mTLR-2基因的克隆及其在毕赤酵母中的表达

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。

Cloning of mouse TLR-2 gene and its expression in Pichia pastroris

AIM: To clone the mouse TLR-2 gene and to express it in Pichia pastroris. METHODS: Full-length gene encoding mouse Toll-like receptor 2 (mTLR-2) was amplified by RT-PCR, cloned into pUCm-T vector, and confirmed by sequencing. The target gene was then inserted into Pichia pastroris expression vector pPICZalphaC, which was transformed into Pichia pastroris. The recombinant Pichia pastroris was confirmed by PCR and RT-PCR. Expressed protein was identified by SDS-PAGE and Western blot. RESULTS: The full-length mTLR-2 gene(GenBank accession No.AY179346) was cloned. The homology of the cloned gene to published mTLR-2 gene reached 99.84%. The recombinant expression plasmid pPICZ- mTLR-2 was constructed successfully. SDS-PAGE analysis showed that the relative molecular mass(M(r)) of recombinant protein was about 97 000. Western blot analysis showed expressed product can react to rabbit anti- mTLR-2 antibody. CONCLUSION: The full-length mTLR-2 gene is cloned and the recombinant protein can be expressed in Pichia pastroris correctly.