粉尘螨 HSP16-1 原核表达体系构建与温度应激响应功能鉴定

目的 建立原核表达体系,在蛋白水平确认粉尘螨 HSP16-1 蛋白的温度应激响应功能。 方法 基于前期RNA-seq 获得的粉尘螨 HSP16-1 基因序列,设计特异性引物,PCR 扩增克隆测序;利用生物信息学软件,分析理化性质,预测亚细胞定位和三维结构;通过构建 pET32a / HSP16-1 原核表达质粒,转化大肠杆菌感受态细胞 BL21;采用 1 mmol / L IPTG 在 16、28 和 37 ℃ 三个温度诱导 HSP16-1 重组蛋白表达,分别在 2、4、6 和 8 h 取样进行 SDSPAGE 分析;绘制热胁迫和冷胁迫下重组菌与空载菌生长曲线。 结果 测序获得粉尘螨SP16-1 完整 CDS 为462 bp,编码 153 个氨基酸;BLAST 比对显示粉尘螨与近缘物种屋尘螨核苷酸和氨基酸序列相似度分别为 84. 63%和 87. 58%;亚细胞定位在细胞核;保守区预测含有 α 晶体 HSP23 超家族保守区。 菌落 PCR 及双酶切鉴定 pET32a /HSP16-1 重组质粒构建成功。 SDS-PAGE 分析显示,HSP16-1 蛋白被成功诱导表达,37 ℃ 诱导 6 h 是最佳诱导条件。 细菌生长曲线显示,pET32a / HSP16-1 重组菌在热胁迫下的生长均优于 pET32a 空载菌,而冷胁迫下则相反,空载菌生长要优于重组菌。 结论 粉尘螨 HSP16-1 蛋白仅在热胁迫下发挥应激响应功能,而在冷胁迫未表现出相似的功能。

Construction of prokaryotic expression system of HSP16-1 of Dermatophagoides farinae and functional identification of temperature stress response

Objective To establish a prokaryotic expression system and confirm the temperature stress response function of HSP16-1 of Dermatophagoides farinae at protein level.Methods Based on the HSP16-1 gene sequence of D.farinae obtained by previous RNA-seq,specific primers were designed,amplified by PCR,cloned and sequenced.Bioinformatics software was used to analyze the physical and chemical properties,subcellular location and three-dimensional structure.The prokaryotic expression of plasmid pET32a/HSP16-1 was constructed and transformed into E.coli BL21.The expression of HSP16-1 recombinant protein was induced by 1 mmol/L IPTG at 16℃,28℃and 37℃,and the samples were collected for SDS-PAGE analysis at 2 h,4 h,6 h and 8 h,respectively.The bacteria growth curves of the recombinant strains(pET32a/HSP16-1)and control strains(pET32a)under heat and cold stress were drawn.Results The sequencing results showed that the complete coding sequence(CDS)of D.farinae HSP16-1 was 462 bp,which encoded 153 amino acids.BLAST alignment demonstrated that the nucleotide and amino acid sequences of D.farinae HSP16-1 were in similarity by 84.63%and 87.58%,respectively,with the closely related species D.pteronyssinus.Subcell were localized in the nucleus,and conservative region prediction revealed conservative region ofαcrystallin HSP23 superfamily.The recombinant plasmid pET32a/HSP16-1 was successfully constructed and identified by bacteria liquid PCR and double restriction enzyme digestion.SDS-PAGE analysis showed that HSP16-1 protein was successfully expressed,and the best induction condition was 37℃for 6 h.The bacterial growth curve indicated that the growth of pET32a/HSP16-1 recombinant strains was better than that of pET32a control strains under heat stress,yet the growth of control strains was better than that of recombinant strains under cold stress.Conclusion HSP16-1 protein of D.farinae only generate stress response function under heat stress,yet does not show similar function under cold stress.