美洲大蠊抗菌肽AMPPA13的表达及纯化

Expression and purification of antimicrobial peptide AMPPA13 of Periplaneta americana

[目的]预测美洲大蠊新的抗菌肽基因,构建原核表达体系并纯化表达产物。[方法]通过生物信息学方法,预测分析出潜在的美洲大蠊抗菌肽。构建pET32a重组质粒,优化诱导表达条件,通过亲和层析,分子筛等手段获得纯化的抗菌肽,并进行Western Blot鉴定。[结果]预测出美洲大蠊新的抗菌肽基因AMPPA13,成功构建重组质粒pET32aAMPPA13。优化诱导表达条件得最佳IPTG浓度为0.1 mmol/L,最佳诱导时间为4 h,最佳诱导温度为37℃。纯化后AMPPA13浓度为268μg/m L。[结论]克隆出美洲大蠊抗菌肽基因,并成功构建原核表达体系,得到1 m L纯化的AMPPA13,经Western Blot鉴定,表达产物正确。

[Objective]To predict new antimicrobial peptides of Periplaneta americana and purify it by prokaryotic expression system. [Methods]Using bioinformatics strategies,antimicrobial peptide candidates of Periplaneta americana were predicted from the transcriptome. Recombinant plasmid of pET32 a was constructed. The induced condition was optimized,and antimicrobial peptide was purified by affinity chromatography and molecular sieve chromatography. The product of purification was identified by Western Blot. [Results] The new antimicrobial peptide gene of Periplaneta americana,AMPPA13,was predicted. Reccombinant plasmid pET32 a-AMPPA13 was constructed. The optimum concentration of IPTG was 0. 1 mmol/L,optimum time was 4 h,and optimum temperature was 37 ℃ to expression. The concentrate of AMPPA13 was 268 μg/m L after purification.[Conclusion]The antimicrobial peptide gene of Periplaneta americana was cloned,and its prokaryotic expression system was constructed. Finally,we obtained 1 m L AMPPA13 which was identified successfully by Western Blot.