雞IL-2二聚體形成機制的研究
發(fā)布時間：2018-07-18 13:41
【摘要】：白細胞介素-2（interleukin-2，IL-2），是特異性抗原或分裂原刺激活化的T淋巴細胞分泌的一種具有廣泛重要作用的細胞因子，對T淋巴細胞、B淋巴細胞、單核細胞和自然殺傷細胞的復制與分化起重要作用，且在后天獲得性免疫防御和先天免疫防御系統(tǒng)的激活和維護中起積極作用。白細胞介素中已發(fā)現(xiàn)IL-6、IL-8、IL-12等多種蛋白能夠形成具有重要生物學活性的二聚體。人、魚、倉鼠和小鼠等多個物種中發(fā)現(xiàn)IL-2二聚體形式的存在。對內(nèi)源性chIL-2進行純化發(fā)現(xiàn)其有兩個洗脫峰，分子量大小相差二倍左右，大分子量條帶可能為chIL-2二聚體，鑒于二聚體形式IL-2的重要性，本試驗進行chIL-2二聚體形成機制的研究。 將366bp的chIL-2基因克隆到原核表達載體pET-28a中，在C-端融合6×His親和純化標簽。再以重組載體pET-28a-chIL-2為模版，擴增融合蛋白的ORF，亞克隆到pFastBac-Dual真核細胞表達載體的P10啟動子下，表達與原核表達蛋白氨基酸序列一致的融合蛋白。將chIL-2基因克隆到pTYB1原核表達載體，使chIL-2基因與1362bp的Intein基因融合在同一ORF，編碼分子量約為71kDa的chIL-2-Intein融合蛋白，變性洗脫純化得到chIL-2-Intein融合蛋白，免疫大白兔制備抗chIL-2多克隆抗體，非變性切割獲得無標簽chIL-2蛋白；制備的兔源多抗用于原核和真核純化的chIL-2-His-tag融合蛋白以及無標簽chIL-2蛋白的檢測。結(jié)果獲得效價為4.01×106的兔抗chIL-2特異性抗體，Western blot檢測結(jié)果表明，真核和原核表達純化的chIL-2-His-tag融合蛋白以及無標簽chIL-2蛋白都有二聚體形式存在。 實驗同時探究溫度對不同表達體系來源蛋白二聚體形成的影響。Western blot檢測結(jié)果表明，4℃條件下，真核表達純化的chIL-2-His-tag二聚體的形成隨時間的增加無顯著性變化（P0.05），37℃孵育24h時，融合蛋白完全以二聚體的形式存在；而原核表達純化的融合蛋白在4℃、37℃孵育不同時間時，兩種形式的蛋白比例均無顯著性變化（P0.05），且37℃孵育不能完全以二聚體的形式存在。結(jié)果表明，真核表達純化的chIL-2融合蛋白二聚體的形成對溫度十分敏感，而原核表達純化的chIL-2融合蛋白二聚形成對溫度不敏感。 為了闡明chIL-2二聚體形成的分子間作用力，本研究采用硫氰酸根（SCN-）、膽酸鹽、Triton X-100、脲素、鹽酸胍、EDTA、二硫蘇糖醇（DTT）和β-巰基乙醇（β-mercaptoethanol，β-ME）等多種不同性質(zhì)的試劑作用于二聚體化的chIL-2蛋白，以確定蛋白分子間相互作用力的類型。結(jié)果表明，硫氰酸根（SCN-）、膽酸鹽、Triton X-100、脲素、鹽酸胍、EDTA等試劑均不能破壞chIL-2二聚體結(jié)構，而40mmol/L二硫蘇糖醇和8%β-巰基乙醇可將chIL-2二聚體完全破壞形成單體。推測chIL-2-His-tag蛋白二聚體的形成可能是chIL-2分子間半胱氨酸殘基相互作用形成共價二硫鍵的結(jié)果。 為了驗證chIL-2分子中4個半胱氨酸殘基在蛋白二聚體形成的作用，對重組載體pET-28a-chIL-2中的半胱氨酸對應的密碼子分別進行單點、兩點、三點和四點突變，誘導表達純化獲得各突變體蛋白，Western blot檢測chIL-2二聚體的形成情況。結(jié)果，單點、兩點和三點突變體蛋白都能形成二聚體結(jié)構，而四點突變蛋白不能形成二聚體。表明chIL-2-His-tag蛋白二聚體的形成可能是chIL-2分子間半胱氨酸殘基相互作用形成共價二硫鍵的結(jié)果，，且每個半胱氨酸殘基都能單獨發(fā)揮作用形成分子間二硫鍵。該實驗為chIL-2二聚體結(jié)構和功能的研究提供理論基礎。
[Abstract]:Interleukin -2 (interleukin-2, IL-2), a cytokine secreted by specific antigen or split prickly activated T lymphocytes, plays an important role in the replication and differentiation of T lymphocytes, B lymphocytes, mononuclear cells and natural killer cells, and acquired immune defense and innate immunity in the future. The activation and maintenance of the defense system play an active role. In interleukin, IL-6, IL-8, IL-12 and other proteins have been found to form two polymers with important biological activity. The presence of IL-2 two polymers in human, fish, hamster and mice is found. The purification of endogenous chIL-2 has two elution peaks. The magnitude difference is about two times, and the large molecular weight strip may be chIL-2 two polymer. In view of the importance of the form of two polymer IL-2, the mechanism of the formation of chIL-2 two polymer is studied in this experiment.
