本文選題：無細(xì)胞蛋白質(zhì)合成系統(tǒng)　切入點(diǎn)：抗菌肽　出處：《浙江大學(xué)》2006年博士論文

【摘要】：近年來一種以外源mRNA或DNA為模板,通過在細(xì)胞抽提物的酶系中補(bǔ)充底物和能量物質(zhì)來合成蛋白質(zhì)的體外蛋白質(zhì)合成系統(tǒng)越來越引起重視。與傳統(tǒng)的體內(nèi)系統(tǒng)相比,無細(xì)胞系統(tǒng)具有眾多的優(yōu)越性:1)反應(yīng)簡便,只需加入抽提物、DNA模板和各種必需的反應(yīng)底物,就可以表達(dá)目的蛋白;2)可用于表達(dá)在體內(nèi)系統(tǒng)中難于表達(dá)的蛋白質(zhì),如對細(xì)胞有毒害作用的抗菌肽和膜蛋白等;3)能夠直接以PCR產(chǎn)物作為線性模板在微孔板上同時平行合成多種蛋白質(zhì),能夠滿足高通量藥物篩選和蛋白質(zhì)組學(xué)研究等的需要。 人β-防御素是近年來發(fā)現(xiàn)的具有廣譜抗菌活性并在機(jī)體抵御外來微生物入侵中起防御作用的一類陽離子抗菌肽。有望成為解決致病菌耐藥性問題的新型內(nèi)源性抗生素,并被證明具有抑制艾滋病病毒復(fù)制的作用。 分別對從人體炎癥皮膚組織中通過RT-PCR方法克隆獲得的人β-防御素-2(HBD-2)人源基因、經(jīng)密碼子優(yōu)化后的HBD-2人工合成基因構(gòu)建了HBD-2的表達(dá)載體,得到了一系列不同密碼子來源或帶有不同融合標(biāo)簽的HBD-2的體外表達(dá)載體。 將不同密碼子來源的HBD-2的表達(dá)載體在大腸桿菌無細(xì)胞蛋白質(zhì)合成系統(tǒng)中進(jìn)行表達(dá),對比實(shí)驗(yàn)結(jié)果表明,密碼子優(yōu)化后合成基因的蛋白質(zhì)表達(dá)量與人源基因表達(dá)量相當(dāng)。 進(jìn)一步將帶有不同融合標(biāo)簽(如硫氧化還原蛋白,trxA和綠色熒光蛋白,GFP)的HBD-2表達(dá)載體在大腸桿菌無細(xì)胞蛋白質(zhì)合成系統(tǒng)中進(jìn)行表達(dá)研究。結(jié)果表明,融合標(biāo)簽的添加明顯提高了HBD-2的表達(dá)量,其中trxA對HBD-2表達(dá)的促進(jìn)作用最為明顯。添加GFP融合標(biāo)簽的HBD-2基因的表達(dá)水平雖然略低于添加trxA融合標(biāo)簽的,但GFP融合標(biāo)簽有利于實(shí)時檢測該融合蛋白的表達(dá)。在無細(xì)胞蛋白質(zhì)合成系統(tǒng)中表達(dá)的融合蛋白可溶性高,避免了在大腸桿菌表達(dá)抗菌肽時易形成包涵體的難題。 比較了反應(yīng)操作模式對無細(xì)胞系統(tǒng)中目標(biāo)蛋白質(zhì)表達(dá)水平的影響。研究表明,與間歇式操作(Batch)相比,連續(xù)交換式(CECF,Continuous Exchange Cell-free)無細(xì)胞反應(yīng)方式能夠明顯提高目標(biāo)蛋白質(zhì)的表達(dá)水平,目標(biāo)蛋白的表達(dá)量最高達(dá)到了2毫克/毫升。這主要是因?yàn)樵贑ECF模式下,能夠不斷補(bǔ)充底物和能量,并同時移走抑制性副產(chǎn)物,從而大大延長了反應(yīng)時間,提高了產(chǎn)物產(chǎn)量。 對無細(xì)胞系統(tǒng)表達(dá)的目標(biāo)蛋白質(zhì)(trxA-HBD-2)的分離純化進(jìn)行了研究,建立了一條高效分離目標(biāo)蛋白質(zhì)的純化工藝,總收率達(dá)到50%,目標(biāo)蛋白質(zhì)純度達(dá)到95.2%。所得產(chǎn)物經(jīng)溴化氰(CNBr)消化后,采用固體平板擴(kuò)散法進(jìn)行抑菌活性的測定,測得結(jié)果具有明顯抑菌活性。
[Abstract]:In recent years, an in vitro protein synthesis system using exogenous mRNA or DNA as template, which can synthesize proteins by adding substrate and energy to the enzyme system of extracellular extracts, has attracted more and more attention.Compared with the traditional in vivo system, the cell-free system has many advantages: 1) the reaction is simple and simple, only by adding the extract DNA template and all kinds of necessary reaction substrates.It can be used to express proteins that are difficult to express in the body system.For example, antimicrobial peptides and membrane proteins, which are toxic to cells, can directly use PCR products as linear templates to parallel synthesize a variety of proteins on microporous plates at the same time, and can meet the needs of high-throughput drug screening and proteomics research.Human 尾 -defensin is a kind of cationic antimicrobial peptide which has broad spectrum antibacterial activity and plays a defensive role in the body against the invasion of foreign microorganisms.It is expected to be a new endogenous antibiotic to solve the drug resistance of pathogenic bacteria, and has been proved to inhibit the replication of HIV.The expression vector of human 尾 -defensin-2 (HBD-2) gene, which was cloned from human inflammatory skin tissue by RT-PCR method, was constructed by codon optimized HBD-2 synthetic gene.A series of HBD-2 expression vectors with different codon sources or different fusion tags were obtained.The expression vector of HBD-2 from different codon sources was expressed in the acellular protein synthesis system of E. coli. The results of comparative experiments showed that the protein expression of the synthesized gene after codon optimization was equal to that of human gene.Furthermore, HBD-2 expression vectors with different fusion tags (such as sulfur redox protein trxA and green fluorescent protein GFP) were expressed in acellular protein synthesis system of Escherichia coli.The results showed that the expression of HBD-2 was significantly increased with the addition of fusion tags, and the effect of trxA on the expression of HBD-2 was the most obvious.Although the expression level of HBD-2 gene with GFP fusion tag was slightly lower than that with trxA fusion tag, GFP fusion tag was helpful to detect the expression of the fusion protein in real time.The fusion protein expressed in acellular protein synthesis system is highly soluble and avoids the difficulty of forming inclusion bodies when expressing antibacterial peptides in Escherichia coli.The effect of reaction mode on the expression of target protein in cell-free system was compared.The results showed that the cell-free reaction mode of continuous Exchange Cell-freewas significantly higher than that of batch operation, and the expression of target protein was up to 2 mg / ml.This is mainly because in the CECF mode, the substrate and energy can be continuously replenished and the inhibitory byproducts can be removed at the same time, thus greatly prolonging the reaction time and increasing the yield of the products.The purification of the target protein, trxA-HBD-2, expressed by cell-free system, was studied. An efficient purification process was established. The total yield and purity of the target protein were 50 and 95.2, respectively.The antibacterial activity of the product was determined by solid plate diffusion method after digested by CNBr. the results showed that the antibacterial activity was obvious.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：Q78;R346

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