人松弛素H2类似物在毕赤酵母中的高效表达及其活性

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

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。

Objective To highly express human relaxin-2 analogue （HR2） in Pichiapastoris, and determine the bioactivity of expressed product. Methods HR2 gene was synthesized by modification of codons, based on which recombinant plasmid pPICZaA- HR2 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 pPICZotA-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 ceils. Conclusion Recombinant HR2 was highly expressed in P. pastods GS115, which showed a certain bioactivity.