[胃癌相关基因MPS-1的融合表达及抗GST-MPS-1兔抗体的制备]
[Fusion expression of gastric cancer-related gene MPS-1 and preparation of rabbit antibody against GST-MPS-1].
作者信息
Wang Yun-wei, Zhu Zheng-gang, Gu Qin-long, Li Jian-fang, Qu Ying, Liu Bing-ya, Lin Yan-zhen
机构信息
Department of Surgery, Ruijin Hospital, Shanghai Second Medical University, Shanghai Institute of Digestive Surgery, Shanghai 200025, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Jan;22(1):60-3.
AIM
To express the fusion protein GST-MPS-1 in E.coli and prepare rabbit antibody against GST-MPS-1.
METHODS
MPS-1 cDNA was inserted into the vector of pGEX-5X. The recombinant was then transformed into E.coli BL21. After induction with the IPTG, the fusion protein GST-MPS-1 was expressed in E.coli. The purified fusion protein was then used to immunize the New Zealand rabbits to obtain the polyclonal antibody against GST-MPS-1. Specificity of the antibody was tested by Western blot and immunofluorescent staining.
RESULTS
The cDNA sequence of MPS-1 in the recombinant was confirmed by restriction endonuclease digestion and sequence analysis. SDS-PAGE result showed that fusion protein was highly expressed in E.coli with a molecular weight of 36 kDa. The titer of the rabbit serum against GST-MPS-1 was as high as 1x10(5) in ELISA analysis. The polyclonal antibody were found to specifically bind to MPS-1 protein in Western blot and immunofluorescent staining.
CONCLUSION
The preparation of the polyclonal antibody against MPS-1 lay a foundation for the further study of MPS-1 expression at protein levels and its function.
目的
在大肠杆菌中表达融合蛋白GST-MPS-1，并制备抗GST-MPS-1的兔抗体。
方法
将MPS-1 cDNA插入pGEX-5X载体。然后将重组体转化到大肠杆菌BL21中。用IPTG诱导后，融合蛋白GST-MPS-1在大肠杆菌中表达。纯化后的融合蛋白用于免疫新西兰兔以获得抗GST-MPS-1的多克隆抗体。通过蛋白质印迹法和免疫荧光染色检测抗体的特异性。
结果
通过限制性内切酶消化和序列分析确认重组体中MPS-1的cDNA序列。SDS-PAGE结果显示融合蛋白在大肠杆菌中高表达，分子量为36 kDa。ELISA分析中，兔抗GST-MPS-1血清的效价高达1×10⁵。在蛋白质印迹法和免疫荧光染色中发现多克隆抗体能特异性结合MPS-1蛋白。
结论
抗MPS-1多克隆抗体的制备为进一步研究MPS-1在蛋白质水平的表达及其功能奠定了基础。
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