人重组IL-22蛋白的表达及其生物学特性的初步分析

发布时间：2018-01-12 08:20

本文关键词：人重组IL-22蛋白的表达及其生物学特性的初步分析　出处：《苏州大学》2009年硕士论文　论文类型：学位论文

【摘要】： 白细胞介素22作为IL-10细胞因子家族的成员之一最初发现于2000年。其在体内的主要来源是活化后的Th1细胞以及NK细胞。IL-22通过其异源二聚体的受体复合物表现其生物学活性。其受体属于Ⅱ型细胞因子受体超家族,分别为IL-22R1和IL-10R2。静息状态下或者活化后的免疫细胞中均不表达IL-22R1,而包括T细胞、B细胞、NK细胞等在内的免疫细胞对IL-22的激发也不产生相应的作用。而在皮肤、肾、消化道、呼吸系统等组织中的细胞是IL-22的靶细胞。IL-22的生物学功能包括提高机体抵抗微生物入侵、保护组织抵御损伤以及修复非免疫系统组织等。除此以外IL-22还可以介导肿瘤细胞对血清淀粉样蛋白、结合珠蛋白、α-1抗胰凝乳蛋白酶等急性反应期蛋白表达上调。这些发现表明IL-22是一种新型的免疫分子,是由免疫细胞产生但起着调节组织抵御损伤和外源病原体感染的保护作用。 1.人白细胞介素22的克隆及原核表达根据NCBI数据库中的人IL-22基因序列设计合成了去信号肽IL-22成熟链基因片段的特异性引物。采用RT-PCR的方法从人外周血PHA刺激活化的T细胞中克隆扩增了人IL-22去信号肽基因片断。应用BamHI单酶切酶切载体和目的基因片断,胶回收酶切产物后以T4连接酶连接。连接产物转化大肠杆菌Top10,经酶切和煮菌PCR验证阳性克隆。测序正确后,抽提质粒重新转化至宿主工程菌株M15。在LB细菌培养基中大量扩增M15工程表达菌,经IPTG诱导表达产生hIL-22/His重组蛋白。经过SDS-PAGE电泳鉴定重组蛋白以包涵体的形式存在。包涵体经过超声波裂解和离心,沉淀变性和复性后经亲和层析柱纯化。实验结果表明,纯化后的IL-22/His融合蛋白纯度可达94%以上。应用SDS-PAGE和Western Blotting对融合蛋白进行鉴定,在相对分子量约为18kd的位置为人IL-22特异性条带。 2.人重组IL-22蛋白生物学特性的初步分析根据NCBI数据库中人Gadd45b(Growth arrest and DNA-damage 45 beta)、Gadd45g(growth arrest and DNA-damage 45 gamma)、Serpin A3(serpin peptidaseinhibitor, clade A, member 3)以及SAA2(serum amyloid A2)基因序列设计合成Real-Time PCR引物。对人肝癌细胞株HepG2、人结肠癌细胞株SW480分别加入终浓度为100ng/ml的人重组IL-22及商品化人IL-22蛋白进行体外激发。于规定时间点收集细胞并使用Trizol抽提细胞Total RNA进行逆转录。以相当于100ng Total RNA逆转录的cDNA为模板进行PCR检测在上述细胞株上Gadd45b、Gadd45g、Serpin A3、SAA2 mRNA的表达。结果显示通过原核表达并纯化的人重组IL-22与商品化人IL-22蛋白均能刺激上述细胞中相关基因的表达上调。由此表明,本实验所研制的人重组IL-22具有良好的生物学活性。综上所述,本项研究我们成功地应用原核表达系统表达出了人IL-22重组蛋白,并进行了生物学活性的初步研究。所表达的蛋白具有良好的生物学活性,为进一步研究人IL-22分子在肿瘤中的作用提供了有价值的生物材料,具有潜在运用价值。
[Abstract]:Interleukin 22 as a member of the IL-10 cytokine family originally found in 2000. It is the activation of Th1 cells and NK.IL-22 cells showed its biological activity by heterologous receptor complex two dimers in vivo. The main source of the type II receptor belongs to the cytokine receptor superfamily, and IL-22R1 respectively. IL-10R2. resting or activated immune cells was not detected in IL-22R1, including T cells, B cells, NK cells and other immune cells to stimulate IL-22 do not produce the corresponding effect. In the skin, kidney, gastrointestinal tract, respiratory system and other tissue cells are the biological function of.IL-22 cells IL-22 include improving the anti microbial invasion, protect the tissue against injury and repair of non immune tissues. In addition IL-22 can also be mediated by tumor cells to serum amyloid protein binding These findings indicate that IL-22 is a new immune molecule, which is produced by immune cells, but plays a protective role in regulating tissue against injury and exogenous pathogen infection. These findings indicate that -1 is an up-regulated expression of anti chymotrypsin and other acute phase proteins.
Cloning and prokaryotic expression of interleukin 22 in 1. people
According to the specific primers to signal peptide IL-22 mature gene was designed and synthesized human IL-22 gene sequence in the NCBI database. From human peripheral blood PHA stimulated by RT-PCR method in T cells clone human IL-22 signal peptide gene fragment. The application of BamHI single enzyme digested vector and target gene fragment plastic recycling, the digested products by T4 ligase. The products were transformed Escherichia coli Top10 by enzyme digestion and boiled bacteria PCR verification positive clones. After sequencing, the plasmid was transformed into the host strain M15. re engineering in LB bacteria culture medium amplification M15 engineering bacteria for expression induced by IPTG, expression of hIL-22/His recombinant protein identification of recombinant SDS-PAGE. After electrophoresis of proteins in the form of inclusion body. The inclusion body after ultrasonic lysis and centrifugation, precipitation after denaturation and renaturation were purified by affinity chromatography. The experimental results show that the pure The purity of IL-22/His fusion protein reached over 94% after the treatment. The fusion protein was identified by SDS-PAGE and Western Blotting, and the specific IL-22 band at the relative molecular weight of 18kD.
Preliminary analysis of the biological characteristics of the recombinant IL-22 protein of 2. people
According to the NCBI database in Gadd45b (Growth arrest and DNA-damage 45 beta), Gadd45g (growth arrest and DNA-damage 45 gamma), Serpin A3 (serpin peptidaseinhibitor, clade A, member 3) and SAA2 (serum amyloid A2) design and synthesis of Real-Time PCR gene sequence. The primer on human hepatocellular carcinoma cell line HepG2, human colon cancer cell line SW480 as the concentration of recombinant human IL-22 and human IL-22 protein product 100ng/ml in vitro stimulated. In time were collected and used to extract Trizol Total RNA cells by reverse transcriptase. Equivalent to 100ng Total RNA cDNA PCR as the template for reverse transcription detection in these cell lines Gadd45b, Gadd45g, Serpin, A3, expression SAA2 mRNA. The results showed that the prokaryotic expression and purification of recombinant human IL-22 and commercial IL-22 protein could up regulate the expression of related genes were stimulated the cells by reason. This shows that the human recombinant IL-22 developed in this experiment has good biological activity.
In summary, in this study we successfully using prokaryotic expression system to express recombinant human IL-22 protein, and carried out a preliminary study on the biological activity. The recombinant protein has good biological activity, provides valuable biological materials for the further study of human IL-22 molecules in tumor cells, with potential application value.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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