Pattern Recognition And Effectors Of Innate Immunity In Chinese Shrimp (Fenneropenaeus Chinensis)

Chinese shrimp (Fenneropenaeus chinensis) was mainly distributed in Yellow and Bo sea of China and western coast of Korean Peninsula as an economically and nutritionally important shrimp. Excessive capture and infection by many pathogens such as bacteria and virus seriously embarrassed survival of the animal. Recent years, marine aquaculture of F. chinensis was carried out widely around the coast of China and so the production of cultured animals were far more than the captured ones. However shrimp cultivation has been suffering serious problems due to the outbreak of the white spot syndrome since 1993. The high mortality of F. chinensis in cultivation was either due to the serious infectious activity of the the white spot syndrome virus or to the low immune activity of the animal. So, it is necessary to study the immune system of F. chinensis. This investigation could make us know more about the immune defence mechanisms of invertebrate. On the other hand, knowledge on immune system of shrimp could be helpful in F. chinensis cultivation through improving its immune activity and consequently reduce economical loss.The panning of T7 phage display library, homology cloning and suppression subtractive hybridization (SSH) were used to obtain immune related genes in the studies. A peritrophin, a lipopolysaccharide andβ-1,3-glucan binding protein (LGBP) and three crustin genes were cloned from F. chinensis, and characterized.1. Identification and molecular characterization of a peritrophin-like proteinfrom fleshy prawn (F. chinensis)T7 phage display library was constructed with mRNA from S. aureus and Vibrio anguillarum challenged shrimp. After three rounds of panning a fragment of peritrophin gene was obtained from the library. Modified SMART (Switching Mechanism At 5' end of RNA Template) cDNA and the long distance PCR (LD-PCR) procedures were used to complete the 5' and 3' ends of the gene with gene-specific primers and adaptor primers. The full length of the cDNA of peritrophin of F. chinensis (Fc-PTPH) was 1048 bp including an 822 bp open reading frame encoding a 274-residue protein. There was a 17 bp untranslated region in the 5' end and a 209 bp untranslated region in the 3' end. The molecular weight of the deduced protein was 30.6 kDa and the theoretical pI was 4.92. The deduced protein, with a signal peptide (1-20 residues), contained four peritrophin A-like domains and the latter three domains was chitin-binding domains (CBDs) (residues 83-145, 146-199 and 201-258) by SMART analysis.The results of Northern blot and RT-PCR demonstrated that Fc-PTPH constitutively expressed in ovaries but did not express in hepatopancreas at all. Otherwise, in other tissues including haemocytes, heart, stomach, gills, intestine and spermary the gene was induced expression. The time course expression patterns of Fc-PTPH after bacteria-challenge were examined in intestine and ovary with Northern blot. The results showed that Fc-PTPH is up-regulated in intestines after bacterial challenge: expression of the gene was detected by 16 h after infection, and at 24 h post-infection the transcription of the gene reached the highest level. A very strong signal was detected in ovaries and the signal intensity did not change between the 4-24 h infection periods. The mRNA of Fc-PTPH was clearly located in the cytoplasm of oocytes of ovaries and a fraction of the hemocytes in challenged shrimp.Basing on full sequence a pair of primers were synthesized to amplify the mature Fc-PTPH cDNA fragment. Then the fragment was subcloned into pET-30(+) vector and the recombinant plasmid was transformed into competent cells of E.coli BL21(DE3) for recombinant expression. Because inclusion bodies were formed denaturation and refolding were introduced before purification with His Bind resin chromatography. Polyclonal antiserum was obtained by injection purified recombinant expressed FC-PTPH (rFC-PTPH) into rabbit and the titer and specificity was examined with double immunodiffusion and Western blot.Binding activities of rFC-PTPH was analyzed and the results indicated that rFC-PTPH could tightly bind to chitin and E.coli but could not bind to 5. aureus at all.2. Molecular cloning and characterization of a lipopolysaccharide andβ-1, 3-glucan binding protein from fleshy prawn (F. chinensis)Two pairs of degenerate primers were designed to obtain a fragment of the LGBP gene from haemocytes of F. chinensis (Fc-LGBP) utilizing nested PCR and then the full length cDNA was obtained with modified SMART cDNA and the LD-PCR procedures. The full-length LGBP cDNA fragment (1253 bp) of F. chinensis was obtained by overlapping three cDNA fragments and included a 7 bp 5' untranslated region (UTR), a 1101 bp open reading frame and a 129 bp untranslated region in the 3' UTR with a 16 bp polyA tail. The ORF encoded a 366 amino acid protein with a 17 amino acid signal peptide. The mature Fc-LGBP, therefore, consisted of 349 amino acid residues with a calculated molecular mass of 39.8 kDa and a predicted isoelectric point of 4.43. The mature protein included two putative glycosylation sites(Asn-Xaa-Ser) for N-linked carbohydrate chains and a putative cell adhesion site (integrin binding site), Arg-Gly-Asp (RGD). Additionally, a glycosyl hydrolase domain was identified (Ser 94 to Ala 312).The expression profiles of bacteria-challenged haemocytes, heart, hepatopancreas, stomach, gills and intestine were analyzed by Northern hybridization. The result demonstrated that Fc-LGBP only expressed in haemocytes and was down-regulated by bacteria-challenge post 24 h injection. This result was confirmed by RT-PCR. Because of more sensitivity of RT-PCR gene expression could also be detected in hepatopancreas and gills although the signals were weak. Utilizing in situ hybridization, we investigated