本文關(guān)鍵詞：鼠源Fcε-Fcγ融合蛋白的瞬時(shí)基因表達(dá)、大量制備及其對(duì)小鼠的抗過(guò)敏作用　出處：《華東理工大學(xué)》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 過(guò)敏 Fcε-Fcγ 瞬時(shí)基因表達(dá) 中國(guó)倉(cāng)鼠卵巢細(xì)胞 被動(dòng)皮膚過(guò)敏實(shí)驗(yàn)

【摘要】：過(guò)敏性疾病普遍存在于人們的日常生活中,隨著全球范圍內(nèi)的空氣和環(huán)境的惡化,發(fā)病率呈逐年上升的趨勢(shì)。哮喘是一種最典型的過(guò)敏性疾病,也是在工業(yè)化國(guó)家最常見(jiàn)的慢性疾病,被世界衛(wèi)生組織列為疾病中的四大頑癥之一。哮喘的治療是世界公認(rèn)的醫(yī)學(xué)難題,人們一直在尋找安全有效的哮喘治療藥物,除了基于傳統(tǒng)藥物的改良和優(yōu)化外,更多地把希望寄托在新的治療靶點(diǎn)和生物藥物的開(kāi)發(fā)上。目前處于臨床研究中的治療哮喘的生物藥物有十多種,另有更多種臨床前研究中的候選藥物。阻斷IgE-多價(jià)抗原復(fù)合體與肥大細(xì)胞和嗜堿性粒細(xì)胞表面的受體FcεRI的結(jié)合可以從源頭上阻止過(guò)敏反應(yīng)的發(fā)生,是治療過(guò)敏疾病的主要方向。 抗過(guò)敏融合蛋白(AAFP)由人Fcε和Fcγ構(gòu)成,可以交聯(lián)肥大細(xì)胞和嗜堿粒細(xì)胞表面的介導(dǎo)過(guò)敏反應(yīng)的Fcε受體Ⅰ(FcεRI)和抑制性Fcγ受體Ⅱb (FcγRⅡb),抑制肥大細(xì)胞和嗜堿粒細(xì)胞的活化及過(guò)敏炎性介質(zhì)的釋放,阻斷過(guò)敏的發(fā)生。受體的特異性決定AAFP只作用于人的細(xì)胞,這就給AAFP的功能和作用機(jī)理的研究帶來(lái)材料來(lái)源的限制。鑒于此,本文設(shè)計(jì)了鼠源的AAFP (mAAFP),利用瞬時(shí)基因表達(dá)(Transient gene expression, TGE)技術(shù)快速大量制備目的蛋白,并采用Protein A親和層析純化mAAFP。通過(guò)小鼠的被動(dòng)皮膚過(guò)敏反應(yīng)實(shí)驗(yàn),研究了mAAFP抗過(guò)敏藥物的藥效,為評(píng)估AAFP可以作為治療人類過(guò)敏性疾病藥物的前期開(kāi)發(fā)研究奠定了基礎(chǔ)。 瞬時(shí)基因表達(dá)技術(shù)可以在短時(shí)間內(nèi)獲得一定量的目的蛋白而被廣泛研究和應(yīng)用。但這種技術(shù)的應(yīng)用效果常因蛋白而異。因此,本實(shí)驗(yàn)需要針對(duì)mAAFP建立一個(gè)快速高產(chǎn)的瞬時(shí)基因表達(dá)體系。TGE過(guò)程復(fù)雜,許多因素影響其轉(zhuǎn)染效率和目的蛋白的產(chǎn)率,如宿主細(xì)胞、表達(dá)載體、轉(zhuǎn)染試劑和轉(zhuǎn)染過(guò)程等等。目前,大部分動(dòng)物細(xì)胞表達(dá)的蛋白藥物都是在CHO細(xì)胞中生產(chǎn)的,采用CHO細(xì)胞作為T(mén)GE的宿主細(xì)胞,可以確保前期研發(fā)樣品與最后臨床應(yīng)用產(chǎn)品在分子結(jié)構(gòu)上的一致性(如糖基化等)。 本實(shí)驗(yàn)首次成功使用兩種無(wú)血清培養(yǎng)基CDAGT-CHO和PF-CHO進(jìn)行了瞬時(shí)基因表達(dá)。前者的特點(diǎn)是可以提供高轉(zhuǎn)染效率,后者的特點(diǎn)足促進(jìn)蛋白的表達(dá)。針對(duì)mAAFP的特性,我們?cè)?孔板內(nèi)優(yōu)化了其瞬時(shí)轉(zhuǎn)染體系。首先對(duì)比了兩種不同商業(yè)化載體在轉(zhuǎn)染效率上的差別,選擇了pID作為后續(xù)實(shí)驗(yàn)的表達(dá)載體。之后,從轉(zhuǎn)染試劑、轉(zhuǎn)染時(shí)DNA/PEI復(fù)合物的共沉淀、轉(zhuǎn)染時(shí)DNA與PEI的用量、轉(zhuǎn)染時(shí)的細(xì)胞密度等多方面對(duì)其轉(zhuǎn)染過(guò)程進(jìn)行了優(yōu)化。結(jié)果表明,利用PEI介導(dǎo)載體pID-EG瞬時(shí)轉(zhuǎn)染表達(dá)mAAFP的最優(yōu)條件是：DNA (4μg/106 cells)與PEI (DNA:PEI=1:2.5)在20%培養(yǎng)體積的DMEM/F12培養(yǎng)基中共沉淀5 min形成DNA/PEI復(fù)合物,加入細(xì)胞密度為2×106 cells/ml的CHO-S細(xì)胞培養(yǎng)體系中進(jìn)行轉(zhuǎn)染,6 h后補(bǔ)加等體積的PF-CHO培養(yǎng)基完成轉(zhuǎn)染并進(jìn)行后期培養(yǎng)。 