The chIL-2 gene of 366bp was cloned into the prokaryotic expression vector pET-28a, and a 6 x His affinity purification label was fused at the C- end. Then the recombinant vector pET-28a-chIL-2 was used as a template to amplify the ORF of the fusion protein. Subcloned to the P10 promoter of the eukaryotic expression vector of pFastBac-Dual, the fusion protein was expressed in accordance with the amino acid sequence of the prokaryotic expression protein. The chIL-2 gene was cloned to the pTYB1 prokaryotic expression vector. The chIL-2 gene was fused with the Intein gene of 1362bp in the same ORF, the molecular weight of the chIL-2-Intein fusion protein was about 71kDa, and the chIL-2-Intein fusion protein was purified by denaturing elution. The immunized white rabbit was prepared for anti chIL-2 clon antibody, and the unlabeled chIL-2 protein was obtained by non denaturing cutting. The rabbit source polyclonal antibody was used for the detection of chIL-2-His-tag fusion protein and unlabeled chIL-2 protein used in prokaryotic and eukaryotic purification. The results obtained the titer of 4.01 * 106 Rabbit anti chIL-2 specific antibody. The Western blot detection results showed that both eukaryotic and prokaryotic expression and purification of chIL-2-His-tag fusion protein and unlabeled chIL-2 protein were all available. The form of two polymer exists.
The experiment also explored the effect of temperature on the formation of protein two polymer of different expression systems. The results of.Western blot detection showed that the formation of chIL-2-His-tag two polymer expressed and purified by eukaryotic expression was not significantly changed with time (P0.05) at 4 C. The fusion protein existed in the form of two polymer when incubated at 37 C; and the prokaryotic cell was prokaryotic. When the purified fusion protein was incubated at 4 and 37, the proportion of the two forms of protein had no significant change (P0.05), and the incubation at 37 C could not be completely in the form of two polymer. The results showed that the formation of the chIL-2 fusion protein two polymer expressed by eukaryotic expression and purification was very sensitive to the temperature, and the chIL-2 melting of the prokaryotic expression and purification was fused. The formation of the protein two is insensitive to temperature.
In order to elucidate the intermolecular force formed by chIL-2 two polymer, this study uses a variety of different properties, such as thiocyanate (SCN-), cholate, Triton X-100, urea, guanidine hydrochloride, EDTA, two thiasanol (DTT) and beta mercaptoethanol (beta mercaptoethanol (beta -mercaptoethanol, beta -ME) and other different properties, to determine the intermolecular interaction between the protein molecules. The results show that the reagent of thiocyanate (SCN-), cholate, Triton X-100, urea, guanidine hydrochloride and EDTA can not destroy the structure of chIL-2 two polymer, and 40mmol/L two thulfyl alcohol and 8% beta mercaptoethanol can completely destroy the monomer of chIL-2 two polymer. It is presumed that the formation of chIL-2-His-tag protein two polymer may be between chIL-2 molecules. Cysteine residues interact to form covalent two sulfur bonds.
