基于哺乳动物细胞系的重组乙酰胆碱酯酶表达载体构建研究

Construction of recombinant acetylcholinesterase expression vector based on mammalian cell line

目的 构建具有不同信号肽、载体骨架元件以及乙酰胆碱酯酶编码基因序列的真核表达载体,为基于哺乳动物细胞系表达重组乙酰胆碱酯酶提供前期研究基础。方法 通过NCBI数据库查询苍蝇头部、电鳗鱼电器官来源的乙酰胆碱酯酶基因序列(mdAChE和eleelAChE),经密码子优化后,选择Humaninsulin、小鼠重链、Human CD33 3种信号肽序列,将其与合成的上述目的基因连接后分别与pCMV3、pcDNA3.1(+)、pcDNA3.4 3种载体骨架元件进行组合,构建重组乙酰胆碱酯酶的表达载体,随后分别在CHO-S、Expi293F细胞系进行瞬时转染表达,产物采用镍柱亲和纯化后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)与蛋白免疫印迹(Western blot)分析鉴定。结果 经菌液聚合酶链式反应(polymerase chain reaction, PCR)及DNA测序鉴定,成功构建了4种重组mdAChE酶的表达载体以及3种重组eleelAChE酶的表达载体。SDS-PAGE、Western blot的结果表明,基于pcDNA3.1(+)和pcDNA3.4载体骨架元件的重组表达载体可实现重组mdAChE和eleelAChE酶的可溶性表达;以p CMV3为载体骨架元件的表达载体,仅在以Human CD33为信号肽时,能在CHO-S细胞系中实现重组mdAChE酶的可溶性表达,其余情况下均未见有效表达。结论 实现了昆虫、鱼类来源的重组乙酰胆碱酯酶在哺乳动物细胞系的可溶性表达,发现载体骨架元件、信号肽类型对其重组表达具有重要影响, CHO-S以及Expi293F细胞均适合重组乙酰胆碱酯酶的表达,后续还需对影响其酶活、农药敏感性的关键限制性因素开展深入研究。

Objective To construct eukaryotic expression vectors with different signal peptides, carrier components and acetylcholinesterase coding gene sequences, and provide preliminary research basis for expressing recombinant acetylcholinesterase based on mammalian cell lines. Methods Through the NCBI database, the acetylcholinesterase gene sequences(mdAChE and eleelAChE) derived from the head of flies and electric eels power generation organs were queried, and after codon optimization, 3 kinds of signal peptide sequences of Humaninsulin,mouse heavy chain and Human CD33 were selected, and they were linked with the synthesized above-mentioned target genes with pCMV3, pcDNA3.1(+) and pcDNA3.4, respectively. The expression vector of recombinant acetylcholinesterase was constructed by combining the 3 kinds of carrier components, and then transiently transfected and expressed in CHO-S and Expi293F cell lines. The products were purified by nickel column affinity and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot. Results The expression vectors of 4 kinds of recombinant mdAChE enzymes and 3 kinds of recombinant eleelAChE enzymes were successfully constructed by colony polymerase chain reaction(PCR) and DNA sequencing. The results of SDS-PAGE and Western blot showed that the recombinant expression vectors based on the carrier components of pcDNA3.1(+) and pcDNA3.4 could realize the soluble expression of recombinant mdAChE and eleelAChE. The expression vector with pCMV3 as the framework element achieved soluble expression of the recombinant mdAChE enzyme in the CHO-S cell line only when Human CD33 was used as the signal peptide, and no effective expression was observed in other cases. Conclusion The soluble expression of recombinant acetylcholinesterase from insects and fish in mammalian cell lines is realized. It is found that the carrier component and signal peptide type have an important influence on its recombinant expression. CHO-S and Expi293F cells are suitable for the expression of recombinant acetylcholinesterase. The key limiting factors affecting its enzyme activity and pesticide sensitivity need to be further studied.