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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。
Objective: To construct the expression vector of human serum albumin (HSA) - thymopentin (TP5) fusion protein and express in Pichia pastoris to clarify its biological activity. Methods: HSA-TP5 fusion gene was constructed by gene recombination technique and transfected into Pichia pastoris so as to construct its eukaryotic expression system. The eukaryotic recombinant expression of PPICZαC-HSA-TP5 was obtained by agarose gel electrophoresis and purification by kit The HSA-TP5 genetically engineered bacteria were fermented at high density by two-step fermentation method. The supernatant protein of fermentation broth was concentrated by precipitation, separated by SDS-polyacrylamide gel electrophoresis, cation exchange chromatography and hydrophobic chromatography The protein was purified and the activity of this fusion protein to promote lymphocyte proliferation was detected by MTT assay. Results: The length of HSA gene fragment was 1845bp. The fragment length of the fusion plasmid HSA-TP5-pPICZαC was 707bp. Sequence analysis revealed that the HSA and TP5 sequences of the target genes were exactly the same as those published in GenBank, and were positively linked and fused. PCR method identified PPICZαC-HSA-TP5 eukaryotic recombinant plasmid and yeast genomic DNA integration, compared with the control group, the transformation group appeared gene fragment length of 1 860bp. SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein gradually increased with the induction time within 72 hours after methanol induction. The HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system. MTT assay, HSA-TP5 fusion protein and TP5 protein consistent with lymphocyte proliferation activity. Conclusion: HSA-TP5 fusion protein can be obtained by constructing HSV-TP5 eukaryotic expression system and has biological activity.