【作者】 宋照雷；

【导师】 洪岸； 孙奋勇； 谢秋玲；

【作者基本信息】 暨南大学 ， 遗传学， 2002， 硕士

【摘要】 目的： 针对目前基因工程发酵生产中一些外源蛋白表达量过低的问题，寻找一种切实可行的方法能高效表达这些外源蛋白。目前基因工程发酵生产的方法一般是按照分子克隆的方法，将一段外源基因克隆到一种表达载体中，然后转化到大肠杆菌中进行发酵生产。但经常会出现表达量太低或者根本不表达的情况。尤其在大规模的工业化生产中，对于表达水平有较高要求，必须有高表达的菌株才能产生规模效益。对决定表达水平的一些重要因素以及相关的构建上的一些方法作一些研究，对于以研究为目的所需或者工业化生产各种外源蛋白，且对于继续探索在酵母细胞、哺乳动物细胞等真核细胞中高效表达外源蛋白都有十分重大的意义。 材料和方法： 以本所正在开发的产品——纳豆激酶作为试验对象来试验本课题中所研究的新方法的实用性。主要考虑两种因素：翻译起始区(TIR)的自由能及大肠杆菌的密码子偏好性。按照最大可能增大TIR区自由能和最大可能符合大肠杆菌密码子偏好性的原则，利用摇摆碱基的特性，对外源基因前35个碱基的一些摇摆碱基位点进行突变。G+C含量是决定TIR区标准自由能变化(△G~0)的决定性因素，降低G+C含量即可增大TIR区△G~0。在一些碱基位点上，以上两个原则发生冲突时，此位点摇摆碱基随机化，随机化的结果将产生很多种序列组合。用计算机软件DNASIS2.5分析每种组合TIR区自由能，计算其△G~0，按△G~0高低排序，选取前10条序列设计10条长引物。此长引物包含各个突变的位点的一种组合。用这种长引物和共同的下游引物进行PCR扩增，然后按分子克隆的一 毕业论义：ECOli中高效表达外源蛋囱的探索及几介纳豆激酶中的应m MS 般方法，分别进行克隆，转化到10块平板上，分别酶切检测出构建成功的克隆， SDS—PAGE测定其目标蛋白产量，选取1株高表达菌株摇瓶扩大生长。纳豆激 酶和纳豆激酶原都用包涵体复性的方法，然后川CLT ik检测其纤溶活性， Superdex200凝胶过滤层析法层析纯化，并测定重组于DNA序列。 结果： 1．用以上所述构建方法，对于纳豆激酶原和纳豆激酶都得到表达量高于总蛋白 量30％的高表达菌株。从转化平板验证各种重组子克险，纳豆激酶原有1株 表达量高于30o，纳豆激酶有3株表达量高于30O。 2．生物活性检测小，纳豆激酶原和纳豆激酶都表现出纤溶活性。 3．凝胶过滤层析方法进行分离提纯，效果显著，纯度达96％左右。 4．测序结火证u重纠了序列是正确的。 结论： 密码于偏好州Z及翻译起始区（TIR）自山能是决定翻译水平的重要因素，而 G ＋ C含量是诀定 RNA二级结构自山能的决定性因素。根孤摇摆碱基的特性， 改变摇摆碱基从而减少G十C含量，增加密码于偏好性。按此原则设计引物构建 克隆将是一种迅速得到高表达菌株的可靠方法。＊＊＊SISZ．5的计算结果和实验 得到的结果一定程度上吻合，暗示计算机辅助分析在将来此类型的研究工作中将 扮演更重要角色，但山于目前有关＊R区二级结构及三级结构的形成机制及能 量参数还不完全明确，计算机软件巡算结果只能作为一种圳助。更多还原

【Abstract】 Aims: To improve exogenous protein expression in E.coli, a new method was studied and applied to Nattokinase and pro-Nattokinase. We usually clone exogenous proteins using regular methods of molecular cloning, but the expression rate is often too low and even no expression. In the large-scale production of exogenous proteins, some high-expression strains must be established. In this experiment, we studied most of the factors influencing the protein expression, and then designed a new method of molecular-cloning.Methods: Among all of the factors influencing expression, we focused on two factors: the Free Energy of the Translation Initiation Region (TIR) and the Codon Preference of E.coli. During the cloning, there are two principles of increasing the A G?of the TIR region and adopting more preferred codons. The A G?of the TIR region is mainly decided by the content of G+C in TIR. In general, the less content of G+C, the higher A G?of the TIR region. Based on the two principles mentioned above and the trait of wobbling bases of triplet code, we mutated the wobbling bases of the head 35 bases of pro-NK and NK cDNA. When contradiction occurred based on the two principles in some sites, we randomly changed them, and it would result in varied combinations in the randomly changed sites. Computer software DNASIS 2.5 was utilized to analyse the A G?of all the sequences. The first ten sequence with the high A G were chosen for subsequent cloning. Using synthetic primers containing the changed sites, ten sequences with different wobbling sites were amplified by PCR and then cloned to pET3c respectively. E.coli strain BL21(DE3) was transformed and induced to express recombinant NK. It was proved by SDS-PAGE that the highest expression rate was above 30%. The target proteins existed as inclusion body and were recovered. Then the proteins were purified by Superdex-200 gel-filtrationchromatography. The enzyme activity of nattokinase and pro-nattokinase was determinated by CLT method. The recombinant with the highest expression rate was sequenced. Results:1. We obtained high-expression strains for both pro-NK and NK. From the ten clone plates, we picked clones and analysed them. For pro-NK, there were 1 clone whose expression rate was above 30%; for mature NK, there were 3 high-expression clones whose expression rate was above 30%.2. The proteins were purified by gel-filtration chromatography. The purity reached about 96%.3. The enzyme activity of the pro-Nattokinase solution after ultra-filtration was about 150U/ml, and that of the Nattokinase solution was 220U/ml.Conclusion: Codon preference and the A G?of the TIR region were important factors influencing the expression rate. The reduction of G+C content will decrease the stability of secondary structure. According to these principles, the TIR was mutated and the resulted sequences were analysed using DNASIS 2.5. Ten primers were designed and cloned. This should be a rapid and reliable method to obtain high expression strain of exogenous protein.更多还原