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

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

【摘要】：補體激活造是炎癥反應(yīng)性疾病的病理反應(yīng)之一。如何能夠控制補體的激活是當前科學研究的一個熱點。 促衰變因子(DAF)屬膜補體調(diào)節(jié)蛋白，，是一種錨定于細胞膜表面的蛋白質(zhì)。可以促進經(jīng)典、旁路途徑C3、C5轉(zhuǎn)化酶的解離并防止其組裝，這對其宿主細胞起到了重要的保護作用�？扇苄訢AF的表達在過去已經(jīng)有過非常多的嘗試，但多為帶有真核細胞分泌信號肽的方式表達。由于DAF有4個短的保守重復序列(SCR)，含有多達8對二硫鍵，原核核細胞表達都以包涵體告終。本研究擬采用大腸桿菌表達Trx融合蛋白的方式來表達大鼠DAF的4個SCR，以期得到可溶性的表達產(chǎn)物。 首先通過PCR的方法，將編碼大鼠DAF四個SCR的基因片段亞克隆到Trx融合原核表達載體pET32a(+)。再將構(gòu)建好的載體轉(zhuǎn)入大腸桿菌表達宿主BL21(DE3)pLysS。 通過比較3種不同的誘導溫度和IPTG濃度，經(jīng)Western Blot檢測，發(fā)現(xiàn)低溫誘導確實能夠增加可溶性蛋白的量，但效果并不是很明顯。通過Ni~(2+)親和層析分離出了可溶性組分，經(jīng)Western Blot檢測，進一步確認了該可溶性成分確實為帶有His標簽的融合蛋白。 按標準的37℃，1mmol／L IPTG誘導4h的方案，得到了大量包涵體。通過包涵體復性，得到了具有抑制致敏綿羊紅細胞發(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DAF的4個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.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R363

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