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pS2融合蛋白的原核表达及其性质鉴定 |
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刘彩云 杨蔚 孙素莲 丁桂风
摘 要 目的：获得pS2融合蛋白。方法：RT-PCR法从乳腺癌组织中扩增pS2片段，定向克隆到PMS-31b载 体上，重组质粒PMS-pS2转化温度敏感型细菌POP2136，使之高效表达融合蛋白MS2-pS2。结果：免疫组织化学试验(IHC)结果表明：纯化的融合蛋白MS2-pS2能 与抗pS2进口单抗特异反应。酶联免疫吸附试验(ELISA)及Western blotting试验表明：MS2 -p S2能与pS2 C端31个氨基酸合成肽抗血清特异反应。以此融合蛋白为免疫原得到的抗血清可 用于免疫组织化学分析。结论：pS2融合蛋白表达是正确的，亦为进一步研究pS2生物学功能及制 备抗pS2单抗奠定了基础。
关键词 pS2融合蛋白 原核表达 温度诱 导 免疫组织化学
中国图书分类号 R392-33
Expression in prokaryote and identification of pS2 fusi on protein
LIU Cai-Yun,YANG Wei,SUN Su-Lian et al.
(The School of Oncol o gy,Beijing Medical University,Beijing Institute for Cancer Research,Beijing 100034)
Abstract Objective:To experss pS2 fusion protein.Methods: pS2 fragment was amplificated by RT-PCR from human breas t cancer tissue.Recombinant plasmid PMS-pS2 was constructed by inserting a Eco RI/BamHI fragment from pS2 cDNA into expression vector PMS-31b and was transferred into competent E coli P OP2136 cells containing a temperature-sensitive mutant.MS2-pS2 fusion protein was synthesized in POP2136 at 42℃.Results:The purified MS2-pS2 fusion protein recognized s pecifically antiserum raised against the synthetic peptide corresponding to C-e nd 31 amino acids of deduced pS2 protein sequence in the ELISA and Western blot ting.The fusion protein reacted with the pS2 monoclonal antibody (DAKO product ) in immunohistochemistry.The MS2-pS2 fusion protein was served as immunogen,t h e antiserum was effective immunohistochemically on formalin-fixed rountinely pr ocessed section of human breast tumors.Conclusion:The pS2 fusion protein expressed in prokaryote cells was correct and estabilished the basis for preparating anti-pS2 monocl onal antibody.
Key words pS2 fusion protein Prokaryotic expression Tem perature induction Immunohistochemistry(IHC)
pS2基因是三叶草因子家族(Trefoil factor family，TFF)中的第一成员， 首先是在人乳腺癌细胞系MCF7中借助雌激素调节而发现的［1］，现命名为TFF1 ［2］。 pS2基因定位于第21号染色体，含有3个外显子和2个内含子，编码一含有信号肽 的84个氨基酸的多肽，成熟后为一[1] [2] 下一页
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