鸡IL-2二聚体形成机制的研究
发布时间：2018-07-18 13:41
【摘要】：白细胞介素-2（interleukin-2，IL-2），是特异性抗原或分裂原刺激活化的T淋巴细胞分泌的一种具有广泛重要作用的细胞因子，对T淋巴细胞、B淋巴细胞、单核细胞和自然杀伤细胞的复制与分化起重要作用，且在后天获得性免疫防御和先天免疫防御系统的激活和维护中起积极作用。白细胞介素中已发现IL-6、IL-8、IL-12等多种蛋白能够形成具有重要生物学活性的二聚体。人、鱼、仓鼠和小鼠等多个物种中发现IL-2二聚体形式的存在。对内源性chIL-2进行纯化发现其有两个洗脱峰，分子量大小相差二倍左右，大分子量条带可能为chIL-2二聚体，鉴于二聚体形式IL-2的重要性，本试验进行chIL-2二聚体形成机制的研究。 将366bp的chIL-2基因克隆到原核表达载体pET-28a中，在C-端融合6×His亲和纯化标签。再以重组载体pET-28a-chIL-2为模版，扩增融合蛋白的ORF，亚克隆到pFastBac-Dual真核细胞表达载体的P10启动子下，表达与原核表达蛋白氨基酸序列一致的融合蛋白。将chIL-2基因克隆到pTYB1原核表达载体，使chIL-2基因与1362bp的Intein基因融合在同一ORF，编码分子量约为71kDa的chIL-2-Intein融合蛋白，变性洗脱纯化得到chIL-2-Intein融合蛋白，免疫大白兔制备抗chIL-2多克隆抗体，非变性切割获得无标签chIL-2蛋白；制备的兔源多抗用于原核和真核纯化的chIL-2-His-tag融合蛋白以及无标签chIL-2蛋白的检测。结果获得效价为4.01×106的兔抗chIL-2特异性抗体，Western blot检测结果表明，真核和原核表达纯化的chIL-2-His-tag融合蛋白以及无标签chIL-2蛋白都有二聚体形式存在。 实验同时探究温度对不同表达体系来源蛋白二聚体形成的影响。Western blot检测结果表明，4℃条件下，真核表达纯化的chIL-2-His-tag二聚体的形成随时间的增加无显著性变化（P0.05），37℃孵育24h时，融合蛋白完全以二聚体的形式存在；而原核表达纯化的融合蛋白在4℃、37℃孵育不同时间时，两种形式的蛋白比例均无显著性变化（P0.05），且37℃孵育不能完全以二聚体的形式存在。结果表明，真核表达纯化的chIL-2融合蛋白二聚体的形成对温度十分敏感，而原核表达纯化的chIL-2融合蛋白二聚形成对温度不敏感。 为了阐明chIL-2二聚体形成的分子间作用力，本研究采用硫氰酸根（SCN-）、胆酸盐、Triton X-100、脲素、盐酸胍、EDTA、二硫苏糖醇（DTT）和β-巯基乙醇（β-mercaptoethanol，β-ME）等多种不同性质的试剂作用于二聚体化的chIL-2蛋白，以确定蛋白分子间相互作用力的类型。结果表明，硫氰酸根（SCN-）、胆酸盐、Triton X-100、脲素、盐酸胍、EDTA等试剂均不能破坏chIL-2二聚体结构，而40mmol/L二硫苏糖醇和8%β-巯基乙醇可将chIL-2二聚体完全破坏形成单体。推测chIL-2-His-tag蛋白二聚体的形成可能是chIL-2分子间半胱氨酸残基相互作用形成共价二硫键的结果。 为了验证chIL-2分子中4个半胱氨酸残基在蛋白二聚体形成的作用，对重组载体pET-28a-chIL-2中的半胱氨酸对应的密码子分别进行单点、两点、三点和四点突变，诱导表达纯化获得各突变体蛋白，Western blot检测chIL-2二聚体的形成情况。结果，单点、两点和三点突变体蛋白都能形成二聚体结构，而四点突变蛋白不能形成二聚体。表明chIL-2-His-tag蛋白二聚体的形成可能是chIL-2分子间半胱氨酸残基相互作用形成共价二硫键的结果，，且每个半胱氨酸残基都能单独发挥作用形成分子间二硫键。该实验为chIL-2二聚体结构和功能的研究提供理论基础。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
本文编号：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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
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