【作者】 张峰；

【导师】 汪世华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 为了深入了解黄曲霉毒素B1对小鼠肝脏蛋白的影响,本研究之前我们已经采用双向电泳及质谱分析等方法来分析不同浓度的黄曲霉毒素B1对小鼠肝脏蛋白的影响。结果发现有35个蛋白质点变化差异显著,分析其中新出现和明显上调的5个蛋白质点以及缺失的和明显下调的7个蛋白质点。其中有5个蛋白属于引发小鼠肝脏发生病变甚至是癌变的蛋白,7个蛋白属于参与小鼠肝脏能量与物质代谢的蛋白,这些蛋白对小鼠肝脏都有一定的影响。因而推测,黄曲霉毒素B1可能通过作用于这12个蛋白质而进一步影响小鼠肝脏,使其发生病变甚至是癌变。为排除上述实验中可能发生的错误,并在转录水平上探索这些基因的表达情况,我们利用荧光定量PCR对黄曲霉毒素诱导后的小鼠肝脏atp5b、idh3α和prxⅡ三个基因的mRNA量进行了相对定量。结果发现atp5b先增后减,idh3α有增加的趋势,prxⅡ则近似增加,这与双向电泳的结果一致。随后,我们将atp5b、idh3α和prxⅡ这三个基因构建到原核表达载体pET28a上,并转化到E.coli BL21(DE3)宿主菌中。通过100 mmol/L的IPTG诱导阳性菌,成功表达了ATP5B、IDH3α、PRXⅡ三个蛋白。最后,我们还将atp5b构建到哺乳动物细胞表达载体pCDNA3.1hisB上并成功插入了报告基因gfp。这些工作为揭示atp5b、idh3α和prxⅡ三个基因在黄曲霉毒素B1致癌中的作用奠定了基础。更多还原

【Abstract】 In order to further understanding the effect of proteins induced by aflatoxin B1 in the mouse liver, in previous study, two-dimensional electrophoresis and mass spectrometry analysis methods were used to analyze different concentrations of Aflatoxin B1 on the impact of proteins in the mouse liver. The result showed that 35 protein spots changed significantly, of which 5 appeared protein and 7 disappeared proteins were analyzed. The results indicated that these 5 proteins maybe cause liver disease or even cancer, and 7 proteins were involved with the material and energy metabolism in the mouse live, thus all these proteins have a certain effect on the mouse liver. That is to say, Aflatoxin B1 might interact with these 12 proteins and finally affect the mouse liver, and ultimately lead to lesion or even cancer.In order to excluding the err of the two-dimensional electrophoresis, we explored the mRNA variation of atp5b, idh3αand prxⅡby fluorescence quantitative PCR after infection. As we expected, the results were according with the protein analysis. Followingly, we inserted these three genes into the protokaryon expression vetor pET28a, and transformed them into the the host E.coli BL21 (DE 3), resepectively. After induced by 100 mmol/L IPTG, the positive bacteria successfully expressed these proteins. Finnally, the atp5b and reporter gene gfp were inserted into the mammal cell expression vector pCDNA3.1hisB. These work were helpful for revealing the function of atp5b, idh3αand prxⅡin hepatocarcinogenesis induced by Aflatoxin B1.更多还原