人松弛素H2类似物在毕赤酵母中的高效表达及其活性

目的利用毕赤酵母表达系统高效分泌表达人松弛素H2类似物(Human relaxin-2 analogue,HR2),并检测其生物活性。方法通过密码子优化合成HR2基因,构建重组表达质粒pPICZαA-HR2,转化毕赤酵母菌GS115,甲醇诱导表达,并对表达产物进行Tricine-SDS-PAGE和Western blot分析。表达产物经超滤和柱层析纯化后,切除人工C肽,检测其生物活性。结果经双酶切鉴定和测序证实,重组表达质粒pPICZαA-HR2构建正确;Tricine-SDS-PAGE分析显示,表达的重组蛋白相对分子质量约6 500,表达量占菌体总蛋白的47.6%,为451μg/ml,且为分泌表达;Western blot分析显示,重组蛋白具有良好的反应原性;纯化的重组蛋白纯度达96%;经检测C肽切除的重组HR2对THP-1细胞具有刺激活性。结论成功在毕赤酵母GS115中高效分泌表达了重组松弛素H2类似物,并具有一定的生物活性。

High expression of human relaxin-2 analogue in Pichia pastoris and activity of expressed product

Objective To highly express human relaxin-2 analogue(HR2)in Pichia pastoris,and determine the bioactivity of expressed product.Methods HR2 gene was synthesized by modification of codons,based on which recombinant plasmid pPICZaAHR2 was constructed and transformed to P.pastoris PGS115 for expression under induction of methanol.The expressed product was identified by Tricine-SDS-PAGE and Western blot,then purified by ultrafiltration and column chromatography,and determined for bioactivity after removal of synthetic C peptide.Results Restriction analysis and sequencing proved that recombinant plasmid pPICZαA-HR2 was constructed correctly.Tricine-SDS-PAGE showed that the expressed recombinant protein in a secretory form,with a relative molecular mass of about 6 500,contained 47.6%(451 μg/ml) of total somatic protein.Western blot showed good reactogenicity of the recombinant protein.The purified recombinant protein reached a purity of 96% and,after removal of C peptide,showed stimulating activity to THP-1 cells.Conclusion Recombinant HR2 was highly expressed in P.pastoris GS115,which showed a certain bioactivity.