文档介绍：猪胃蛋白酶原 A 基因密码子优化及在毕赤酵母中表达的研究
摘要
天冬氨酸蛋白酶一般被称为酸性蛋白酶,在酸性条件下可催化水解蛋白质,是目前重要的工业用酶之一。胃蛋白酶属于天冬氨酸家族,是研究蛋白质结构与功能关系的重要模型。胃蛋白酶广泛存在于脊椎动物胃液中。胃蛋白酶经济价值高,用途广泛,胃蛋白酶在制革、方便食品、奶、口香糖加工制造等行业也有一定的用处。目前,商业、医药途径获得的胃蛋白酶主要从动物胃组织中提取,受动物脏器资源的限制,胃蛋白酶价格较高;容易残留一些动物自身的蛋白酶类,如一些组织蛋白酶等。而微生物反应器具有效率高效,操作简单,能够降低成本等优点,所以,通过筛选微生物反应器,,为解决这些问题提供了新的研究方向。
第一,根据Genebank上公布的猪胃蛋白酶原 A 基因序列,进行引物设计,通过RT-PCR克隆获得了湖北白猪胃蛋白酶原 A 基因。
第二,在猪胃蛋白酶原 A 克隆和序列分析的基础上,构建了毕赤酵母胞内表达载体 -pA。研究不同 pH、不同诱导时间、不同启始浓度和甲醇诱导浓度等因素对表达量的影响。
第三,根据毕赤酵母密码子的偏嗜性,利用定点突变,对稀有密码子进行了优化, 希望能够进一步提高重组酵母表达量。
关键词: 胃蛋白酶原 A;毕赤酵母;克隆;表达;优化
Abstract
Aspartic acid proteases, known as acidic proteases, can catalyze the hydrolysis of protein under the acid condition. Pepsin is wildly presented in gastric juice of vertebrate .mercially used pepsin is generally obtained from animal stomach tissue,which is very expensive and un-pure because of the ans resource constraints and other proteases . Therefore,a great interest has been focused on the new method of producing pepA (Pepsinogen A). Among these,the microbial production is supposed to be the most promising method because of its high yield, simple production process and lower cost. Therefore,the objective of this study is trying to produce efficiently binant porcine pepsin with ic engineering technology in microbial system. Aspartic acid proteases are widely applied in the fields of feed additive, vintage, food, leather and medicine. Porcine pepsin A, which is the important model for the study of structure-function relationship of protein, belongs to the family of aspartic protease. Using gene engineering to study the expression of pepsin will supply a new way for solving these questions in this paper.
Firstly, the primers were designed according to the sequence J04601 in Genebank. And then the swine pepsinogen A cDNA, which was cloned by RT-PCR from the Hubei White swine.
Secondly, the binant plasmid of -pA was constructed. Effects of different pH, different