人生长激素基因在酿酒酵母中的同源重组及初步鉴定  

Homologous recombination and identification of human growth hormone gene in saccharomyces cerevisiae:apreliminary study

作　　者：赵慧[1] 郑文岭[1] 崔东[1] 马文丽[2] 

机构地区：[1]广州军区广州总医院肿瘤分子生物学研究所,广东广州510010 [2]第一军医大学分子生物学研究所,广东广州510515

出　　处：《医学研究生学报》2004年第6期481-483,F002,共4页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目 (批准号 :3 9880 0 3 2 );广州市重点科技攻关项目 (批准号 :99 Z 0 2 2 0 1)

摘　　要：目的 :将人生长激素基因构建至pHSS6 mTn 3×HA/lacZ转座文库载体中 ,利用酿酒酵母同源重组稳定高效表达人生长激素。　方法 :通过两步PCR获得带有信号肽的目的基因 ,插入到文库载体的Sse8387I位点中 ,NotI酶切 ,酵母同源重组 ,SC Ura筛选 ,酵母菌落PCR及lacZ基因表达初步鉴定阳性重组子。　结果 :酵母菌落PCR初步鉴定 ,筛选出目的基因正确插入的重组子 ,其中两株有lacZ基因高表达。　结论 :对阳性重组子进行lacZ显色反应 ,筛选出可高水平表达x gal的菌株 ,以此推测重组位点前有强启动子 ,为目的基因的稳定高效表达奠定基础。Objective: Human growth hormone gene was inserted into pHSS6-mTn-3×HA/lacZ library plasmids, which contribute to express human grwoth hormone steadily at high level via homologous recombination in saccharomyces cerevisiae. Methods: The human growth hormone gene with albumin signal peptide upstream obtained by two-step polymerase chain reaction(PCR) was inserted into pHSS6-mTn-3×HA/lacZ at Sse8387I site. After digesting with NotI, the recombinated vector was transfomed into saccharomyces cerevisiae to induce homologous recombination. The positive recombinants were preliminary screened through its expression of URA3 selectable marker and lacZ gene. Results:Several strains were identified to contain the foreign gene at the right site with the right direction, and high levels β-gal activity were assessed in two strains. Conclusion: High level expression of β-gal in yeast show that LacZ gene have strong promoter upstream , which will be useful for the expression of human grwoth gene at high level.

关 键 词：酿酒酵母 人生长激素基因 同源重组 菌落多聚酶链反应 

分 类 号：Q575.11[生物学—生物化学]