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微管蛋白基因(Tubulin)具有高度的保守性,常作为目标基因相对表达量研究时的内参基因。本研究旨在通过构建蓖麻(Ricinus communis L.) RcTUBβ1基因的原核表达载体pET32a(+)-RcTUBβ1并优化表达工艺以表达融合蛋白His-RcTUBβ1。以蓖麻雌花为材料,通过RT-PCR技术,扩增得到含酶切位点Bam HⅠ和XhoⅠ的RcTUBβ1基因(GenBank Accession number:XM_002509734.3)的ORF序列,经双酶切构建原核表达载体pET32a(+)-RcTUBβ1,将pET32a(+)-RcTUBβ1转化到大肠杆菌表达菌株BL21 (DE3)中诱导表达,并考察诱导时间、诱导温度、诱导IPTG浓度对表达的影响。结果表明,所获得的蓖麻RcTUBβ1 ORF序列与GenBank中的序列一致,长度为1 341 bp,未发生移码突变。IPTG可诱导产生分子量约为68 kDa的融合蛋白,最优表达条件为诱导温度28℃、诱导时间7 h和IPTG 0.5 mmol/L,融合蛋白主要以包涵体的形式存在。这为纯化融合蛋白以制备其抗体,用以研究RcTUBβ1的免疫组织化学分布及时空表达模式提供了基础。
Abstract：Tubulin genes are highly conserved and are often used as internal reference genes in the study of relative expression of target genes. In this study, in order to express the fusion protein of beta-Tubulin1 gene(RcTUBβ1) from castoroil plant(Ricinus communis L.), the recombinant prokaryotic expression vector pET32 a(+)-RcTUBβ1 was constructed and the expression conditions were optimized. Open Reading Frame(ORF) sequence of RcTUBβ1(GenBank Accession: XM＿002509734.3) was amplified from the female flowers of castor, through RT-PCR using a pair of specific primers containing restriction endonucleases recognition sites of Bam HⅠand XhoⅠ. ORF sequence of RcTUBβ1 was ligated into pET32 a(+) by double enzyme digestion to construct the recombinant expression vector. This recombinant expression vector was then transformed into E. coli strain BL21(DE3) to induce the expression of fusion protein His-RcTUBβ1. The effects of induction temperature, induction time and concentration of IPTG on induced expression were investigated. The results showed that the obtained RcTUBβ1 ORF sequence was consistent with the sequence in Genbank, with a length of 1 341 bp with no code mutation. The recombinant expression vector was successfully constructed and named as pET32 a( +)-RcTUBβ1. A fusion protein with a molecular weight of approximately 68 kD, inducted by IPTG, was successfully expressed in E.coli BL21. The optimal expression conditions for the fusion protein were induction temperature 28 ℃, induction time 7 h and concentration of IPTG 0.5 mmol/L. The fusion protein existed mainly in forms of inclusion body.The results provided a foundation for purifing the fusion protein and preparing polyclonal antibody, which were the necessary materials for further research of the immunohistochemical distribution and spatio-temporal expression pattern of RcTUBβ1.
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基本信息:
DOI：10.13417/j.gab.040.001764
中图分类号:S565.6
引用信息:
[1]董乐,梁文文,王芳,等.蓖麻RcTUBβ1基因的克隆和原核表达[J].基因组学与应用生物学,2021,40(04):1764-1770.DOI:10.13417/j.gab.040.001764.
基金信息:
国家自然科学基金(31270370)资助
2020-06-19
2020-06-19
2020-06-19