發(fā)布時(shí)間：2018-06-24 10:11

  本文選題：蜂毒素與基因變構(gòu)IL-2嵌合蛋白 + 表達(dá)�。� 參考：《青島大學(xué)》2007年碩士論文

【摘要】： ①目的：將含重組質(zhì)粒pGEX-4T-2／melittin-~(88)ArgIL-2的大腸桿菌進(jìn)行表達(dá)發(fā)酵條件的優(yōu)化，得到可溶性表達(dá)的重組蛋白；分離純化該重組蛋白，得到高純度的、足量的、具有生物學(xué)活性的蜂毒素與基因變構(gòu)IL-2fmelittin-~(88)ArgIL-2)嵌合蛋白，探索建立該蛋白的純化工藝；研究此嵌合蛋白在體外增強(qiáng)免疫功能和抗腫瘤的生物學(xué)活性。②方法：將含重組質(zhì)粒的大腸桿菌DH5a，經(jīng)IPTG誘導(dǎo)進(jìn)行高效表達(dá)，經(jīng)反復(fù)實(shí)驗(yàn)摸索表達(dá)出可溶性蛋白；經(jīng)過(guò)親和層析、凝血酶Thrombin酶切和離子交換層析進(jìn)行純化，純化的重組melittin-~(88)ArgIL-2嵌合蛋白，經(jīng)SDS-PAGE電泳鑒定蛋白純度并進(jìn)行定量；用液相測(cè)定法研究嵌合蛋白的抑菌活性，用MTT法研究嵌合蛋白對(duì)外周血單個(gè)核細(xì)胞PBMC增殖、NK細(xì)胞殺傷活性、人卵巢癌細(xì)胞SKOV_3和Hela細(xì)胞生長(zhǎng)抑制的作用。③結(jié)果：在25℃和IPTG0.8mmol／L條件下，表達(dá)出了可溶性蛋白；經(jīng)過(guò)一系列的分離純化步驟，純化后的重組melittin-~(88)ArgIL-2嵌合蛋白純度達(dá)95％，蛋白濃度大約200mg／L，建立了該嵌合蛋白的純化工藝；純化后的嵌合蛋白在體外能抑制金黃色葡萄球菌和大腸桿菌的生長(zhǎng)，能促進(jìn)PBMC細(xì)胞增殖，能增強(qiáng)NK細(xì)胞的殺傷活性，能抑制人卵巢癌細(xì)胞SKOV3和Hela細(xì)胞的生長(zhǎng)增殖。④結(jié)論：建立了melittin-~(88)ArgIL-2嵌合蛋白的純化工藝，，該嵌合蛋白在體外具有一定的抑菌作用，具有一定的增強(qiáng)免疫功能和抗腫瘤活性。嵌合蛋白具有能協(xié)同基因變構(gòu)IL-2與蜂毒素增強(qiáng)細(xì)胞免疫的作用，并且其抗腫瘤活性得到激活并顯著增強(qiáng)。為其在真核細(xì)胞系統(tǒng)的表達(dá)、純化、體內(nèi)的生物學(xué)活性研究以及大規(guī)模制備的中試放大研究和臨床前期研究奠定了基礎(chǔ)。
[Abstract]:Objective: to optimize the fermentation conditions of Escherichia coli containing recombinant plasmid pGEX-4T-2 / melittin-88 ArgIL-2, to obtain soluble recombinant protein, to isolate and purify the recombinant protein, and to obtain high purity and sufficient amount of recombinant protein. The bioactive chimeric protein of melittin-88 (IL-2fmelittin-88) chimeric protein with biological activity was studied and the purification process of the protein was explored. To study the enhancement of immune function and biological activity of the chimeric protein in vitro. Methods: the recombinant plasmid containing E. coli DH 5a was highly expressed by IPTG, and the soluble protein was expressed by repeated experiments. The purified recombinant melittin-88 ArgIL-2 chimeric protein was purified by Thrombin digestion and ion exchange chromatography. The protein purity was identified by SDS-PAGE electrophoresis, and the bacteriostatic activity of the chimeric protein was studied by liquid chromatography. MTT assay was used to study the cytotoxicity of chimeric protein on PBMC proliferation and NK cell proliferation of peripheral blood mononuclear cells. The results showed that soluble protein was expressed at 25 鈩

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