摘要
为了获得足够量的单克隆非特异性抑制因子-β蛋白(monoclonal nonspecific supressor factor β,MNSFβ)及其抗体用于探讨MNSFβ在着床中的作用机理,本研究构建了表达质粒pBV220/MNSFβ-hCGβ,在大肠杆菌中表达了融合蛋白MNSFβ-hCGβ,用抗hCGβ抗体对表达产物进行鉴定,结果表明融合蛋白MNSFβ-hCGβ得到了正确表达,且分子量和理论值相近。表达产物MNSFβ-hCGβ经初步纯化后用于免疫Balb/C小鼠,制备抗体。同时,我们还构建了表达质粒pGEX-4T-2/MNSFβ,在大肠杆菌中表达了融合蛋白GST-MNSFβ。用融合蛋白GST-MNSFβ对融合前的免疫小鼠回忆刺激并进行检测,制备了抗MNSFβ多克隆抗体和单克隆抗体。应用所制备的多克隆抗体进行免疫组化研究,对MNSFβ在小鼠子宫内膜上进行了组织定位,结果显示,MNSFβ在着床日(受精后第4.5天)小鼠子宫非着床位点的表达和着床位点相比明显提高。
In order to explore the role of MNSFβ in the process of implantaion, MNSFβ and its antibodies are required. The expression plasmid pBV220/MNSFβ-hCGβ was constructed, and then transferred into E coli to express the fusion protein MNSFβ-hCGβ. The anti-hCGβ antibody was used to identify the fusion protein. The result demonstrated that MNSFβ-hCGβ was expressed correctly and its molecular weight was consistent with the anticipated one. Finally the expression product MNSFβ-hCGβ was preliminarily purified and used to immunize Balb/C mouse to generate the antibodies. In the meantime, the expression plasmid pGEX-4T-2/ MNSFβ was also constructed and transferred into K. coli to express the fusion protein GST-MNSFβ. GST-MNSFβ was purified and used to stimulate the immunized mouse before the preparation of hybridomas cells. The prepared polyclonal and monoclonal antibodies against MNSFβ were checked and measured by fusion protein GST-MNSFβ. The prepared polyclonal antibody was then used to perform the immunohistochemistry analysis. The result suggested that the level of MNSFβ in interimplantation sites was significantly higher as compared with implantation sites in the mouse uterine on Day 4. 5 of pregnancy.
出处
《实验生物学报》
CSCD
北大核心
2002年第2期89-97,共9页
Acta Biologiae Experimentalis Sinica
基金
WHO资助项目(97826)
国家重点基础研究规划项目(G1999055903)
