本文關(guān)鍵詞：大鼠DAF功能區(qū)的重組表達(dá)純化和功能鑒定　出處：《第四軍醫(yī)大學(xué)》2006年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： DAF 包涵體 蛋白復(fù)性 Trx融合蛋白 可溶性表達(dá)

【摘要】：補(bǔ)體激活造是炎癥反應(yīng)性疾病的病理反應(yīng)之一。如何能夠控制補(bǔ)體的激活是當(dāng)前科學(xué)研究的一個(gè)熱點(diǎn)。 促衰變因子(DAF)屬膜補(bǔ)體調(diào)節(jié)蛋白，，是一種錨定于細(xì)胞膜表面的蛋白質(zhì)�？梢源龠M(jìn)經(jīng)典、旁路途徑C3、C5轉(zhuǎn)化酶的解離并防止其組裝，這對(duì)其宿主細(xì)胞起到了重要的保護(hù)作用�？扇苄訢AF的表達(dá)在過(guò)去已經(jīng)有過(guò)非常多的嘗試，但多為帶有真核細(xì)胞分泌信號(hào)肽的方式表達(dá)。由于DAF有4個(gè)短的保守重復(fù)序列(SCR)，含有多達(dá)8對(duì)二硫鍵，原核核細(xì)胞表達(dá)都以包涵體告終。本研究擬采用大腸桿菌表達(dá)Trx融合蛋白的方式來(lái)表達(dá)大鼠DAF的4個(gè)SCR，以期得到可溶性的表達(dá)產(chǎn)物。 首先通過(guò)PCR的方法，將編碼大鼠DAF四個(gè)SCR的基因片段亞克隆到Trx融合原核表達(dá)載體pET32a(+)。再將構(gòu)建好的載體轉(zhuǎn)入大腸桿菌表達(dá)宿主BL21(DE3)pLysS。 通過(guò)比較3種不同的誘導(dǎo)溫度和IPTG濃度，經(jīng)Western Blot檢測(cè)，發(fā)現(xiàn)低溫誘導(dǎo)確實(shí)能夠增加可溶性蛋白的量，但效果并不是很明顯。通過(guò)Ni~(2+)親和層析分離出了可溶性組分，經(jīng)Western Blot檢測(cè)，進(jìn)一步確認(rèn)了該可溶性成分確實(shí)為帶有His標(biāo)簽的融合蛋白。 按標(biāo)準(zhǔn)的37℃，1mmol／L IPTG誘導(dǎo)4h的方案，得到了大量包涵體。通過(guò)包涵體復(fù)性，得到了具有抑制致敏綿羊紅細(xì)胞發(fā)生溶血的蛋白。 綜上所述，本研究采用的Trx融合蛋白的方式表達(dá)DAF的4個(gè)SCR
[Abstract]:Complement activation is one of the pathological reactions of inflammatory response diseases. How to control the activation of complement is a hot spot in the current scientific research.
Decay accelerating factor (DAF) is a membrane complement regulatory protein, is a membrane anchored to the cell surface. The protein can promote the classical pathway, C3, dissociation of the C5 convertase and prevent their formation, which is to protect the important role of the host cell. The expression of soluble DAF in the past has been try very much, but is a eukaryotic secretory signal peptide expression. Because DAF has 4 short consensus repeat (SCR), containing up to 8 to two disulfide bonds, the prokaryotic cells have ended in inclusion. This study used Escherichia coli Trx fusion protein expression to 4 SCR expression of DAF in rats, in order to obtain the soluble expression product.
First, the gene fragment encoding four SCR of rat DAF was subcloned into Trx fusion prokaryotic expression vector pET32a (+) by PCR method. Then the constructed vector was transferred into Escherichia coli to express host BL21 (DE3) pLysS..
Through the comparison of 3 different induction temperature and IPTG concentration by Western Blot detection found induced by low temperature can increase the soluble protein content, but the effect is not very obvious. The Ni~ (2+) affinity chromatography to isolate the soluble components by Western Blot detection, further confirmed the soluble components indeed a fusion protein with His tag.
A large number of inclusion bodies were obtained according to the standard 37 degree temperature, 1mmol / L IPTG and 4H induction. A lot of inclusion bodies were obtained by inclusion body renaturation, and a protein that inhibited hemolysis of sensitized sheep erythrocytes was obtained.
To sum up, 4 SCR of DAF were expressed by the Trx fusion protein used in this study.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R363

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