Functional Study Of C-type Lectin And Its Ligand Spiroplasma Glycosylation Protein Of Macrobrachium Nipponense

Macrobrachium nipponensis, known as freshwater prawn or river prawn, is one of the most promising species in many Macrobrachium species. However, as its farming scale continues to expand and the density continues to increase, the risk of its disease is also greatly increased. There are many types of pathogens in Macrobrachium,including bacterias and viruses. The diseases are becoming more and more serious. In recent years, Spiroplasma as a new type of pathogens was widely attentioned.Spiroplasma eriocheiris can cause the Chinese mitten crab (Eriocheir sinensis)suffering from "Tremor disease". Professor Wang further confirmed that the S.eriocheiris is not only the pathogen of the "Tremor disease" of the crab, but also the pathogen of aquatic crustaceans such as Procambarus clarkii, Penacus vannamei and Macrobrachium rosenbergii and so on. The scope of their host is gradually expanding.Because of lack of immunoglobulin, the defense mechanism of M. nipponensis is completely dependent on the innate immune system. C-type lectin as a class of very important immune factors are well known. Therefore, it is very important to study the infection mechanism of S. eriocheiris and the immune defense mechanism of M.nipponensis lectins. In this paper, firstly, three kinds of C-type lectins were cloned from the M. nipponensis, and studied on the immunoassay mechanism and functional characteristics. Secondly, three kinds of S. eriocheiris glycosylated proteins interacted with C-type lectins were screened by Far-western blotting, and studied on the immunoassay mechanism by in vitro recombinant expression, polyclonal antibody preparation and MTT cytotoxicity test. The study includes the following three aspects:1. Study on the Sequence Characteristics and Function of C-type LectinIn this part, three kinds of C-type lectins were obtained from M. nipponense,named MnCTLDcp1, MnCTLDcp2 and MnCTLDcp3. Sequence analysis showed that the three kinds of C-type lectins all contained a CTLD domains. RT-PCR showed that MnCTLDcpl and MnCTLDcp2 was expressed the highest in heart, while MnCTLDcp3 was expressed in the hepatopancreas. After the prawns were challenged by Staphylococcus aureus?Aeromonas hydrophila and S.eriocheiris, the expression level of them were all significantly up-regulated. After RNAi and stimulated by A.hydrophila, and expression level of MnCTLD were significantly down-regulated,indicating that the three kinds of C-type lectins were involved in the anti-bacterial reaction. In addition, those recombinant proteins could agglutinate and bind to various Gram-positive and Gram-negative bacteria. All those results demonstrated that C-type lectin plays a very important role in the innate immune of M. nipponense.2. Screening S. eriocheiris glycosylated protein using C-type lectinsIn this experiment, the total protein of the S. eriocheiris was extracted, then separated by SDS-PAGE and transferred onto the PVDF membranes. Three recombinant C-type lectins were incubated respectively, the bands were identified by mass spectrometry after TMB staining. The results showed that there were three S.eriocheiris glycosylated proteins interacted with C-type lectins, designated as ATP synthase subunit beta, molecular chaperone DnaK, fructose-bisphosphate aldolase.Three S. eriocheiris glycosylated proteins were cloned and recombinant expressed in E.coli BL21. The interaction between the three C-type lectins and S. eriocheiris glycosylated protein were verified by Far-western blotting. The results showed that the combination of the three glycosylated proteins with MnCTLDcp3 was the best and the binding ability to MnCTLDcpl was the weakest.3. Functional characteristics analysis of S. eriocheiris glycosylated proteinIn this research, the co-transfection experiments and immunoprecipitation experiments demonstrated that MnCTLDcp3 has a good interaction with the three glycosylated proteins. In addition, three S. eriocheiris protein were abundantly distributed on the cell membrane by incubated with polyclonal antibody. In the MTT cytotoxicity experiment showed that S. eriocheiris glycosylated proteins played an important role in the process of the S. eriocheiris infection.