【作者】 孙章辉；

【导师】 蔡谨；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 硕士

【摘要】 PCR是分子生物学中最重要的工具之一。在PCR技术中,DNA聚合酶起着关键性作用。Taq DNA酶是从嗜热杆菌中分离纯化得到的第一个用于PCR的热稳定性DNA聚合酶。Pfu DNA酶具有3’-5’外切酶活性,是保真度最高的DNA聚合酶之一。本文主要研究从重组工程菌中分别提取Pfu DNA聚合酶及Taq DNA聚合酶。 以重组表达质粒p-Taq转化宿主菌E. coli DH5á,获得转化子。利用Taq酶的热稳定性,将破胞液在75℃水浴1h,然后用硫酸铵沉淀法获得纯化的表达蛋白。对重组E.coli的培养条件进行优化,优化培养条件为:在OD₆₀₀=0.8时加入IPTG诱导,IPTG的终浓度为125mg/L,诱导培养时间为9h,诱导培养温度为37℃。在优化条件下,1L发酵液可得50,000单位的聚合酶,比活可达10,000U/mg蛋白质。 用Pfu DNA聚合酶的表达质粒pET-Pfu（表达载体为pET11）转化大肠杆菌BL21（DE3）（含有pLysS质粒）得到重组工程菌。在Pfu酶的纯化过程中,用溶菌酶裂解细胞,利用Pfu酶的热稳定性,采用热变性法（冻融法）除去绝大部分杂质蛋白,得到Pfu酶粗溶液。然后尝试使用磷酸纤维素P11、CM-Sepharose CL-6B、DEAE-Sepharose FF、JK110等几种离子交换树脂,及葡聚糖凝胶G-200、Sephacry1 S-200、Sephadex G-100、Sephadex G-75等几种凝胶树脂,来进一步纯化Pfu酶。研究用无毒价廉的乳糖代替IPTG作诱导剂,并对诱导条件进行了优化,优化条件为:培养液接种后立即加入1g/L的乳糖,诱导培养8h后收获菌体。用优化后的培养条件及纯化技术,在实验室环境中制备重组Pfu DNA聚合酶。将破胞液在75℃水浴20 min,然后依次过JK110离子交换柱和Sephadex G-75凝胶柱得到纯化Pfu酶。在PCR扩增实验中,纯化产品具有与商品酶类似的活性和扩增效果,从500ml培养液中共得到3×10⁵U的聚合酶,纯化Pfu酶比活达到22,200U/mg,浓度为10U/ì 1。更多还原

【Abstract】 PCR is one of the most important tools in molecular biology. DNA polymerase takes a critical role in the PCR technology. Taq DNA polymerase is the first one which is applied in the PCR. Pfu DNA polymerase, having 3’-5’ exonuclease activity, is one of the DNA polymerases that have the highest fidelity. This study concentrated on extracting and purification of Taq and Pfu DNA polymerase in the recombinant strains.The expression plasmid of p-Taq was transformed in E. coli DH5a. Using the thermostable ability of Taq DNA polymerase, the protein was immerged in a 75℃ water-bath for 1h, and then obtained by the method of ammonium sulfate precipitation. The culture conditions of recombinant E.coli were studied. The optimal culture conditions were that: induced at OD₆₀₀=0.8 with 125mg/L IPTG, and grown at 37℃ for 9h. Then 50,000 unit of polymerase was obtained from 1L culture, with a specific activity of 10,000U/mg.The recombinant strain of Pfu DNA polymerase was constructed by transforming the expression plasmid pET-Pfu into BL21 （DE3 ） containing plasmid pLysS. During the purification of Pfu DNA polymerase, the cells were lysed by lysozyme, and a heat treatment step was performed to eliminate most contaminant proteins, using the thermostable ability of Pfu DNA polymerase. Then further purification of target protein by chromatography was studied, including cellulose phosphate （P11）, CM-Sepharose CL-6B, DEAE-Sepharose FF, JK110, Sephacryl S-200, Sephadex G-100 and Sephadex G-75. Lactose, instead of IPTG, was used as the inducer, and the induce conditions were optimized. The optimized conditions were that: adding 1g/L lactose into the culture immediately after inoculation and the cells were harvest after grown for 8h. The purified protein was obtained by a heat-treatment at 75℃ for 20min and a JK110 chromatography and Sephadex G-75 gel filtration. In the PCR amplification, our purified polymerase had similar result to the commercial one. We obtained 3×10⁵U of purified Pfu DNA polymerase from 500ml culture, with a specific activity of 22,200U/mg and a concentration of 10U/（?）1.更多还原