| Part I Construst of substruct cDNA libray of schistosomiasis mouseIn almost any area of biology or medicine question arise from differential gene expression. Transformation in the normal- schistosomiasis sequence result from the expression changes of specific gene(s) too. The construction of differential expression cDNA libray of schistosomiasis, Genes expressed exclusively or preferentially in schistosomiasis identification and characterization will, hopefully, shed light on the mechanisms of disease development and provide useful genetic markers for early diagnosis and therapy.There exists a large repertoire of techniques that aim at producing an inventory of differential transcripts between two populations of mRNAs. This is the basis to construct schistosomiasis related differential cDNA libraries. Despite the fact that all these methods have proven successful in isolation of differentially expressed genes,they all possess some specific intrinsic drawbacks: requiring a large amount of initiating materials, inefficient to obtain rare transcripts, significant incidence of false positive, intensive labor and so on. Suppression subtractive hybridization (SSH)is a new method recently described by Diatchenko in 1996. It combines subtractive hybridization with suppression PCR to generate a population of PCR fragments enriched for differentially expressed sequences. The particular aspect of the technique relies on the so-called normalization that equalizes the wide range in abundance of different transcripts. This allows the detection of rarely transcribed genes, on one hand, and prevents the excessive isolation of genes that are highly abundance, on the other hand.The SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization, so it only need 1-2 u g mRNA. But the base of screening genes related to disease is you must assure that there is good pair-matching between tester and driver, Thus can get better result from screening.During the process of schistosomiasis, the main host cell response to schistosoma is T lymphocyte in spleen, and hepatic stellate cells (HSCs) are the target cell of fibrosis. So we choose spleen and HSC as material can assure the specity of investigation.1. Material and methods:BALB/C mice challenged by cercariae of Schistosoma japonicum (S. japonicum) were served as animal model. In this study, SSH was performed to screen the differentially expressed genes between schistosoma japonica infected mice and normal mice. We obtaine spleen six weeks after challenge and obtain hepatic stellate cells (HSC) sixteen weeks after challenge as tester. And take normal spleen and HSC that obtain simultaneously as driver. Use SSH to construct subtractive libraies. The PCR products obtain from subtractive cDNA were clone to pGEM-T Easy vector through T/A clone. After Virtual Northern Blot and sequencing, take cDNA compareto GenBank with BLAST.102. ResultsWe constructed hepatic fibrosis of schistosomiasis mice and isolated HSC successfully. Constructed of substruct cDNA libray of schistosomiasis mouse with SSH. 76 ESTs were obtained by sequencing, by BLAST programme, results showed that there were some ESTs correlated with immunity in DS-NS library. And some ESTs correlated with cell differentiation and protein binding to growth factor, in NH-DH, it suggested that they were related to HSC activation and proliferation. Some ESTs known their function suggested the efficient of the cDNA libraries. Constructed these two libraries have provide the basis for identification genes related to schistosomiasis hepatic fibrosis.3.Conclusion(1) Isolation HSC from schistosomiasis hepatic fibrosis mouse. Using SSH, we first time have contracted two subtracted cDNA libraries, NS-DS and NH-DH, as mention above. Sequence and homology some differentially expressed genes and analysis their significance, also we found some new ESTs.(2) Subtract efficiency analysis, cDNA libraries screening and Virtual Northern Blot results to ESTs that... |
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