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目的 :构建高效表达人睾丸前列腺素D合成酶 (htL PGDS)的重组表达质粒。 方法 :以经测序证实为人L PGDS的人睾丸L PGDScDNA为模板进行PCR扩增 ,得到编码L PGDS的基因片段 ,用限制性内切酶BamHⅠ和EcoRⅠ进行酶切后 ,连接到经相同内切酶处理的原核表达载体pGEX 2T中。重组质粒pGEX 2T/htL PGDS被进一步转化感受态大肠杆菌JM10 3 ,转化物接种到含氨苄青霉素的LB平板 ,37℃培养 12～ 16h后 ,挑取氨苄青霉素抗性菌落 ,并接种到含氨苄青霉素的LB液体培养基中 ,37℃、16 0r/min培养 12～ 16h后 ,提取质粒DNA供PCR和双酶切鉴定。 结果 :共筛选出氨苄青霉素抗性菌落 10 9个 ,经PCR和双酶切鉴定出 10个重组质粒pGEX 2T/htL PGDS。结论 :重组表达质粒pGEX 2T/htL PGDS的构建及正确鉴定 ,为以后的重组抗原的表达、纯化及单克隆抗体的制备奠定了基础。
Objective: To construct a recombinant plasmid expressing human testosterone prostaglandin D synthase (htL PGDS) efficiently. METHODS: The gene fragment encoding L PGDS was obtained by PCR amplification of L PGDS cDNA from human testis LPL-labeled human L PGDS. After digested with restriction endonucleases BamHI and EcoRI, Treated prokaryotic expression vector pGEX 2T. The recombinant plasmid pGEX 2T / htL PGDS was further transformed into competent E. coli JM103. The transformants were inoculated into ampicillin-containing LB plates and cultured at 37 ° C for 12-16h. Ampicillin-resistant colonies were picked and inoculated into ampicillin- Of LB liquid medium, 37 ℃, 16 0r / min cultured 12 ~ 16h, plasmid DNA extracted for PCR and double enzyme digestion. Results: A total of 10 9 ampicillin-resistant colonies were screened. Ten recombinant plasmids pGEX 2T / htL PGDS were identified by PCR and double enzyme digestion. Conclusion: The construction and correct identification of recombinant plasmid pGEX 2T / htL PGDS laid the foundation for the future expression and purification of recombinant antigens and the preparation of monoclonal antibodies.