Isolation of Tanichthys albonubes beta actin gene and production of transgenic Tanichthys albonubes.

A beta actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5'UTR and a 564-bp 3'UTR. Genomic DNA containing the transcription region and 5'-flanking region was cloned based on the beta actin cDNA by Genome walker. A 3,000-bp beta actin gene promoter was then produced by PCR according to the sequences of the 5'-flanking region and the first intron. This promoter consisted of a 1,800-bp 5'-flanking region, and a 1,200-bp 5'-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5'-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The beta actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs. Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.

Cloning and characterization of the tiger shrimp lysozyme.

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.

Molecular cloning of two C1q-like cDNAs in mandarin fish Siniperca chuatsi.

C1q, a subunit of the C1 complex, plays a key role in the recognition of immune complexes to initiate the classical complement pathway. In this study, we reported two C1q-like cDNAs from mandarin fish (Siniperca chuatsi), mC1q-like-1 (mC1qL1) and mC1q-like-2 (mC1qL2). The full-length cDNA of mC1qL1was 990bp, containing a 71bp 5'-untranslated region (UTR), an open reading frame (ORF) of 723bp, and a 196bp long 3'-UTR. mC1qL2 cDNA was 1193bp, containing a 100bp 5'-UTR, followed by an ORF of 756bp and a 3'-UTR of 337bp. mC1qL1 and mC1qL2 share 29% identity in amino acid sequence. Both mC1qL1 and mC1qL2 contained three parts: a short amino-terminal region, a collagen-like region and a carboxyl-terminal globular C1q domain. The phylogenetic analysis showed that mC1qL1 clustered with two Danio rerio hypothetical proteins and further grouped with C1q proteins, while mC1qL2 clustered with C1qA proteins from other species. In healthy mandarin fish, mC1qL1 and mC1qL2 were expressed in all tissues tested, including liver, spleen, head kidney, caudal kidney, intestine and gill. mC1qL1 was highly expressed in head kidney, while mC1qL2 was mainly expressed in spleen. The expression level of mC1qL1 and mC1qL2 in liver were not changed obviously and mC1qL2 was significantly changed (p<0.05) in spleen after infectious spleen and kidney necrosis virus (ISKNV) infection. Mandarin fish C1q may play a role in response to ISKNV infection.

[Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.