水稻苯丙氨酸氨解酶基因在大肠杆菌BL21 DE3中的表达  

Expression of phenylalanine ammonia-lyase cDNA from rice in E. coli BL21DE3

作　　者：蔡朱男[1] 余应年[1] 罗建红[1] 钱羽力[1] 陈星若 

机构地区：[1]浙江大学医学院病理生理教研室,浙江杭州310031

出　　处：《中国病理生理杂志》2000年第1期12-16,共5页Chinese Journal of Pathophysiology

基　　金：浙江省自然科学基金资助 (39746 3)

摘　　要：目的 :探索水稻苯丙氨酸氨解酶基因在大肠杆菌中的表达及其规律 ,为治疗苯丙酮尿症奠定基础。方法 :应用基因工程技术 ,将水稻苯丙氨酸氨解酶的cDNA(rPAL - 1-cDNA)重组入大肠杆菌高效表达质粒pET - 2 8c ,通过异丙基硫代 - β -D -半乳糖苷 (IPTG)诱导 ,在大肠杆菌中获得表达。 结果 :SDS -聚丙烯酰胺凝胶电泳表明 ,诱导 1,3,5 ,7h后 ,表达蛋白量占菌体总蛋白分别为 2 1.40 % ,30 .6 0 % ,35 .40 % ,35 .43% ,融合蛋白His6 -rPAL分子量为 78.6kDa ,主要以包涵体形式存在。结论AIM: To study the expression and its kinetics of rice phenylalanine ammonia-lyase gene encoding into E. coli as the basis of treatment for phenylketouria. METHODS: The phenylalanine ammonia-lyase-1-cDNA(rPAL-1-cDNA) from rice was recombined into E. coli high expression vector pET-28c and transformed into E. coli host strain BL21DE3. Engineering bacteria was then inducted by isopropyl-β-D-thiogalactoside (IPTG) for 1, 3, 5, 7 hours, in order to obtain high level expression. RESULTS: After induction, the expression level of fusion protein was 21.40%, 30.60%, 35.40%, 35.43% respectively. The fusion protein exhibited a band of 78 6 kD on SDS-PAGE analysis,but was not found in controls.The target protein was mainly existed in the form of inclusion body. CONCLUSION:Rice PAL gene expressing E. coli was established by gentic engineering technique.

关 键 词：质粒 大肠杆菌 苯丙酮尿症 苯丙氨酸氨解酶 

分 类 号：R589.3[医药卫生—内分泌] R378.21[医药卫生—内科学]