV infection, and the analysis of their roles and mechanism in pathogen resistance. Our study can provide a theoretical basis for the establishment of an effective method to control shrimp disease.