利用優(yōu)化的瞬時(shí)轉(zhuǎn)染條件,在1.3 L生物反應(yīng)器中瞬時(shí)表達(dá)了mAAFP, (?)且蛋白表達(dá)量只有2 mg。為了進(jìn)一步提高蛋白表達(dá)量,采用葡萄糖限制性流加及降低溫度控制策略對(duì)轉(zhuǎn)染后的培養(yǎng)過(guò)程進(jìn)行了調(diào)控,提供足夠的營(yíng)養(yǎng)物質(zhì)給轉(zhuǎn)染后的細(xì)胞,以延長(zhǎng)反應(yīng)器中細(xì)胞的生長(zhǎng)和存活率維持時(shí)間。結(jié)果顯示,采用降低溫度流加培養(yǎng)工藝對(duì)轉(zhuǎn)染后的培養(yǎng)過(guò)程進(jìn)行控制,與原來(lái)的分批培養(yǎng)相比,培養(yǎng)時(shí)間延長(zhǎng)了一倍,蛋白總產(chǎn)量提高了14倍,達(dá)到30 mg。這一結(jié)果表明,對(duì)瞬時(shí)轉(zhuǎn)染后的培養(yǎng)過(guò)程進(jìn)行調(diào)控可以顯著提高蛋白的表達(dá)。將此過(guò)程進(jìn)一步成功放大到5L生物反應(yīng)器中,最終蛋白濃度達(dá)到了25 mg/L。利用rProtein A Sepharose Fast Flow介質(zhì)純化mAAFP,通過(guò)優(yōu)化蛋白的洗脫條件,收獲的mAAFP純度達(dá)到98%,收率達(dá)到85%以上。通過(guò)SDS-PAGE、Western blot對(duì)純化得到的mAAFP進(jìn)行了驗(yàn)證。 被動(dòng)皮膚過(guò)敏反應(yīng)(PCA)實(shí)驗(yàn)可以用于檢測(cè)抗原或者評(píng)價(jià)抗過(guò)敏藥物的效果。在本研究中,PCA結(jié)果成功揭示了mAAFP可以抑制IgE介導(dǎo)的I型過(guò)敏反應(yīng),其抑制效率可以達(dá)到96%。在6.13mg/kg的mAAFP劑量下,其抑制效果在體內(nèi)可以持續(xù)24天甚至更久。這一現(xiàn)象說(shuō)明,mAAFP一旦注射入體內(nèi)迅速與肥大細(xì)胞或嗜堿性粒細(xì)胞表而的受體結(jié)合后,其在體內(nèi)不易被清除,其半衰期長(zhǎng)于其結(jié)構(gòu)中的任一免疫球蛋白。這一結(jié)果顯示了AAFP蛋白具有治療過(guò)敏性疾病的潛力,并為其深層次的分子機(jī)理研究以及AAFP在人體內(nèi)的藥效研究奠定了基礎(chǔ)。
[Abstract]:Allergic diseases are common in people's daily life, with the deterioration of global air and environment, the incidence rate is increasing year by year. Asthma is one of the most typical allergic diseases, is the most common chronic disease in industrialized countries, the WHO is listed as one of the four major chronic diseases. The treatment of asthma is recognized worldwide medical problem, people have been searching for a safe and effective drug for the treatment of asthma, in addition to improving and optimizing the traditional medicine based on more put their hopes in the development target and biological treatment. The new biological drugs currently in clinical research in the treatment of asthma more than 10 otherwise, more in preclinical studies of candidate drugs. Blocking IgE- binding multivalent antigen complex with mast cells and basophilic cell surface receptor Fc epsilon RI from the source to stop The occurrence of anaphylaxis is the main direction for the treatment of allergic diseases.
