AChR_(α211)的分泌表达及其对肌无力患者血清中AChRAb的清除作用

The secreting expression of recombinant AChR_(α211) and its removal role for AChRAb in myasthenia gravis patient serum

目的 在毕赤酵母(P .pastoris)中分泌表达AChRα2 1 1 ,研究其对重症肌无力患者血清中AChRAb的清除作用。方法 以质粒pUC AChRα2 1 1 为模板,PCR扩增人AChRα2 1 1 DNA片段,插入真核表达载体pPIC9K中。重组质粒转入P .pastorisGS115后,经甲醇诱导分泌表达AChRα2 1 1 ,用SepharoseQ离子交换柱和Superdex 75分子排阻层析进行纯化。Westernblot和ELISA进行免疫活性分析后,将目的蛋白偶联于CNBr Sepharose 4B树脂上,研究该免疫吸附剂对肌无力患者血清中AChRAb的清除作用。结果 双酶切鉴定和序列分析表明,AChRα2 1 1 DNA片段已正确插入到pPIC9K载体中。重组菌P .pastorisGS115 pPIC9K AChRα2 1 1 经甲醇诱导可分泌表达目的蛋白AChRα2 1 1 ,经阴离子交换柱和分子排阻层析可得95 %纯AChRα2 1 1 ,并制备成免疫吸附剂。Westernblot和ELISA分析表明,重组蛋白具有与天然AChRα亚基相似的免疫活性。免疫吸附实验表明该重组蛋白可特异性地去除肌无力患者血清中的AChRAb。结论 在P .pastoris中分泌表达并纯化重组蛋白AChRα2 1 1 ,制备成一种新的免疫吸附剂AChRα2 1 1 Sepharose ,可清除肌无力患者血清中的AChRAb。

Objective To express recombinant AChR α211 a nd investigate its removal function to AChRAb existed in myasthenia gravis (MG ) patient serum. Methods Human AChR α211 DNA fragment was amplified by PCR with plasmid pUC-AChR α211 as the template and inserted into the vector pPIC9K at the sites of EcoRⅠ and Not Ⅰ. The recombinant pPIC9K-AChR α211 was transferred into P. pastoris GS115, and the target protein was expressed as the secrete form in the resultin g recombinant strain by the induction with methanol. The recombinant AChR α2 11 was purified by Sepharose Q ion exchange change chromatography and Superdex 75 gel filtration chromatography. After the immunoreactivity was identified by the analysis of Western blot and ELISA, the AChR α211 was coupled with CNB r-Sepharose 4B as an immunoadsorbent. The elimination of AChRAb in MG patient s erum was performed by mixing with the immunoadsorbent at 37℃ for 3?h. Results It was shown by the results of double enzymatic digestion and DNA sequence analysis that the correct coding region of the target protein had been inserted into the vector pPIC9K. After transferring into P. pastoris GS115, the target protein was expressed as detected by SDS-PAGE and Western blot analysis. AChR α211 with 95% purity was obtained by ion exchange chro matography and gel filtration chromatography, and the product has its immunoreac tivity with McAb35 or MG patient serum as shown in the ELISA, which implies that the target protein has its natural conformation and immunoreactivity. Furthermo re, AChR α211 coupled with CNBr-Sepharose 4B specifically remove AChRAb f rom MG patient serum. Conclusion The recombinant AChR α211 extracellularly expressed in P. pastoris was purified and coupled with CNBr-Sepharose 4B as an immunoadsorbent which can potentially be used as an effective therapeutic agent to eliminate AChRAb existed in MG patient serum.