a variety of tissues types to localize Fc-LGBP mRNA in challenged fleshy prawn. Our data suggested that hybridized signal was only detected in haemocytes, with strong signals noted in the cytoplasm of granular haemocytes.The sequence coding for mature protein was amplified and ligated into expression vector pET30a(+) . The recombinant vector was transformed into competent E. coli BL21(DE3) host cells. After denature and refolding the recombinant FC-LGBP (rFC-LGBP) was purified with His Bind resin chromatography. Polyclonal antiserum was obtained from rabbit after continuous injection of rFC-LGBP.With polyclonal antibodies we located FC-LGBP in haemocytes. Goat anti-rabbit IgG conjugated with FITC was used as second antibody. The result confirmed FC-LGBP membrane localization in most haemocytes, with granule-like signals noted in some areas of haemocyte membrane. With polyclonal antibodies we also studied the nature protein in different tissues including plasm, haemocytes, heart hepatopancreas, gills. After incubation with second antibody (HRP-conjugated goat anti-rabbit IgG) and visualization the natural FC-LGBP was characterized as a 40kDa protein in haemocytes and hepatopancreas. But in other tissues no signal could be detected at all.Gram-negative bacteria (E. coli; Klebsiella peneumoniae), Gram-positive bacteria (S. aureus; Bacillus thuringiensis; Micrococcus luteus; Bacillus cereus; Bacillus megaterium) and yeast (Pichia pastoris) were utilized to analyze FC-LGBP binding activity. Our data suggested that FC-LGBP proteinwas able to strongly bind to Gram-negative bacteria (K. peneumoniae) and relatively less to E. coli, Gram-positive bacteria (M. luteus, B. megaterium) and yeast (P. pastoris).We noted no binding activity to S. aureus, B. thuringiensis or B. cereus.3. Molecular cloning and characterization of three crustin-like proteins from fleshy prawn (F. chinensis)In the haemocyte SSH library of Chinese shrimp a fragment of a crustin-like gene was obtained. With gene specific primers, the 3' end of the crustin cDNA was obstained from SMART-cDNA and the gene was named Fc-crustin I. During cloning the 5' end of Fc-crustin I a crustin-like fragment that did not overlap with Fc-crustin I very well was obstained and named Fc-crustin II. A gene specific primer was synthesized to get the full cDNA of Fc-crustin II gene. In the result of 3' end cloning of Fc-crustin I a band other than the expected one was detected. The fragment exhibited high similarity with crustin-like protein and then named Fc-crustin III. The full cDNA was also got from SMART-cDNA with a gene specific primer.There was a complete WAP (whey acidic protein) domain in the obtained fragment of Fc-crustin I. The domain was near the C terminal of the protein and composed of 49 amino acid residues.The full length of the cDNA of Fc-crustin II of F. chinensis was 473 bp including a 390 bp open reading frame that encoding 130 amino acids. There was a 4 bp untranslated region in the 5' end and a 79 bp untranslated region in the 3' end. The molecular weight of the deduced protein was 14.0 kDa and the theoretical isoelectric point was 8.60. The former 17 amino acids formed a signal peptide. The deduced protein also contained a WAP domain (residues 83-124).The full-length Fc-crusin III cDNA of F. chinensis was obtained by overlapping two cDNA fragments and composed of 574 bp including a 38 bp 5' untranslated region (UTR), a 462 bp open reading frame and a 74 bp untranslated region in the 3' UTR1. The ORF encoded a 154 amino acid protein with a 20 amino acid signal peptide. The deduced WAP domain was compsed of residues from 62 to 127. The molecular weight of the deduced protein was 17.79 kDa and the pI was 4.64.Haemocytes, heart, hepatopancreas, stomach, gills, intestine, ovary and spermary of normal and bacteria challenged F. chinensis were selected to study the expression patterns of three crustin-like genes with RT-PCR method. The data demonstrated that Fc-crustin I expressed in all the selected tissues at different levels. There was a strongest signal in haemocytes and weakest signal in spermary. Bacteria-challenge could not change expression levels of the gene evidently in most tissues. But the expression of Fc-crustin I was up-regulated obviously in stomach. Expression pattern of Fc-crustin II was similar to that of Fc-crustin I. The gene also expressed in all the tissues and the highest expression level was also detected in haemocytes. After bacteria-challenge expression of Fc-crustin II was up-regulated obviously in heart, gills and ovary, but there were no obviously changes in other tissues. The exprssion pattern of Fc-crustin III differ distinctly from the former two genes. The signals could only be detected in some tissues including normal heart, ovary and challenged stomach, ovary. Otherwise there were weak signals could be detected in challenged heart and intestine.The cDNA fragments encoding for mature proteins of Fc-crustin II and Fc-crustin III were subcloned into pET30a(+) and expressed in E. coli. The mature proteins purified with His Bind resin chromatography were used to analyze the antimicrobial activities against Gram-positive and Gram-negative bacteria. But no activities could be detected. And then the fragments coding for all the amino acids including signal peptide were amplified with new primers and expressed in E. coli. The antimicrobial activities of recombinant expressed FC-crustin III could not be detected. But the antimicrobial activities of recombinant expressed FC-crutin II against two Gram-pssitive bacteria including Micrococcus luteus and Staphylococcus aureus could be detected with cylinder-plate method.In conclusion, Peritrophin, LGBP and crustin genes had been successfully cloned from Chinese shrimp and their functions and features were characterized. These works accumulated our knowledge on immune system of shrimp.