Establishment Of Two Kinds Of Target Cells Model Of Eriocheir Sinensis And Its Preliminary Screening Of Mutation - Related Proteins

The freshwater Chinese mitten crab, Eriocheir sinensis, is an economically important species cultured in China in the last few decades. With the development of intensive aquaculture, various diseases caused by bacteria, viruses and parasite have emerged, and started to threaten the sustainability of the aquaculture populations of E. sinensis, severely affecting its production. The tremor disease (TD) is the most devastating disease of E. sinensis. A spiroplasma was previously 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, but the research on the study about S. eriocheiris in vitro infecting the host cells was blank. Proteins, which working during the infection process were not known. In this article, we build two infection models about S. eriocheiris infected mouse 3T6 cell and primary culture hemocytes of E. sinensis. We find a receptor protein on host cells by Far-western blot method.1. S. eriocheiris infected mouse 3T6 cell and pathological researchAccording to the literature, Spiroplasma mirum grew in the mouse 3T6 cells. Due to the similarities between the two Spiroplasmas, S. mirum and S. eriocheris, S. eriocheiris BY-10 was selected to establish an infection model of mouse 3T6 cells. By immunofluorescence labeling spiroplasma, The S. eriocheiris enter 3T6 cell with a single form. After entering the 3T6 cells for 48 hours, spiroplasmas formed aggregative bodies wrapped by membrane. With a transmission electron microscopy, the bodies are detected as intracellular inclusion bodies. S. eriocheiris-infected 3T6 cells exhibited vacuolization. Entry of the spiroplasma into the 3T6 cells was analyzed quantitatively by oxtetracycline protection assays and qualitatively by immunofluorescence microscopy and TEM.2. Primary culture hemocytes of E.sinensis and its S. eriocheiris infectionIn order to investigate the interaction between hemocytes of E. sinensis and S. eriocheiris, a primary culture system for in vitro culture of the hemocytes of E. sinensis with high viability was developed. Sterile anticoagulant citrate dextrose solution B (ACD-B) was employed. Separate cells with haemolymph Mixture by Centrifuge speed 1000 rpm10 min. In this context, a modified method of hemocytes culture from this crustacean has been standardized by employing L-15 growth medium supplemented with 15% Fetal Bovine Serum (FBS) along with 0.15% glucose,0.75% NaCl and antibiotics (100 U·ml-1 penicillin,100 U·ml-1 streptomycin) and a suitable pH of 7.20-7.40, incubated at 28℃ without 5% carbondioxide. Three types of hemocytes cells of E. sinensis in L-15 medium observed by inverted phase contrast microscope, Such as: granulocytes (GC), semi granulocytes (SGC) and hyalinocytes (HC). The susceptibility of the primary hemocytes culture was investigated by challenging with S. eriocheiris. Cytopathic effects (CPE) were observed as early as 36 hours post-inoculation, with cell debris and cellular exudates present in inoculated cultures. The green fluorescent Alex-488 was marked on the S. eriocheiris in fluorescence immunoassay to study the adhesion and infection of the spiroplasmas to the host cell. The results showed at about 24h post-inoculation, a large number of spiroplasmas were observed infected into hemocytes cells, which presented as a slug distribution. With a transmission electron microscopy, intracellular inclusion body was observed. And we also observed that a large number of spiroplasmas released from the ruptured cells.3. Screening the host receptor protein by Far-Western Blot method and a preliminary study of interaction S. eriocheiris with the host receptor proteinAn in vitro protein overlay assay identified one significant binding activity between S. eriocheiris proteins and host proteins from E. sinensis hemocytes. One hemocytes protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as Beta-Actin. We use immunofluorescence method to determine the intracellular localization of S. eriocheiris and receptor protein. The results showed that the green fluorescent labeling spiroplasma attached to the red fluorescent labeling Actin proteins.