HSA-TP5融合基因表达载体的构建及其真核表达

发布时间：2018-11-19 08:54

【摘要】：目的:构建人血清白蛋白(HSA)-胸腺五肽(TP5)融合蛋白表达载体,并在毕赤酵母中表达,阐明其生物学活性。方法:利用基因重组技术构建HSA-TP5融合基因,并转染至毕赤酵母中从而构建其真核表达体系,通过琼脂糖凝胶电泳分离及试剂盒纯化获得PPICZαC-HSA-TP5真核重组表达质粒;采用两步发酵法对HSA-TP5基因工程菌进行高密度发酵,对发酵液上清蛋白沉淀浓缩,通过SDS-聚丙烯酰胺凝胶电泳法、阳离子交换层析及疏水层析等方法分离纯化蛋白;采用MTT法检测该融合蛋白促淋巴细胞增殖活性。结果:PCR法获得HSA目的基因片段长度为1 845bp。酶切鉴定融合质粒HSA-TP5-pPICZαC得到片段长度为707bp。测序分析,目的基因HSA和TP5序列与GenBank公布的基因序列完全一致,并正向连接融合。PCR法鉴定PPICZαC-HSA-TP5真核重组质粒与酵母基因组DNA整合,与对照组比较,转化组出现基因片段长度为1 860bp。SDS-PAGE分析,在甲醇诱导后72h内,随着诱导时间延长,HSA-TP5融合蛋白表达量逐步升高。利用阳离子交换层析及AKTA多功能蛋白纯化系统纯化得到HSA-TP5融合蛋白。MTT法检测,HSA-TP5融合蛋白与TP5蛋白具有一致的促淋巴细胞增殖活性。结论:通过构建HSA-TP5毕赤酵母真核表达体系可获得HSA-TP5融合蛋白并具有生物学活性。
[Abstract]:Aim: to construct a fusion protein vector of human serum albumin (HSA) (HSA) thymus pentapeptide (TP5) and express it in Pichia pastoris to elucidate its biological activity. Methods: HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct its eukaryotic expression system. The eukaryotic expression plasmid of PPICZ 伪 C-HSA-TP5 was isolated by agarose gel electrophoresis and purified by kit. High-density fermentation of HSA-TP5 genetically engineered bacteria was carried out by two-step fermentation. The protein was precipitated and concentrated on the fermentation broth. The protein was separated and purified by SDS- polyacrylamide gel electrophoresis, cation exchange chromatography and hydrophobic chromatography. The MTT assay was used to detect the proliferative activity of the fusion protein. Results: the length of target gene fragment of HSA obtained by PCR was 1 845 BP. The length of the fusion plasmid HSA-TP5-pPICZ 伪 C was 707bp. Sequence analysis showed that the sequence of HSA and TP5 was identical with that of GenBank, and the recombinant plasmid of PPICZ 伪 C-HSA-TP5 was identified by PCR, and compared with the control group. The gene fragment length was 1 860bp.SDS-PAGE analysis in the transformed group. Within 72 hours after methanol induction, the expression of HSA-TP5 fusion protein increased gradually with the prolongation of the induction time. HSA-TP5 fusion protein was purified by cationic exchange chromatography and AKTA multifunctional protein purification system. MTT assay showed that HSA-TP5 fusion protein and TP5 protein had the same lymphocyte proliferation activity. Conclusion: HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of HSA-TP5 Pichia pastoris and has biological activity.
【作者单位】：北华大学生命科学研究中心;北京大学人民医院血液研究所骨髓移植病房;北华大学附属医院骨外1科;吉林大学药学院生物工程教研室;
【基金】：吉林省教育厅“十二五”科学技术研究项目资助课题(吉教科合字[2014]第173号) 吉林省科技厅分子老年医学重点实验室项目资助课题(20130624003JC) 吉林省吉林市科技计划项目资助课题(201437024,2015233007)
【分类号】：Q78;R3416

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