本文選題：rhTRAIL + 工業(yè)生產(chǎn)��； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：1.pHS-TRAIL表達質(zhì)粒的構(gòu)建 人腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(human TNF-Related Apoptosis Inducing Ligand, hTRAIL)是一種跨膜蛋白,由281個氨基酸組成,其中1-14位氨基酸為胞內(nèi)區(qū),15-40位氨基酸為跨膜區(qū),41-281位氨基酸為胞外區(qū)。由于114-281位氨基酸的胞外區(qū)片段完全具有促細胞凋亡活性,同時完整的rhTRAIL蛋白分子在體外較難獲得表達,因此我們選擇114-281位氨基酸所在肽段進行重組表達質(zhì)粒的構(gòu)建。先前的研究發(fā)現(xiàn),rhTRAIL表達系統(tǒng)的選擇具有一定的難度,若使用真核系統(tǒng)對rhTRAIL蛋白進行表達,容易形成沒有生物學(xué)活性的二聚體形式；當(dāng)采用常規(guī)的原核系統(tǒng)例如T7表達體系和溫度誘導(dǎo)性表達體系時,rhTRAIL蛋白又容易形成包涵體,這些表達系統(tǒng)的問題會給后期純化工藝、產(chǎn)品質(zhì)量等均會帶來問題,因而我們選用了pBR322載體,并以此為基礎(chǔ)構(gòu)建了一個色氨酸啟動子驅(qū)動目的基因的載體,隨后在色氨酸啟動子的下游插入編碼hTRAIL114-281片段的基因序列,再將該重組質(zhì)粒導(dǎo)入宿主菌W3110。構(gòu)建獲得表達載體,確認目的蛋白的正確表達,并初步摸索了其在搖瓶水平影響表達的條件。 2. rhTRAIL的發(fā)酵及純化工藝研究 上一章節(jié)的研究中,通過基因工程技術(shù)將人腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(Recombinant Human TNF-related apoptosis-inducing ligand、rhTRAIL)編碼胞外部分114-281aa的基因片段插入到表達載體pHS色氨酸啟動子的下游,得到重組質(zhì)粒pHS-TRAIL,而后將此質(zhì)粒轉(zhuǎn)化到W3110菌株內(nèi),基因測序結(jié)果顯示插入的目的基因序列正確。由于rhTRAIL的臨床使用劑量可能較大(通常百毫克級),因此對于表達系統(tǒng)、高密度發(fā)酵工藝以蛋白純化工藝的選擇和優(yōu)化以及質(zhì)量控制均提出了較高的要求。通常,影響大腸桿菌高密度發(fā)酵的因素主要包括細胞新陳代謝所需的營養(yǎng)的供給和控制、毒副產(chǎn)物(乳酸和氨)的控制或排出、氧氣的供給以及關(guān)鍵參數(shù),如pH值、溶氧濃度和溫度等的實時控制問題。本章就rhTRAIL的搖瓶培養(yǎng)、發(fā)酵生產(chǎn)工藝以及純化工藝進行了初步的研究和優(yōu)化,通過對培養(yǎng)基的組成、高密度發(fā)酵條件的優(yōu)化以及純化工藝的摸索,從而研究如何在保證生物制品產(chǎn)品質(zhì)量的前提下提高目的蛋白表達量和純化工藝的收率,并最終提高rhTRAIL的產(chǎn)量。 3. rhTRAIL的體外及體內(nèi)藥效學(xué)及機理研究 在前面的研究中,我們通過對培養(yǎng)基的組成、高密度發(fā)酵條件的優(yōu)化以及純化工藝的摸索,在保證rhTRAIL蛋白品質(zhì)的前提下提高了蛋白產(chǎn)量,獲得了足夠量的rhTRAIL蛋白純品。本章節(jié)我們進一步對獲得的重組蛋白進行體外及體內(nèi)藥效學(xué)及機理研究。由于重組TRAIL蛋白具有廣譜抗腫瘤活性,因此我們將HCT-116、Colo-205、A549及MCF-7等12種腫瘤細胞作為體外藥效活性研究對象,并且構(gòu)建了人結(jié)腸癌HCT-116和Colo-205裸小鼠模型和非小細胞肺癌95D裸小鼠模型,采用單獨用藥、聯(lián)合用藥的方式進行了體內(nèi)藥效學(xué)研究；最后以A549細胞為實驗對象,通過Annexin-PI雙染和Western blot的實驗手段研究了rhTRAIL蛋白的誘導(dǎo)細胞凋亡途徑。 4. rhTRAIL在大鼠體內(nèi)的藥動學(xué)和組織分布研究 在前述章節(jié)中,我們已經(jīng)利用基因工程手段獲得了具有生物學(xué)活性的rhTRAIL純品；本章中,我們研究了大鼠靜脈注射5、10和30mg/kg rhTRAIL后,血漿濃度經(jīng)時變化曲線,并研究了大鼠靜脈注射10mg/kg rhTRAIL后,各組織中的rhTRAIL分布情況。結(jié)果顯示在5-30mg/kg范圍內(nèi),主要藥物動力學(xué)參數(shù)AUC及Cmax與劑量呈線性相關(guān),CL和V1在三個劑量組沒有顯著性差異(P0.05),雌雄大鼠間各動力學(xué)參數(shù)沒有顯著性差異,同時,大鼠靜脈注射rhTRAIL后,其迅速分布到各組織。腎臟為最主要的分布器官,其次為脾、血、卵巢、肝臟和肺,心、腦、脂肪、胃、肌肉、腸、胸腺和睪丸組織分布較低。上述組織中rhTRAIL含量隨著時間的延長迅速下降,未觀察到蓄積現(xiàn)象。 