Study On The Function Of Several Key Proteins In Phospholipid Metabolism And Arginine Metabolism Of Eriocheir Sinensis

Chinese mitten crab (Eriocheir sinensis) is an economically important species cultured in China in recent years. With the expansion and intensification of aquaculture scale, various diseases caused by bacterium, viruses and parasites have become more common and severe, which directly led to magnificent economic lost to the aquaculture industry. The tremor disease (TD) is the most devastating disease of E. sinensis. A kind of spiroplasma is identified as a novel causative pathogen of the disease and given the name of Spiroplasma eriocheiris. The biochemical and biological properties, evolutional analysis, identified the pathogen and antibacterial test had been studied. Infection models of mouse 3T6 cells and the hemocytes of E. sinensis had been established successfully. However, the studies of molecular biology and protein function of S. eriocheiris are still in the early stage. This research is based on the genomic sequencing results of S. eriocheiris, and aims to reveal the mechanism of infection and energy metabolism during infection by screening and identifing for virulence factors involving in pathogenesis and energetic infection. Those works will lay the foundation for further researches on molecular mechanism of pathogenesis.1. Bioinformatics analysis, Clone, expression, purification and Enzyme analysis of Lysophospholipase in S. eriocheirisThe genome alignment shows S. eriocheiris genome only contain one phospholipase-Lysophospholipase (SE-LsyoPL). The DNA fragment of 927 bp encoding LysoPL was was discovered by sequencing the genomic DNA of S. eriocheiris. The DNA sequence and deduced amino sequence encodes 308 amino acid residues with a theoretical molecular weight of 35 kDa and pI of 9.64. Based on bioinformatics analysis, the SE-LysoPL protein sequence included the active site serine motif, GXSXG, which are both characteristics for lipolytic enzymes. The catalytic triad of SE-LysoPL can be assigned to Ser115, Asp254, and His288. SE-LsyoPL gene was cloned, point mutated, eukaryotic expressed and purified, recombinant enzyme activity was examined with p-NP palmitate as substrate, the optimal pH and optimal temperature of recombinant SE-LsyoPL was at pH 7.0 and 30 ℃, respectively. The recombinant enzyme was stable in the temperature range between 0-30℃, while no activity was detected under 50 ℃, suggested that SE-LsyoPL was a neutral and mild phospholipase. Analysis of effects on different divalent cations revealed that Ca2+ and Mn2+ activated the enzyme. Hydrolysis analysis on substrates showed that SE-LsyoPL performs higher activity against long-chain fatty acid than short-chain ones.2. Function study of of Lysophospholipase in S. eriocheirisThis experiment we successfully preparated the SE-LsyoPL polyclonal antibody using the SE-LsyoPL recombinant protein which has been purified. The indirect ELISA method was used to detect the titer of antiserum, the SE-LsyoPL antiserum titer at around 1:70000. The super high speed centrifugal method was used to extract the membrane protein of S. eriocheiris. Western blotting indicated SE-LsyoPL is a membrane protein in S. eriocheiri.In this experiment, we use the S. eriocheiri to infect the mouse embryonic fibroblasts (3T6 cell) in vitro, use the antibody neutralization test to close the SE-LsyoPL enzyme located on the surface of S. eriocheiri. Oxytetracycline assay showed the S. eriocheiri infection rate decreased during the process of infection on 3T6 cells in the early stage when SE-LsyoPL antibody concentration of 20 μg/mL. When the spiroplasma infected after 48h, the infection rate between neutralizing antibody group and spiroplasma infection group without difference (P>0.05). It is found that when the SE-LsyoPL antibody concentration of 2 μg/mL, spiroplasma infected rate did not change significantly. Further flow cytometry analysis on apoptosis against neutralized group and infected group revealed that neutralized subjects had lower apoptosis rate than infected subjects in early stage after 48 h of infection, the former subjects also appeared more likely normal on cell morphology, as well as larger in the number of them than infected group. The results of indirect immunofluorescence localization showed that S. eriocheiris sporadically distributed on the membrane of