山羊重组促卵泡素长效类似物基因在毕赤酵母中表达
DOI:
作者:
作者单位:

作者简介:

通讯作者:

中图分类号:

基金项目:

江苏省自然科学基金项目资助 (No. BK2005050)。


Expression of Goat Follicle-stimulating Hormone Analogous Gene in Pichia pastoris
Author:
Affiliation:

Fund Project:

the Natural Science Foundation of Jiangsu Province(No. BK2005050).

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    为了获得长效FSH制剂, 利用重叠PCR技术将山羊FSH的a, b亚单位, 通过hCGb亚单位羧基末端延长肽(CTP)基因序列的连接, 构建成单链长效类似物基因FSHb-CTP-a。将其克隆至表达载体pPIC9K, 重组载体线性化处理后电转化至毕赤酵母GS115中, 经判型和G418筛选后获得高拷贝菌株His+Mut+。经甲醇诱导表达后, 进行SDS-PAGE和Western blot分析表明: 重组FSHb-CTP-a的转化子可以正确有效地表达目的蛋白, 分子量约为29 kD。放射免疫分析法(RIA)测定表达上清, 高拷贝转化子的平均表达量为91.849 mIU/mL, 显著高于低拷贝转化子的平均37.419 mIU/mL的表达量, 为FSH的结构研究和长效FSH制剂的生产奠定了基础。

    Abstract:

    In order to obtain the long-acting FSH preparation, the single strand long-acting analogous gene FSHb-CTP-a was successfully constructed by the C-terminal peptide(CTP) of carboxyl-terminal region of human chorionic gonadotropin with the goat FSHα-subunit and b-subunit genes, then it was inserted into pPIC9K vector.The recombinant plasmid pPIC9K FSHb-CTP-a was transformed into Pichia pastoris GS115 by electroporation. The multi-copy inserts His+Mut+ were gained by the screening of pheno-type and hyper-resistance to G418.After methanol induction, the supernatant was analysised by SDS-Polyacrylamide Gen Electro-phoresis and Western blot. The results show that the transformants of FSHb-CTP-a could express the objective protein successfully and the molecular weight is about 29 kD. The concentration of supernatant was detected by Radio-immunoassay and the average expression of multi-inserts is 91.849 mIU/mL and the low-inserts is 37.419 mIU/mL. The expression of multi-inserts is higher than the low-inserts significantly. This research lay the foundation for studying the structure of FSH and the production of long-acting FSH preparation.

    参考文献
    相似文献
    引证文献
引用本文

杨欢利,赵西彪,宋晓娟,丁家桐. 山羊重组促卵泡素长效类似物基因在毕赤酵母中表达[J]. 生物工程学报, 2008, 24(3): 409-414

复制
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2007-06-26
  • 最后修改日期:
  • 录用日期:
  • 在线发布日期:
  • 出版日期:
您是第位访问者
生物工程学报 ® 2024 版权所有

通信地址:中国科学院微生物研究所    邮编:100101

电话:010-64807509   E-mail:cjb@im.ac.cn

技术支持:北京勤云科技发展有限公司