摘要
酪氨酸酶基因（mel）编码的酪氨酸酶是合成黑色素的关键酶。用鸟枪法分离嗜麦芽假单胞菌（PseudomonasmaltophiliaAT18）的mel基因：以pUC18为载体，E．coliHB101为受体菌，在加有一定量的Amp和L－tyr的酪素平板上筛选到分泌可溶性黑色素的转化子，所含重组质粒pWSY约700bp的外源DNA片段上携有mel基因，该片段无BamHI、HindIII、EcoRI、BclI等酶的识别位点。Southern杂交证实此片段确实来源于P．maltophilia。克隆片段的核酸序列分析鉴定ORF504为酪氨酸酶编码序列，SDS－PAGE电泳也显示工程菌HB101／pWSY额外表达一分子量约为18kD的蛋白，因此证明了这一蛋白是重组子表达的酪氨酸酶。
The enzyme tyrosinase, encoded by tyrosinase gene (mel), is responsible for melanin formation. In a shotgun cloning experiment a SalI-digested DNA fragment coding for tyrosinase was cloned from Pseudomonas maltophilia DNA into plasmid vector (pUC18) to generate the hybrid plasmid (pWSY). The recombinant plasmid imparted the ability of melanin synthesis to an E. coli host (HB101). The foreign DNA fragment (0.7kb) possessed no recognition sites for BamHI, HindIII, EeoRI or BclI. Hybridization studies confirmed that the small fragment cloned in pWSY was from P. maltophilia DNA. Nucleotide sequence analysis identified an ORF of 504nt coding tyrosinase. SDS-PAGE analysis also revealed an additional protein of 18kDa, which was equal to the putative tyrosinase according to the size of mel fragment was expressed in the E. coli recombinant carrying the plasmid pWSY.
基金
湖北省科委资助
关键词
mel基因
克隆
表达
酪氨酸酶基因
mel, Pseudomonas maltophilia, Cloning, Sequencing, Expression