neutralized group and generated slightly green fluorescence, on the contract, infected samples filled with S. eriocheiris internally, much of them have become inclusion bodies with high intensity of fluorescence. This study showed when the spiroplasma SE-LsyoPL enzyme located on surface was blocked by the antibody, the spiroplasma infection rate decreased obviously.3. Growth characterization and arginine utilization of S. eriocheirisThis experiment we use a simple medium R8-1 to study the growth characteristics of S. eriocheiris, our results showed that spiroplasma could grow well in R8-1 medium. In this medium, the optimum growth temperature for the S. eriocheiris is 30-32 ℃, the maximum growth temperature of S. eriocheiris is 42℃, its optimum pH is 7.2-7.6, meanwhile its tolerance of pH range from 5.0-10.0. It was showed that approximately 20 mM supplement of arginine increased growth rate of S. eriocheiris significantly, and the stationary phage prolonged compare with those without arginine supplied. We use high performance liquid chromatography (HPLC) method to analysis the utilization of arginine of S. eriocheiris, the study showed spiroplasma couldn’t use of arginine during the the early stage of the culture, while it can gradually began to use the arginine only when it growth to stationary phages, meanwhile the two amino acids citrulline and ornithine aer also increased significantly, this suggests that spiroplasma can use of arginine as a source material.4. Bioinformatics analysis, Clone, expression, purification and Function study of Arginine deiminase (ADI) system in S. eriocheirisBased on the genomic analysis of S. eriocheiris, a typical ADI system, which contained the complete ADI gene cluster (ADI, arginine deiminase; OTC, ornithine carbamyl transferase; AOA, arginine ornithine transferase; CK, cabarmyl kinase CK) was discovered by bioinformatics analysis. Three key genes (ADI, OTC and CK) of the gene cluster were cloned, point mutated, expressed and purified. Consequently, the polyclonal antibodies of purified recombinant proteins were prepared. Western blotting analysis found that two enzymes CK and OTC are distributed in S. eriocheiris membranes. Through the different pH, temperature, mediums were supplemented with/without arginine and glucose, using of RealTime PCR and Western blotting technology to study under different environmental conditions. Our study shows that, the gene and protein expressions in ADI system are all increased significantly under the high temperature and acid (pH=5) and other odious conditions. But when cultured in the presence of high concentration of sugar in the medium conditions, the ADI system will be obvious inhibition. Those indicated that ADI system played an important role both in energy metabolism and stress resistance.5. Bioinformatics analysis, Clone, expression, purification and Function study of Agmatine deiminase (AgDI) system in S. eriocheirisGenomic analysis revealed that S. eriocheiri genome also contains a complete agmatine deiminase (AgDI) system. Gene cluster containing complete genes include agmatine deiminase (AgDI), agmatine-putrescine transporter (A/P) and putrescine Carbamoyltransferase (PTC). We studied two kinds of key prote inincluding agmatine deiminase AgDI system (AgDI) and putrescine Carbamoyltransferase (PTC) by gene cloning, point mutations, recombinant protein prokaryotic expression, protein purification and preparation of polyclonal antibody. RealTime-PCR and Western blot under different conditions, including different pH, temperatures and under the differen condition of glucose, were performed to study the transcriptional level and protein expression level of those key enzymes in AgDI system. Our study showed that, in acidic (pH=5) adverse environment, gene expression levels and protein expression levels of AgDI system increased significantly, however, under the environment of high temperature, expression of AgDI enzyme did not significantly increased, which indicated the AgDI system is not obvious activation under high temperature. When cultured in the presence of high concentration of sugar in the medium conditions, the AgDI system will be obvious inhibition. Results showed AgDI system in the S. eriocheiri plays an important role in energy metabolism and resist the pH acidic adverse environment, but it showed no obvious change under high temperature conditions.