An Early Manifestation Of The Adaptive Immune System And Cloning And Expression Of Ikaros Gene In Fish

Fish has been living in the water from the stage of fertilized eggs, however, at the same time, all kinds of pathogens also lives around them and attack them all the time. The adult fish will carry out innate immune system firstly when they are attacked, and if pathogens have invaded into body, fish will begin to start the adaptive immune system (AIS). Then how do the embryos resist attack from pathogens successfully and protect themselves? When do the AIS functionally mature and what is the protection mechanism of immune system? The issues have been focused for a long time. To be close to the answers, the following research is carried out. Firstly, the expression of some marker genes involved in the AIS and response to LPS in zebrafish embryos/larvae are detected, and then Ikaros gene which is a transcription factor related to blood cell differentiation is cloned and analyzed about its structure and expression pattern.The first section of this paper dealt with the early manifestation of the AIS in zebrafish. The ontogenetic and differential expressions of seven key genes (Rag2, AID, TCRAC, IgLC-1, mIg, sIg and Mznf297) that are the suitable markers for indicating the maturation of AIS and their responses to the challenge with LPS during development of D. rerio are demonstrated. Apart from Rag2 and Mznf297, the remaining five genes (AID, TCRAC, IgLC-1, mIg and sIg) display generally similar trends of expression with the expression levels being relatively low at early stages and then rising subsequently. Rag2 and Mznf297 are both expressed at relatively high levels at the gastrula stage, decrease dramatically later, followed by a slight steady rise. In contrast, all these genes are capable of responding to the challenge with LPS in the embryos/larvae by up-regulating their expression levels at least from 23 dpf (day post fertilization) onward in a fashion similar to that of adult D. rerio, which has a mature function in AIS. It is therefore suggested that the AIS in D. rerio larvae has been founded during the course of ontogeny development, slightly earlier than considered previously. The cDNA of Ikaros from half-smooth tongue sole codes for a protein of 430 amino acids. To investigate the relationship between Ikaros from half-smooth tongue sole and other species, a phylogenetic tree is constructed using the amino acid sequence of Ikaros and that of other representative Ikaros from 14 species and other family members including Helios, Aiolos, Eos. It is found that Ikaros from half-smooth tongue sole formed a cluster together with that of some fish including Japanese medaka, Japanese amberjack, trout and zebrafish. Further comparison between some species and Ikaros reveals that the regions that show higher homology focus zinc finger domains at N and C-terminal. This is consistent with previous results. Using the method of CLUSTAL W in DNAstar, the identity in structure between half-smooth tongue sole and other species is analyzed, and the result is similar to that of phylogenetic tree. Ikaros from half-smooth tongue sole shows higher identity with Japanese medaka, Japanese amberjack, trout and zebrafish, the percent identity is 80.0%, 76.7%, 66.7% and 57.3%, respectively. In many species, Ikaros has been proved to be a transcription factor related to immune system, and to test this, a series of experiments are carried out. Firstly, the expression of Ikaros gene in different organs is detected. According to the result of QRT-PCR, Ikaros gene shows higher expression levels in thymus, liver and spleen, and moderate expression levels in kidney, testis and heart; however, in intestine, skin or gill, we hardly detected expression of Ikaros gene. This is consistent with that of skate, lamprey and axolotl. Ikaros gene shows different expression levels in organs related to immune and conservative expression pattern, which imply that Ikaros from half-smooth tongue sole probably has the same function with that of other species. Then, macrophage cells from the head kidney of half-smooth tongue sole are isolated and have been cultured for 7 days. At the seventh day, the cells are challenged by LPS or LTA, and then detected by Q-PCR technology. Compared to the control, the expression levels of Ikaros gene are up-regulated significantly in cells post LPS and LTA challenge. This indicates Ikaros gene which has showed expression in macrophage cells from head kidney of half-smooth tongue sole is a factor related to immune.