[1]向延芳,何畏,梁志清,等.小鼠FSH-Occludin重组多价避孕疫苗的构建及表达[J].陆军军医大学学报(原第三军医大学学报),2007,29(19):1834-1837.
 XIANG Yan-fang,HE-Wei,LIANG Zhi-qing,et al.Construction of a mouse multivalent contraceptive vaccine based on follicle stimulating hormone and occludin genes[J].J Amry Med Univ (J Third Mil Med Univ),2007,29(19):1834-1837.
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小鼠FSH-Occludin重组多价避孕疫苗的构建及表达(/HTML )
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陆军军医大学学报(原第三军医大学学报)[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第19期
页码:
1834-1837
栏目:
论著
出版日期:
2007-10-15

文章信息/Info

Title:
Construction of a mouse multivalent contraceptive vaccine based on follicle stimulating hormone and occludin genes
作者:
向延芳何畏梁志清史常旭
第三军医大学西南医院妇产科
Author(s):
XIANG Yan-fang HE-Wei LIANG Zhi-qing SHI Chang-xu
Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University, Chongqing 400038,China
关键词:
卵泡刺激素闭锁因子毕赤酵母
Keywords:
follicle stimulating hormone occludin Pichia pastoris
分类号:
R169.41; R392.13; R394-33
文献标志码:
A
摘要:
目的  构建FSH与Occludin重组基因的毕赤酵母分泌型表达载体。  方法  利用RT-PCR技术,从小鼠新鲜脑组织中提取总RNA扩增获得FSH-β亚单位,同法从小鼠新鲜睾丸组织中提取总RNA扩增Occludin,通过双重PCR扩增,获得含FSH-β亚单位特定基因序列和闭锁因子第2个胞外环序列的耦合片段, 构建两种重组质粒pPIC9K FSH-Occludin;经酶切鉴定后电转化毕赤酵母菌株GS115,YPD-G418平板筛选高拷贝转化子,甲醇诱导后SDS-PAGE电泳鉴定。  结果  通过双酶切分析、DNA测序鉴定明确特定的FSH-β亚单位和Occludin基因耦合片段序列与设计完全一致。目的蛋白相对分子质量分别约为14×103与12×103 ,与理论预测值相吻合;Western blot检测具有良好的免疫反应性。  结论  耦合片段被成功克隆到巴氏毕赤酵母表达载体pPIC9K中并高效、正确的表达。
Abstract:
Objectives    To construct the recombinant plasmid pPIC9K FSH-Occludin for preparing a multivalent contraceptive vaccine, which interfering the function of Sertoli cells in the mouse testis.     Methods    After isolated the total RNA from mouse brain and testis tissues, the specific region of follicle stimulating hormone-b (FSH-b) subunit and the secondary extracellular domain of occludin were amplified by RT-PCR, respectively. The catenation fragments of FSH-occludin were obtained through double PCR amplification and cloned into the expression vector PPIC9K of P. pastoris to construct 2 different recombinant plasmids (pPIC9K FSH-occludin). The recombinant vector was introduced into P. pastoris strain GS115 by electroporation and selected with G418 for FSH-occludin gene integration 1. Super G418 resistant clones were selected and screened for FSH-occludin gene expression. The FSH-occludin gene was successfully cloned into pPIC9K.     Results    The recombinant plasmids (pPIC9K FSH-occludin) were identified by DNA sequencing and double enzymatic digestion with EcoRⅠ and NotⅠ. It was confirmed that the reconstruct plasmids containing the same sequences as the theoretical designing. The results of  SDS-PAGE showed that the molecular weight of the expressed product was 1.4×103 and 1.2×103.    Conclusion    The results indicate successful construction of recombinant plasmids containing specific regions of FSH and occludin. It can be used for further study on the immune contraceptive effect in vivo.

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更新日期/Last Update: 2008-07-11