pS2融合蛋白的原核表达及其性质鉴定

Expression in prokaryote and identification of pS2 fusion protein

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POP2 136 ,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Westernblotting试验表明 :MS2 pS2能与pS2C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。

Objective:To experss pS2 fusion protein Methods: pS2 fragment was amplificated by RT PCR from human breast cancer tissue Recombinant plasmid PMS pS2 was constructed by inserting a EcoRI/BamHI fragment from pS2 cDNA into expression vector PMS 31b and was transferred into competent E coli POP2136 cells containing a temperature sensitive mutant MS2 pS2 fusion protein was synthesized in POP2136 at 42℃ Results:The purified MS2 pS2 fusion protein recognized specifically antiserum raised against the synthetic peptide corresponding to C end 31 amino acids of deduced pS2 protein sequence in the ELISA and Western blotting The fusion protein reacted with the pS2 monoclonal antibody (DAKO product) in immunohistochemistry The MS2 pS2 fusion protein was served as immunogen,the antiserum was effective immunohistochemically on formalin fixed rountinely processed section of human breast tumors Conclusion:The pS2 fusion protein expressed in prokaryote cells was correct and estabilished the basis for preparating anti pS2 monoclonal antibody