Functional analysis of HCV core protein using HCV core transgenic mouse

为了阐明HCV核心蛋白在体内的功能，我们采用减法和cDNA芯片的方法，综合分析了HCV核心转基因小鼠和野生小鼠肝脏中mrna表达的差异。我们用抑制减法杂交的方法从野生的HCV转基因核心小鼠中减去了50个克隆，从HCV转基因核心小鼠中减去了44个克隆。这些克隆的序列分析显示，没有任何与炎症或致癌相关的基因，这些基因在转基因和野生克隆之间的表达强度在两倍以内。接下来，我们分别用cDNA阵列和DNA芯片分析了转基因小鼠和野生小鼠之间约1000个基因的表达差异。两种方法都揭示了一些基因在转基因小鼠中表达两次以上，这些基因似乎也与炎症或致癌无关。我们将从现在开始分析这些基因的意义。通过northern blotting测定了HCV核心mRNA在转基因小鼠肝脏中的表达，估计mRNA在肝脏中的表达量约为2 pg/μgRNA。HCV核心蛋白并未在本研究中使用的转基因小鼠中进行分析，但之前的结果显示HCV核心蛋白通过western blotting表达。因此，HCV核心蛋白对该转基因小鼠体内基因表达的影响可能不大。

We analyzed comprihensively the difference of mRNAs expressed in the livers of HCV core transgenic mouse and wild mouse in order to clarify the function of HCV core protein in vivo by the method of subtraction and cDNA microarray. We have got fifty clones of HCV core transgenic mouse subtracted from wild one and 44 clones of wild mouse subtracted from HCV core transgenic one by the method of suppression subtractive hybridization. Sequence analysis of these clones showed not any genes associated with inflammation or carcinogenesis, and the intensity of expression of these genes between transgenic and wild ones was within two times. Next, we analyzed the difference of expression of about 1000 genes between transgenic and wild mice by cDNA array and DNA chip, respectively. Both methods revealed several genes expressed more than two times in transgenic mouse, and these genes seem also not associated with inflammation or carcinogenesis. We will analyze the significance of these genes from now.Expression of HCV core mRNA was ascertained in the transgenic mouse used in this study by northern blotting and the amount of mRNA was estimated to be around 2 pg/μgRNA in the liver. HCV core protein was not analyzed in the taransgenic mouse used in this study, but previous results showed the expression of HCV core protein by western blotting. Therefore HCV core protein might have little effect on the expression of genes in this transgenic mouse in vivo.