【作者】 郑淑霞；

【导师】 刘树滔；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 酪氨酸酶（TYR）是一种含铜酶，是黑素代谢和儿茶酚胺合成的关键酶。长期以来，虽然有大量关于酪氨酸酶的研究，但是由于可获得的酪氨酸酶的数量有限，有关酪氨酸酶的生化性质和功能关系的信息仍然非常有限。本文报道了在克隆了小鼠酪氨酸酶基因的基础上，在大肠杆菌表达体系中诱导表达野生型和变异型酪氨酸酶的结果。实验主要包括以下五个方面：基因克隆和表达菌株的构建：利用逆转录-聚合酶链式反应（RT-PCR）方法，从总RNA中扩增得到小鼠酪氨酸酶的cDNA，应用DNA重组技术，将基因分别进行改造，重组到T7启动子控制下的表达载体pET-22b（+）和tac启动子控制下的表达载体pGEX-2T中，先后构建了表达质粒pET-TYR（野生型）和变异型表达质粒pET-Tat-TYR（含双精氨酸分泌信号肽变异型）、pGEX-TYR（缺失C端跨膜区的GST融合变异型）。野生型酪氨酸酶的表达：将构建好的野生型表达质粒pET-TYR转化到大肠杆菌BL21(DE3)中，发现重组蛋白几乎没有表达；转化到Rosetta(DE3)时，表达出的蛋白经N-末端氨基酸序列测定确定是酪氨酸酶。产物为包涵体，没有活性。双精氨酸分泌信号肽变异型表达：将变异型表达质粒pET-Tat-TYR转化到大肠杆菌Rosetta(DE3)中，37℃，IPTG浓度为1.0mmol/L，诱导4小时，可表达出Tat－酪氨酸酶。产物也是包涵体，没有活性。缺失C端跨膜区的GST融合蛋白的表达：变异型表达质粒pGEX-TYR（缺失C端跨膜区）转化到大肠杆菌BL21(DE3)中，可表达出GST－酪氨酸酶（缺失C端跨膜区）。37℃表达时产物也是包涵体，没有活性。从诱导温度、诱导时间和诱导剂的浓度三方面对诱导条件进行优化，结果发现：含表达质粒pGEX-TYR（缺失C端跨膜区）在30℃（或16℃），IPTG浓度为1.0mmol/L时，诱导过夜，大部分表达产物为可溶性蛋白。但也是没有活性。以上结果说明了在大肠杆菌表达系统中，很难获得有活性的重组酪氨酸酶，原因可能是酪氨酸酶无法进行糖基化，这些结果为重组酪氨酸酶的表达和将来结构与功能的分析奠定了一定的基础。更多还原

【Abstract】 Tyrosinase, a copper-containing enzyme,.plays a key role in melanin metabolism and catecholamine synthesis. Tyrosinase was widely investigated for a long time. However, limited information on the relation of biocharacter and function of tyrosinase was obtained because of the limited acquisition of tyrosinase. Here we reported the cloning of the gene from murine tyrosinase, the expression of wild tyrosinase and variants in E.coli. The results were showed in following:Gene cloning and expression plasmid construction: the cDNA encoding murine tyrosinase was amplified from the total RNA by RT-PCR. By using the technology of DNA recombination, the cDNA was reconstructed and ligated into expression vector pET-22b(+) with T7 promoter and pGEX-2T with tac promoter, successfully constructed pET-TYR(wild type) and variant pET-Tat-TYR (Twin Arginine Transport variant type) and pGEX-TYR (GST fusion type with the lack of transmembrane region).Expression of wild tyrosinase: the pET-TYR was transformed into BL21(DE3), but no tyrosinase expressed was discovered. When the pET-TYR was transformed into Rosetta(DE3) the target protein was expressed and identified by amino acid sequencing.Expression in Twin Arginine Transport system: pET-Tat-TYR was transformed into Rosetta (DE3). Then Rosetta (DE3) was induced with IPTG (1.0mmol/L) for 4 hours at 37℃, and the Tat-tyrosinase was successfully expressed.Expression of GST fusion system: pGEX-TYR was transformed into BL21 (DE3), the protein GST-tyrosinase can also be expressed, but the expressed GST-tyrosinase is in the form of inclusion bodies devoid of activity.The optimization of the induce conditions: induce temperature, time and <WP=4>concentrations of IPTG were mainly considered. We found that soluble protein can be expressed by using pGEX-TYR, which was induced at 30℃ (or 16℃) over night. But no activity was discovered. These results indicated that: the active recombinant tyrosinase was hard to be produced by E.coli expression system. Possibly, it is due to failure of glycosylation in E.coli. These findings establish a certain foundation for the expression of tyrosinase and the analysis of structure and function in the future.更多还原