【作者】 吴炳礼；

【导师】 李恩民； 李伍举；

【作者基本信息】 汕头大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 对甲醇营养型毕赤酵母表达系统的研究和应用有近20年的历史。大量文献资料表明，许多外源基因可以在毕赤酵母中成功表达，但表达水平有时相差很大。基因表达的影响因素较多。迄今为止，人们尚不能在实验前对目的基因的表达水平进行有效评估。在本文中，我们分析了外源基因3’末端二级结构与表达水平之间的关系；在此基础上，建立了一个针对pPIC9表达载体在毕赤酵母中高效表达的数学模型。而且以NGAL(Neutrophil Gelatinase-Associated Lipocalin)为目的基因，应用这一模型对NGAL基因是否能在毕赤酵母中高效表达进行了预测和实验验证，初步证明了这一数学模型的准确性。 内容与方法： 1．收集了40例应用pPIC9载体在毕赤酵母GS115株中表达目的基因的数据。获取下列基本信息：基因名称、表达量、目的基因插入载体的酶切位点、基因扩增引物等，拟定以表达量100mg／L为界限，按此界限将40例目的基因表达水平分为高／低两组。 2．从NCBI获取相关目的基因的核酸序列，与基因的3’端扩增引物进行核酸序列比对，确认表达载体的翻译终止密码子前后各100bp的序列，据此对每个数据构建200bp的片段。 3．从上述每个基因的200bp片段，截取9，000个区间，截取方式为[X，Y]，X和Y在200bp片段的范围为1≤X≤100，和111≤Y≤200。 4．使用RNAfold软件计算每个区间最低自由能，构建最低自由能矩阵。 5．使用Tclass分类程序和目的基因的分类信息对最低自由能矩阵进行t检验及判别分析，得到理论上可以判别表达水平高低的区间组合，随后重复200次按75％比例随机将所有40例目的基因的自由能数据分为训练集和检验集，通过分类稳定性分析来筛选最佳判别区间组合。 6．利用获得的最佳判别区间组合建立判别函数，将40个数据按75％比例随机分成两组，并利用多数组建立判别函数，此过程重复1000次，最终得到1000个判别函数。 7．构建NGAL基因的200bp片段，利用RNAfold软件计算上述步骤“6”结果中最佳判别区间的最低自由能。更多还原

【Abstract】 Numerous heterologous proteins have been successfully expressed in the methylotrophic yeast, Pichia pastoris. However, we still cannot predict the expression level of heterologous genes before experiments because many factors are involved in affecting the expression level. Here we report a mathematical model for high-level expression using 40 heterologous genes expressed in pPIC9 vector as the samples, and the results showed the 3’-UTR stable local secondary structure is the necessary condition for high-level expression of heterologous genes in pPIC9 vector. To verify this model, human neutrophil gelatinase-associated lipocalin (NGAL) gene was expressed in pPIC9 in pichia pastoris with high-level expression more than 100 mg/L. The model has the following three potential applications. The first is to predict the expression level of heterologous genes before experiments. The second is to optimize the experiment designs to obtain the high level expression. The third is that the conclusions can be referenced qualitatively for other vectors in yeast expression system. Materials and methods:1. Forty cases of foreign genes expressed in pPIC9 vector in Pichia pastoris were collected as samples, the following information were recorded: the gene name, the expression level, the site of the heterologous gene inserted, and the primers. The 40 heterologous genes are classified into two groups: high-level expression group (HEG) with expression level more than 100 mg/L, and low-level expression group (LEG) with expression level less than 100 mg/L.2. The nucleic acid sequences of the sample genes were retrived from the NCBI, and aligned to the 3’ primer to verify the 100bp sequence before and after’ translation stop codon, after then a 200bp segment was extracted for each sample.3. Based on the 200bp segment of each sample, we extracted 9,000 intervals [X, Y], where 1≤ X≤100, and 111 ≤Y≤ 200.4. The RNAfold program was used to calculate the secondary structure free energy of each interval with temperature parameter set as 30℃, and a minimum free energy matrix (MFEM, or profile of RNA secondary structure)更多还原