| The Vibrio parahaemolyticus(Vp) is a zoonosis pathogen. It is not only an important vibriosis pathogeny of aquatic animals, but also a kind of common food-borne pathogenic bacteria. It may cause food poisoning, even can lead to serious disease like gastroenteritis. In addition, the Vp can cause wound infection, what’s worse, it can cause sepsis and even lead to death. It is very harmful to human’s healthy. Zebrafish is one of the most important vertebrate model. There is a particular advantage to study innate immune response on the organ level and individual level for only innate immune system within 3 weeks after fertilization.To test the bacterial virulence The zebrafish was challenged by Vp from different sources. We concluded that Vp13 was the most virulent strain, so it was used to preliminarily explore the Notch moleculars, inflammatory cytokine factor il1 b and il10 expression levels after infected by the Vp13 in zebrafish embryos. In order to further explore the Notch signal mechanism in Vp infected zebrafish embryos, we used CRISPR/Cas9 to get notch1 a and notch1 b mutants, which provided valuable materials for further research about infection mechanism. Achievements of this study were as follows: 1) We established an adult zebrafish model infected by Vp, and used the accumulation method and Bliss method to calculate LD50. The virulence of human pathogenic bacteria: ATCC17802, ATCC33847, and aquatic animal pathogens Vp13, Vp31, Vp41, Vp57 were tested, and discovered the accordant results that the Vp13> ATCC33847> ATCC17802> Vp57> Vp31> Vp41, and the virulence of Vp13 strain was strongest, so we utilizated it for the following study. 2) We infected 1dpf(day post fertilization), 2dpf, 3 dpf and 5dpf wild type of zebrafish embryos with Vp13, and tested the expression levels of Notch1 receptor(notch1a, notch1b), the ligand(delta a,delta b, delta c), inflammatory cytokine factors(il1b, il10 and il6, tnfa and ifnphi), chemokines(ccl-c5 a, cxcl-8a) and the important genes of the TLR signaling pathways(tlr2 and tlr4, myd88, mmp9 and hey1). We infected 1dpf, 2dpf, 3dpf and 5dpf embryos, results indicating that: notch1 a, notch1 b and four kinds of ligands had similar trend about 3dpf zebrafish embryos infected with Vp13, reaching the minimum level at 4hpi. The notch1 a and notch1 b showed a negative regulation role on TLR signaling pathway. The expression levels of notch gene reached to the top after 2dpf zebrafish embryos infected with Vp13 at 2h. Vp13 could activate Notch signaling pathways, and they could participate in innate immune response. 3) In order to further study the role of Notch signaling in innate immune mechanism induced by Vp13, we used the advanced technology CRISPR/Cas9 to knockout notch1 a and notch1 b gene, and successfully got the mutants. It provided a good experimental material for further in-depth study of Notch molecules mechanism involved in the innate immune response. The Cas9 / g RNA system could produce different types of mutations for notch1 a and notch1 b, and the efficiency was above 70%. Compared with ZFN or TALEN, Cas9 / g RNA system was a simple and economic way to edit gene, and it could be more effective to produce indels(insert/deletion) mutations at specific sites.To conclude, the control measures used in the disease caused by Vp directly affected the course of the disease. At the early stage of the disease, the body initiates the innate immune response against infection, which becomes the focus of modern medicine research. In our study, we used Vp as the object to establish an animal infection model of adult and larvae zebrafish. We made the preliminary reseach on expression levels of Notch molecules involved in the innate immune response, so as to got effective means and methods to control the immune response in the moderate range, and provided the basic theory of disease prevention for the Vp infection. |
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