蓖麻毒素B链可溶性表达及其工艺优化、纯化

发布时间：2018-05-14 03:28

本文选题：蓖麻毒素B链 + 表达　；参考：《延边大学》2017年硕士论文

【摘要】：目的:通过大肠杆菌原核表达系统,优化诱导表达条件,实现重组蓖麻毒素B链蛋白的大量可溶性表达,并提高其纯度。目的是用重组蛋白作为抗原,为制备抗体以及蓖麻毒素疫苗的研究提供前期技术支持和理论基础。方法:本实验首先通过PCR扩增出目的基因RTB连接于pMD-18T克隆载体,经序列比对正确后,将目的基因插入到表达载体P300-TrxA-Sumo和pGEX-4T-1中,构建重组表达质粒P300-TrxA-Sumo-RTB及pGEX-4T-1-RTB。将鉴定构建成功的重组表达质粒转化至BL21(DE3)感受态细胞中,建立表达工程菌P300-TrxA-Sumo-RTB/BL21、pGEX-4T-1-RTB/BL21。通过IPTG的诱导,利用单因素试验和正交试验法在不同诱导时间、不同IPTG浓度以及不同温度的情况下对表达条件进行优化,实现对RTB的可溶性表达。通过Ni-Chelating Sepharose亲和层析柱纯化得到纯度相对较高的重组蛋白,最后切去Sumo标签,得到目的蛋白RTB。并通过Western blot和间接ELISA法证明其具有良好的免疫原性。检测RTB作用于RAW264.7细胞的一氧化氮水平。最后用MTT法检测RTB作用于小鼠巨噬细胞时存活情况。结果:成功构建重组表达质粒P300-Trxa-Sumo-RTB以及pGEX-4T-1-RTB。在经过诱导条件的优化后,成功实现重组蓖麻毒素蛋白的大量可溶性表达,表达量分别为11%和8%。通过亲和层析柱纯化得到纯度相对较高的目的蛋白。经过Western blot和ELISA验证目的蛋白具有免疫原性。一氧化氮检测结果显示,RTB组数值较正常细胞组增高,说明RTB能够促进NO的释放,可能作为治疗巨噬细胞相关炎症性疾病的突破口。通过MTT法证明纯化得到的RTB对于小鼠巨噬细胞RAW264.7不具有毒性。结论:经大肠杆菌诱导条件工艺优化后,可实现重组蓖麻毒素B链蛋白可溶性表达。经Ni-NTA、GST标签亲和层析柱纯化后获得具有生物学活性的蛋白,且重组蛋白具有免疫原性并且不具有毒性。
[Abstract]:Aim: to optimize the induced expression conditions of recombinant ricin B chain protein by E. coli prokaryotic expression system, and to improve the purity of recombinant ricin B chain protein. The aim of this study was to use recombinant protein as antigen to provide early technical support and theoretical basis for the preparation of antibody and ricin vaccine. Methods: in this experiment, the target gene RTB was amplified by PCR and ligated to the pMD-18T clone vector. After sequence alignment, the target gene was inserted into the expression vectors P300-TrxA-Sumo and pGEX-4T-1 to construct the recombinant expression plasmids P300-TrxA-Sumo-RTB and pGEX-4T-1-RTB. The recombinant expression plasmid was transformed into BL21DDE3) competent cells, and the expression engineering strain P300-TrxA-Sumo-RTB / BL21 pGEX-4T-1-RTB / BL21 was established. Through the induction of IPTG, the single factor experiment and orthogonal test were used to optimize the expression conditions under different induction time, different IPTG concentration and different temperature to realize the soluble expression of RTB. The recombinant protein with relatively high purity was purified by Ni-Chelating Sepharose affinity chromatography. Finally, the Sumo tag was removed and the target protein was obtained. Its immunogenicity was proved by Western blot and indirect ELISA. The levels of nitric oxide (no) in RAW264.7 cells induced by RTB were measured. Finally, MTT method was used to detect the survival of mouse macrophages treated with RTB. Results: the recombinant plasmid P300-Trxa-Sumo-RTB and pGEX-4T-1-RTBwere successfully constructed. After the optimization of the induction conditions, the soluble expression of the recombinant ricin protein was successfully achieved, with the expression levels of 11% and 8%, respectively. The target protein with relatively high purity was purified by affinity chromatography. The immunogenicity of the target protein was confirmed by Western blot and ELISA. The results of nitric oxide test showed that the value of RTB was higher than that of normal cells, which suggested that RTB could promote the release of no and might be a breakthrough in the treatment of macrophage-related inflammatory diseases. The purified RTB was not toxic to mouse macrophage RAW264.7 by MTT assay. Conclusion: the recombinant ricin B chain protein can be expressed in a soluble way after optimization of the inducing conditions of Escherichia coli. The protein with biological activity was purified by Ni-NTA-GST tag affinity chromatography and the recombinant protein was immunogenicity and non-toxic.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

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