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目的 :克隆mTLR 2基因 ,并在毕赤酵母中的表达其融合蛋白。方法 :采用RT PCR从鼠肝脏中扩增mTLR 2全基因 ,并将其克隆到T载体中 ,测序验证。将目的基因编码序列插入毕赤酵母表达载体pPICZαC中 ,构建重组质粒 ,并转化毕赤酵母。重组酵母以PCR、RT PCR验证 ,表达的重组蛋白用SDS PAGE和Westernblot进行分析。结果 :克隆了mTLR 2全基因(AY179346 ) ,与已发表的mTLR 2基因的同源性为 99.84 %。构建了重组表达质粒pPICZ mTLR 2。SDS PAGE和Westernblot分析显示 ,在相对分子质量 (Mr)为约 970 0 0处出现 1条特异性蛋白带 ,且能与兔抗mTLR 2抗体发生反应。结论 :克隆了mTLR 2全基因 ,并在毕赤酵母中获得表达。
OBJECTIVE: To clone the mTLR 2 gene and express its fusion protein in Pichia pastoris. Methods: The mTLR 2 gene was amplified from murine liver by RT PCR and cloned into T vector and sequenced. The target gene coding sequence inserted into the Pichia expression vector pPICZαC, construct a recombinant plasmid, and transformed into Pichia pastoris. Recombinant yeast was verified by PCR and RT PCR. The expressed recombinant protein was analyzed by SDS PAGE and Western blotting. Results: The mTLR 2 gene (AY179346) was cloned. The homology of the mTLR 2 gene was 99.84%. The recombinant plasmid pPICZ mTLR 2 was constructed. SDS PAGE and Western blot analysis showed that a specific protein band appeared at a molecular weight of about 970 0 0 and was able to react with rabbit anti-mTLR 2 antibody. Conclusion: The mTLR 2 gene was cloned and expressed in Pichia pastoris.