| The transcription factor Pax5(also called B cell activation protein)is one of the members of the Pax family,which are located in the nucleus and have an important role in the process of B cell development from the pro-B to the mature B lymphocytes.In this study of common carp,which is one of the most popular fish in the agriculture of our country,total RNA was extracted from head kidney of carp,and then used for preparation of cDNA as a template.Two Pax-5 ORFs of common carp were obtained from amplification,including 1263 bp of Pax5 X1 and 1191 bp of Pax5 X5.Sequence analysis and evolutionary analysis were finished about both of the two splicesomes..To study the response of Pax5 agaist bacteria infection,the common carp in the experiment were stimulized by immersion or injection of Aeromonas hydrophila,a Gram-negative bacteria,the tissues in relation to the immune function were obtained at different time point after stimulation and then were detected by Real time quantitative PCR for analyzing the gene expression profile during the period of time.The results showed that Pax5 was expressed in the head kidney,spleen,liver,anterior intestine,middle intestine and posterior intestine,with the highest expression in the head and kidney,and the highest expression in each segment of intestinal tissue.After injection with Aeromonas hydrophila,the expression was significantly decreased in the headkidney and spleen.After immersed with Aeromonas hydrophila,the spleen also showed a significant down-regulated expression.The correlation analysis of Pax5 and IgM expression showed that there was no significant correlation between the two types during the early period after immune stimulation.Experiments were also carried out for detecting the expresstion profile of Pax5 of common carp during the early developmental stage from 1 day post fertilization to 29 days post hatch(dph).The quantitative PCR results showed that Pax5 gene expresstion appeared in 1-2 days post fertilization of maternal sexual expression,following reduction in expresstion after hatching.At 14 dph,Pax5 expression continued to increase,with the maximum expression at 29 dph.Since Pax5 begin to act from lymphoid progenitor cells to mature B cells,and then lost function until plasma cells stage,therefore,the early stage of the fertilization and hatching after initial response to the external environment may be associated with maternal plasma cells,Pax5 expression profile are compatiable to the expression pattern of IgM antibody in the developmant stage.Pax5 and IgM expression shows a positive correlation between the expression quantity.In further study,the Pax5 protein induced and purified from E.coli were used to prepare polyclonal antibody of Pax5 for western blot detection,It can be seen from the results,the Pax5 protein can be detected from the samples of larva common carp and the results are consistent with that from quantitative PCR for mRNA detection,the highest expression level of Pax5 protein of common carp during the larvae development stagewas at 29 dph,which is a little bit lower than the amount of fish serum in the expression of Pax5.Specific immune originate from the fish,the lower vertebrates,the study of the immune molecules and immune cells of fish will contribute to further understanding of the origins and evolution of specific immunity,What’s more,transcription factor can be used as not only a marker to distinguish the different development stages of B lymphocytes but also for better understanding of the differentiation mechanismof B lymphocytes,and on the reason,carp Pax5 gene cloning and function are well worth studying,It will help to further study about the mechanism of B lymphocytes development on the basis of these data. |
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