Chinese Shrimp Body Condensation Cloning And Expression Of Immune Genes In The Reaction Process

The molecular mechanisms of the immune system in shrimp, Fenneropenaeus chinensis, areknown limitedly, despite its economic importance as an aquaculture species, especially in China. Itis very important for us to comprehend the immune response process of shrimps includingnon-self recognitions, signal transduction and mode of action. Crustaceans have open circulatorysystems and must seal wounds and keep bacteria from entering the hemocoel using efficientclotting systems. C-type lectin is regarded as a potential molecule involved in immune recognition,cellular interactions, pathogen coagulation and wound healing in crustacean. Transglutaminase(TG), serine proteinase (SP) and serine proteinase inhibitors (SPI) are participants in two differentcoagulation mechanisms in invertebrate. In this article, we cloned a novel C-type lectin gene(Fclectin), a transglutaminase gene (FcTG), two serine proteinase genes (SP-1 and SP-2) and twoserine proteinase inhibitor genes (SPI-1 and SPI-2) from Fenneropenaeus chinensis. Thecharacterization, tissue localization and expression after challenged by different pathogens werealso analyzed.A novel C-type lectin (Fclectin) with two tandem CRDs was cloned from hemocytes ofChinese shrimp Fenneropenaeus chinensis. All of the five residues at Ca2+ binding site 1 areconserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif presented in the twoCRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced thatFclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were mainlyconstitutively expressed in hemocytes by Northern blot and RNA in situ hybridization. Thevariation of mRNA transcription level in hemocytes during artificial infection with bacteria andwhite spot syndrome virus (WSSV) was quantitated by capillary electrophoresis RT-PCR. Anexploration of mRNA expression variation after LPS stimulation was carried out in primarilycultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified afterbacteria/LPS or WSSV challenge.A transglutaminase gene (FcTG) was cloned from haemocytes of Chinese shrimp. It containsa typical transglutaminase/protease-like homologue with all of three catalytic sites. All potentialCa2+-binding sites exist in FcTG except one residue. The deduced amino acids sequence of FcTGshowed the 93% identity with black tiger shrimp STG I. Based on the preservation of residues andsimilarity to other TG, we deduced that FcTG is a member of transglutaminase family. Transcriptsof FcTG were mainly expressed in hemocytes both from circulatory system and lymphoid organ.The challenge of pathogens could not change the expression of FcTG mRNA significantly. But thewound stimulation of inoculation would affect the expression profiles of FcTG mRNA.Two serine proteinase genes (SP-1 and SP-2) were cloned by using cDNA library of Chineseshrimp haemocytes. The full length of them is 1348bp and 1208bp respectively. They are allsecretory proteins. SP-1 is a Serine Proteinase Homologue (SPH) with a pseudo-clip domain. SP-2is a SPH with an intact clip domain, which is the first report of the shrimp. In proPO system, ppAand many other serine proteinase have the similar structure. So SP-2 may be involved in theimportant immune response system in shrimps. Transcripts of SP-1 and SP-2 were mainlyexpressed in hemocytes. SP-1 is also got its high expression level in the lymphoid organ. Bacterialchallenge could enhance the expression of SP-2 but not to SP-1. WSSV challenge could inducethe increase of the expression level of both these two genes.Two serine proteinase inhibitor genes (SPI-1 and SPI-2) were cloned by usingSMART-RACE and cDNA library. They belong to kazal and serpin family respectively. The fulllength of them is 606bp and 1734bp respectively. They are all secretory proteins too. SPI-1 hastwo conserved kazal domains and shows the identity of 76% with black tiger shrimp. It mightinhibit elastase, subtilisin and chymotrypsin according to its P1 position. SPI-2 is the first report ofserpin in shrimps. It shows 42% identity with crayfish serpin. SPI-2 might inhibit trypsin, ppA,thrombin and clotting enzyme according to its P1 position. Transcripts of SPI-1 and SPI-2 weremainly expressed in gill, hemocytes and lymphoid organ. After challenged by bacteria, theexpressions of them were all decreased and then recovered to the original level. InWSSV-challenged group, the expressions of SPI-1 and SPI-2 maintained a steady level before 12hpost-injection and decreased dramatically to the bottom in the end of the experiment. It might becaused by the reproduction of WSSV. The results implied that the activity of SP might increasesignificantly abnormally and the stable immune system balance of the animal might be destroyed.