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  本文選題：蓖麻毒素B鏈 + 表達(dá)��； 參考：《延邊大學(xué)》2017年碩士論文

【摘要】：目的:通過大腸桿菌原核表達(dá)系統(tǒng),優(yōu)化誘導(dǎo)表達(dá)條件,實現(xiàn)重組蓖麻毒素B鏈蛋白的大量可溶性表達(dá),并提高其純度。目的是用重組蛋白作為抗原,為制備抗體以及蓖麻毒素疫苗的研究提供前期技術(shù)支持和理論基礎(chǔ)。方法:本實驗首先通過PCR擴增出目的基因RTB連接于pMD-18T克隆載體,經(jīng)序列比對正確后,將目的基因插入到表達(dá)載體P300-TrxA-Sumo和pGEX-4T-1中,構(gòu)建重組表達(dá)質(zhì)粒P300-TrxA-Sumo-RTB及pGEX-4T-1-RTB。將鑒定構(gòu)建成功的重組表達(dá)質(zhì)粒轉(zhuǎn)化至BL21(DE3)感受態(tài)細(xì)胞中,建立表達(dá)工程菌P300-TrxA-Sumo-RTB/BL21、pGEX-4T-1-RTB/BL21。通過IPTG的誘導(dǎo),利用單因素試驗和正交試驗法在不同誘導(dǎo)時間、不同IPTG濃度以及不同溫度的情況下對表達(dá)條件進(jìn)行優(yōu)化,實現(xiàn)對RTB的可溶性表達(dá)。通過Ni-Chelating Sepharose親和層析柱純化得到純度相對較高的重組蛋白,最后切去Sumo標(biāo)簽,得到目的蛋白RTB。并通過Western blot和間接ELISA法證明其具有良好的免疫原性。檢測RTB作用于RAW264.7細(xì)胞的一氧化氮水平。最后用MTT法檢測RTB作用于小鼠巨噬細(xì)胞時存活情況。結(jié)果:成功構(gòu)建重組表達(dá)質(zhì)粒P300-Trxa-Sumo-RTB以及pGEX-4T-1-RTB。在經(jīng)過誘導(dǎo)條件的優(yōu)化后,成功實現(xiàn)重組蓖麻毒素蛋白的大量可溶性表達(dá),表達(dá)量分別為11%和8%。通過親和層析柱純化得到純度相對較高的目的蛋白。經(jīng)過Western blot和ELISA驗證目的蛋白具有免疫原性。一氧化氮檢測結(jié)果顯示,RTB組數(shù)值較正常細(xì)胞組增高,說明RTB能夠促進(jìn)NO的釋放,可能作為治療巨噬細(xì)胞相關(guān)炎癥性疾病的突破口。通過MTT法證明純化得到的RTB對于小鼠巨噬細(xì)胞RAW264.7不具有毒性。結(jié)論:經(jīng)大腸桿菌誘導(dǎo)條件工藝優(yōu)化后,可實現(xiàn)重組蓖麻毒素B鏈蛋白可溶性表達(dá)。經(jīng)Ni-NTA、GST標(biāo)簽親和層析柱純化后獲得具有生物學(xué)活性的蛋白,且重組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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

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