| 摘要: |
| [目的]在大肠杆菌中克隆和表达胸腺肤β4(thymosin β4,Tβ4)基因,并进行分离纯化及免疫活性测定.[方法]将基因序列拆分成10个互补的小片段,互为模板,采用PCR两步法扩增得到Tβ4基因.将Tβ4基因片段克隆至pTXB1载体的NdeⅠ和XhoⅠ酶切位点之间,将重组质粒转化至E.coli BL21 eodon plus,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达、几丁质(Chitin beads)亲和层析柱纯化Tβ4.然后用MTT法测定Tβ4的免疫活性.[结果]经测序表明获得了序列正确的人胸腺肽β4基因,构建重组质粒pTXB1-Tβ4,经SDS-PAGE电泳分析表明,重组质粒pTXB1-Tβ4在E.coli BL21 codon plus中高效表达,表达量为30%.将表达产物用几丁质亲和层析柱纯化,获得较纯的Tβ4.MTT检测表明,Tβ4有促进淋巴细胞增殖的活性,最适刺激质量浓度为1.6 μg/mL.[结论]获得高效表达的、高纯度的、有生物活性的重组Tβ4,它可促进淋巴细胞的分化、增殖,具有免疫活性,最适刺激质量浓度为1.6 μg/mL. |
| 关键词: 人胸腺肽β4 基因表达 MTT 活性检测 |
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| Cloning and expression in E. coli and biological activity testing of human thymosin β4 |
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| Abstract: |
| 【Objective】 The study cloned and highly expressed the gene encoding of human thymosin β4 (Tβ4) in E.coli,followed by the purification of the protein Tβ4 and the detection of its biological activity.【Method】 The coding sequence of human thymosin β4 was dissected into 10 oligonucleotides of 23 bp each and were amplified by two-step total gene synthesis method.The Tβ4 gene was then inserted into the NdeⅠand XhoⅠrestriction endonuclease sites of an expression plasmid pTXB1 and the recombinant plasmid was transformed to E.coli BL21 codon plus.Then the expression of Tβ4 gene induced by IPTG and recombinant Tβ4 purified with chitin beads.【Result】 The recombinant plasmid was identified and confirmed with DNA sequence analysis.A high level expression of Tβ4 fusion protein was obtained and purified successfully.Biological assay indicated that the Tβ4 could induce lymphocyte proliferation and differentiation.【Conclusion】 Tβ4 is expressed with highly efficiency and purified and Tβ4 is highly pure and bioactive,becoming the foundation of the further study for Tβ4. |
| Key words: human Tβ4 gene expression MTT biological testing |