結(jié)論 腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TNF-related apoptosis inducing ligand, TRAIL)屬于腫瘤壞死因子家族,在體內(nèi)以三聚體的形式可與不同受體結(jié)合后通過不同信號通路介導(dǎo)細胞凋亡。相關(guān)研究證實TRAIL蛋白對多種人腫瘤細胞(如肺癌、肝癌、結(jié)腸癌、白血病細胞等)具有生長抑制和細胞毒效應(yīng)。國內(nèi)外一些制藥企業(yè)均對TRAIL開展了多項臨床前和臨床研究,但進入臨床后的研究進展則較為緩慢,對其原因也未見明確報道。 本研究在構(gòu)建了重組TRAIL蛋白的表達體系并通過工藝優(yōu)化獲得了高純度的重組TRAIL蛋白后,對其體外、體內(nèi)藥效以及體內(nèi)代謝、組織分布性質(zhì)開展了相關(guān)研究。研究結(jié)果顯示重組TRAIL蛋白在體內(nèi)體外對不同腫瘤細胞均具有一定的生長抑制作用,體內(nèi)代謝組織分布結(jié)果顯示其在體內(nèi)主要分布于腎臟。上述結(jié)果一方面提示重組TRAIL蛋白具有良好的成藥性,另一方面可能由于TRAIL蛋白被腎小球快速濾過所致的半衰期較短,提示聯(lián)合小分子用藥或TRAIL蛋白偶聯(lián)小分子藥物可能更適合于該分子作為治療性蛋白藥物的開發(fā)方向。
[Abstract]:Construction of 1.pHS-TRAIL expression plasmid
The human tumor necrosis factor related apoptosis inducing ligand (human TNF-Related Apoptosis Inducing Ligand, hTRAIL) is a transmembrane protein consisting of 281 amino acids, of which 1-14 amino acids are intracellular, 15-40 amino acids are transmembrane regions and 41-281 amino acids are extracellular domain. The extracellular domain of 114-281 amino acids is fully promoted. The cell apoptosis activity, and the complete rhTRAIL protein molecules are difficult to express in vitro, so we choose the 114-281 amino acid peptide segment for the construction of recombinant expression plasmid. Previous studies have found that the selection of rhTRAIL expression system is difficult. If the eukaryotic system is used to express rhTRAIL protein, it is easy to form. There is no biological activity in the form of two polymer; when the conventional prokaryotic system such as the T7 expression system and the temperature induced expression system are used, the rhTRAIL protein is easy to form inclusion bodies. The problems of these expression systems will bring problems to the later purification process and the quality of the products. Therefore, we choose the pBR322 carrier and take this as the basis. The carrier of a tryptophan promoter to drive the target gene was constructed, then the gene sequence of the encoded hTRAIL114-281 fragment was inserted into the downstream of the tryptophan promoter, and the recombinant plasmid was introduced into the host bacterium W3110. to construct the expression vector, to confirm the correct expression of the target protein, and to preliminarily explore the expression of the target protein at the shake flask level. Conditions.