In order to verify the role of the 4 cysteine residues in the protein two polymer in the chIL-2 molecule, the codon corresponding to cysteine in the recombinant vector pET-28a-chIL-2 was given a single, two, three and four point mutation, and the expression and purification of the mutant protein were induced, and the formation of chIL-2 two polymer was detected by Western blot. Point, two and three point mutant proteins can form a two polymer structure, while the four point mutation protein can not form a two polymer. It shows that the formation of the chIL-2-His-tag protein two polymer may be the result of the covalent two sulfur bond formed by the interaction of chIL-2 molecular cysteine residues, and each cysteinic acid residue can play a role as a molecule to form a molecule. The two sulfur bonds between the two groups provide a theoretical basis for the study of the structure and function of chIL-2 two dimers.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831
本文編號：2132078
[Abstract]:Interleukin -2 (interleukin-2, IL-2), a cytokine secreted by specific antigen or split prickly activated T lymphocytes, plays an important role in the replication and differentiation of T lymphocytes, B lymphocytes, mononuclear cells and natural killer cells, and acquired immune defense and innate immunity in the future. The activation and maintenance of the defense system play an active role. In interleukin, IL-6, IL-8, IL-12 and other proteins have been found to form two polymers with important biological activity. The presence of IL-2 two polymers in human, fish, hamster and mice is found. The purification of endogenous chIL-2 has two elution peaks. The magnitude difference is about two times, and the large molecular weight strip may be chIL-2 two polymer. In view of the importance of the form of two polymer IL-2, the mechanism of the formation of chIL-2 two polymer is studied in this experiment.
The chIL-2 gene of 366bp was cloned into the prokaryotic expression vector pET-28a, and a 6 x His affinity purification label was fused at the C- end. Then the recombinant vector pET-28a-chIL-2 was used as a template to amplify the ORF of the fusion protein. Subcloned to the P10 promoter of the eukaryotic expression vector of pFastBac-Dual, the fusion protein was expressed in accordance with the amino acid sequence of the prokaryotic expression protein. The chIL-2 gene was cloned to the pTYB1 prokaryotic expression vector. The chIL-2 gene was fused with the Intein gene of 1362bp in the same ORF, the molecular weight of the chIL-2-Intein fusion protein was about 71kDa, and the chIL-2-Intein fusion protein was purified by denaturing elution. The immunized white rabbit was prepared for anti chIL-2 clon antibody, and the unlabeled chIL-2 protein was obtained by non denaturing cutting. The rabbit source polyclonal antibody was used for the detection of chIL-2-His-tag fusion protein and unlabeled chIL-2 protein used in prokaryotic and eukaryotic purification. The results obtained the titer of 4.01 * 106 Rabbit anti chIL-2 specific antibody. The Western blot detection results showed that both eukaryotic and prokaryotic expression and purification of chIL-2-His-tag fusion protein and unlabeled chIL-2 protein were all available. The form of two polymer exists.
The experiment also explored the effect of temperature on the formation of protein two polymer of different expression systems. The results of.Western blot detection showed that the formation of chIL-2-His-tag two polymer expressed and purified by eukaryotic expression was not significantly changed with time (P0.05) at 4 C. The fusion protein existed in the form of two polymer when incubated at 37 C; and the prokaryotic cell was prokaryotic. When the purified fusion protein was incubated at 4 and 37, the proportion of the two forms of protein had no significant change (P0.05), and the incubation at 37 C could not be completely in the form of two polymer. The results showed that the formation of the chIL-2 fusion protein two polymer expressed by eukaryotic expression and purification was very sensitive to the temperature, and the chIL-2 melting of the prokaryotic expression and purification was fused. The formation of the protein two is insensitive to temperature.
In order to elucidate the intermolecular force formed by chIL-2 two polymer, this study uses a variety of different properties, such as thiocyanate (SCN-), cholate, Triton X-100, urea, guanidine hydrochloride, EDTA, two thiasanol (DTT) and beta mercaptoethanol (beta mercaptoethanol (beta -mercaptoethanol, beta -ME) and other different properties, to determine the intermolecular interaction between the protein molecules. The results show that the reagent of thiocyanate (SCN-), cholate, Triton X-100, urea, guanidine hydrochloride and EDTA can not destroy the structure of chIL-2 two polymer, and 40mmol/L two thulfyl alcohol and 8% beta mercaptoethanol can completely destroy the monomer of chIL-2 two polymer. It is presumed that the formation of chIL-2-His-tag protein two polymer may be between chIL-2 molecules. Cysteine residues interact to form covalent two sulfur bonds.
In order to verify the role of the 4 cysteine residues in the protein two polymer in the chIL-2 molecule, the codon corresponding to cysteine in the recombinant vector pET-28a-chIL-2 was given a single, two, three and four point mutation, and the expression and purification of the mutant protein were induced, and the formation of chIL-2 two polymer was detected by Western blot. Point, two and three point mutant proteins can form a two polymer structure, while the four point mutation protein can not form a two polymer. It shows that the formation of the chIL-2-His-tag protein two polymer may be the result of the covalent two sulfur bond formed by the interaction of chIL-2 molecular cysteine residues, and each cysteinic acid residue can play a role as a molecule to form a molecule. The two sulfur bonds between the two groups provide a theoretical basis for the study of the structure and function of chIL-2 two dimers.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831
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