Anti allergy fusion protein (AAFP) composed of Fc and Fc epsilon gamma, the cross-linking of mast cells and basophils surface mediated allergic reaction Fc epsilon receptor (Fc - RI) and inhibitory Fc receptor gamma (Fc gamma B R II B), inhibition of mast cell and basophil cell activation and allergic inflammatory mediators release, blocking allergies. The receptor specificity AAFP only effects on human cells, which bring source material restrictions on research to the function and mechanism of AAFP. In view of this, this paper designed a murine AAFP (mAAFP), transient gene expression using (Transient gene expression, TGE) technology and rapid preparation of target protein, and purified by using Protein A mAAFP. passive cutaneous anaphylaxis in mice affinity chromatography, pharmacodynamic study of anti allergy drug mAAFP, for the evaluation of AAFP can be used as a pre treatment of human allergic disease development The research laid the foundation.
Transient gene expression can be obtained in a short period of time a certain amount of target protein is widely studied and applied. But the application of this technique for protein varies. Therefore, the need for mAAFP to establish a rapid high transient gene expression system of.TGE process is complicated, many factors affect the transfection efficiency and protein the yield, such as host cell, expression vector, transfection reagent and the transfection process and so on. At present, most of the animal cell expression of protein drugs are produced in CHO cells, using CHO cells as TGE host cells, can ensure the consistency of the early development of sample and finally clinical application of the molecular structure of the product (such as sugar etc.).
This is the first successful use of two kinds of serum free medium CDAGT-CHO and PF-CHO were transient gene expression. The characteristic is that it can provide high transfection efficiency, foot characteristics of the latter promotes protein expression. Aiming at the characteristics of the mAAFP, we optimized the transfection system in 6 hole plate. The comparison of the difference between the two different commercial carriers in the transfection efficiency, choose the pID as the expression vector of the follow-up experiments. Then, from the transfection reagent, transfection of DNA/PEI complex co precipitation, the amount of DNA and PEI transfection, transfected cell density of many aspects such as optimization of the transfection process. The results show that the missile carrier transient transfection of pID-EG expression of mAAFP's optimal conditions is the use of PEI: DNA (4 g/106 cells) and PEI (DNA:PEI=1:2.5) in the 20% volume of culture medium of DMEM/F12 co precipitation in 5 min formation of the DNA/PEI complex, adding cell density The transfection was carried out in the CHO-S cell culture system of 2 x 106 cells/ml. After 6 h, the transfection was supplemented with equal volume PF-CHO medium and later culture was carried out.
Based on Instantaneous Optimization of transfection conditions, in 1.3 L bioreactor transiently expressed mAAFP, (?) and the expression of only 2 mg. in order to further improve the amount of protein expression by glucose restriction and flow and reduce the temperature control strategy on the training process after transfection of regulation, to provide sufficient nutrients for transfection after the cell, to extend the celligen growth and survival duration. The results showed that the lower temperature fed batch culture process to control the process training after transfection, compared with the original batch cultivation, cultivation time doubled, total protein yield increased 14 times, reaching 30 mg. results show that the process of training after transient transfection control can significantly improve the expression of the protein. This process will be further enlarged successfully in 5L bioreactor, the final protein concentration reached 25 mg/L. by rP Rotein A Sepharose Fast Flow medium purifies mAAFP. By optimizing the elution condition of protein, the purity of harvested mAAFP reaches 98%, and the yield is above 85%. Through SDS-PAGE, Western blot, the purified mAAFP is verified.
Passive cutaneous anaphylaxis (PCA) test can be used for the detection of antigen or anti allergy drug evaluation results. In this study, I type PCA allergic reaction results revealed that mAAFP can inhibit IgE mediated, the inhibition efficiency can reach 96%. at the dose of mAAFP 6.13mg/kg, its inhibitory effect in vivo can last for 24 days even more. This phenomenon shows that mAAFP once injected into the body quickly and mast cells or basophilic cell surface receptor binding, the clearance in the body is not easy, the half-life is longer than any immunoglobulin in its structure. The results showed that the AAFP protein has the potential for the treatment of allergic diseases, and laid the foundation for the efficacy of the deep research on the molecular mechanism of AAFP in the body.

【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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