Study on the fermentation and purification process of 2. rhTRAIL
In the last chapter, the recombinant plasmid pHS-TRAIL was inserted into the downstream of the expression vector pHS tryptophan promoter by the gene engineering technique, which was encoded by the human tumor necrosis factor related apoptosis inducing ligand (Recombinant Human TNF-related apoptosis-inducing ligand, rhTRAIL), and then the recombinant plasmid pHS-TRAIL was obtained. The gene sequencing results show that the inserted target gene sequence is correct. Because the clinical dosage of rhTRAIL may be larger (usually 100 milligram), the high density fermentation process has high requirements for the selection and optimization of protein purification process and quality control for the expression system. Usually, the high density fermentation process has a great influence on the expression system. The factors for high-density fermentation of Enterobacteriaceae include the supply and control of nutrients needed for cell metabolism, control or discharge of toxic by-products (lactic acid and ammonia), supply of oxygen and key parameters, such as pH, dissolved oxygen concentration and temperature. This chapter deals with the shake flask culture, fermentation process and purification process of rhTRAIL. The preliminary study and optimization were carried out. Through the composition of the medium, the optimization of the high density fermentation conditions and the exploration of the purification process, the yield of the target protein expression and the purification process was improved on the premise of ensuring the quality of the biological products, and the production of rhTRAIL was finally raised.
Pharmacodynamics and mechanism of 3. rhTRAIL in vitro and in vivo
In the previous study, we improved the production of protein by improving the composition of the medium, the optimization of high density fermentation conditions and the purification process. We obtained a sufficient quantity of rhTRAIL protein in the premise of guaranteeing the quality of rhTRAIL protein. In this chapter, we further studied the pharmacodynamics of the obtained recombinant protein in vitro and in vivo and in vivo. As a result of the broad-spectrum anti-tumor activity of recombinant TRAIL protein, 12 tumor cells, such as HCT-116, Colo-205, A549 and MCF-7, were used as research objects in vitro, and a nude mouse model of HCT-116 and Colo-205 in human colon cancer and a nude mouse model of non-small cell lung cancer were constructed. In vivo pharmacodynamics study was conducted. Finally, the apoptosis pathway induced by rhTRAIL protein was studied by the experimental means of Annexin-PI double staining and Western blot using A549 cells as the experimental object.
Pharmacokinetics and tissue distribution of 4. rhTRAIL in rats
In the foregoing section, we have obtained the biological activity of rhTRAIL pure products using genetic engineering. In this chapter, we studied the time variation curve of plasma concentration after intravenous injection of 5,10 and 30mg/kg rhTRAIL in rats, and studied the distribution of rhTRAIL in each tissue after intravenous injection of 10mg/kg rhTRAIL. The main pharmacokinetic parameters, AUC and Cmax, were linearly related to the dose in the 5-30mg/kg range. There was no significant difference between the three doses of CL and V1 (P0.05). There was no significant difference in the kinetic parameters between the male and female rats. At the same time, after the intravenous injection of rhTRAIL, the rats were rapidly distributed to the tissues. The kidney was the most important distribution organ. The distribution of the spleen, blood, ovary, liver and lung, heart, brain, fat, stomach, muscle, intestines, thymus and testis was low. The content of rhTRAIL in the above tissues declined rapidly with the time, and the accumulation was not observed.
conclusion
The tumor necrosis factor related apoptosis inducing ligand (TNF-related apoptosis inducing ligand, TRAIL) belongs to the tumor necrosis factor family, which can be combined with different receptors in the form of a tripolymer in the body to mediate cell apoptosis through different signaling pathways. The relevant studies have proved that TRAIL egg white has a variety of human tumor cells (such as lung cancer, liver cancer, and colon cancer). A number of pharmaceutical companies both at home and abroad have carried out a number of pre clinical and clinical studies on TRAIL, but the progress of the study after entering the clinic is slow, and the reasons for it have not been clearly reported.
In this study, the recombinant TRAIL protein expression system was constructed and a highly purified recombinant TRAIL protein was obtained by process optimization. In vitro, in vivo, in vivo, in vivo and in vivo metabolism and tissue distribution properties were studied. The results show that recombinant TRAIL protein has a certain growth inhibition on different tumor cells in vivo. The results showed that it was mainly distributed in the kidneys in vivo. The results suggested that the recombinant TRAIL protein had good drug resistance, and on the other hand, the half-life of TRAIL protein was caused by rapid glomerular filtration, suggesting that small molecular drugs or TRAIL protein coupling small molecular drugs can be used. It is more suitable for the development of this molecule as therapeutic protein drug.

【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】：2014
【分類號】：R969.1

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