本文關(guān)鍵詞：人重組IL-22蛋白的表達及其生物學(xué)特性的初步分析　出處：《蘇州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人白細胞介素22 重組蛋白 原核表達 包涵體純化 實時熒光定量PCR

【摘要】： 白細胞介素22作為IL-10細胞因子家族的成員之一最初發(fā)現(xiàn)于2000年。其在體內(nèi)的主要來源是活化后的Th1細胞以及NK細胞。IL-22通過其異源二聚體的受體復(fù)合物表現(xiàn)其生物學(xué)活性。其受體屬于Ⅱ型細胞因子受體超家族,分別為IL-22R1和IL-10R2。靜息狀態(tài)下或者活化后的免疫細胞中均不表達IL-22R1,而包括T細胞、B細胞、NK細胞等在內(nèi)的免疫細胞對IL-22的激發(fā)也不產(chǎn)生相應(yīng)的作用。而在皮膚、腎、消化道、呼吸系統(tǒng)等組織中的細胞是IL-22的靶細胞。IL-22的生物學(xué)功能包括提高機體抵抗微生物入侵、保護組織抵御損傷以及修復(fù)非免疫系統(tǒng)組織等。除此以外IL-22還可以介導(dǎo)腫瘤細胞對血清淀粉樣蛋白、結(jié)合珠蛋白、α-1抗胰凝乳蛋白酶等急性反應(yīng)期蛋白表達上調(diào)。這些發(fā)現(xiàn)表明IL-22是一種新型的免疫分子,是由免疫細胞產(chǎn)生但起著調(diào)節(jié)組織抵御損傷和外源病原體感染的保護作用。 1.人白細胞介素22的克隆及原核表達 根據(jù)NCBI數(shù)據(jù)庫中的人IL-22基因序列設(shè)計合成了去信號肽IL-22成熟鏈基因片段的特異性引物。采用RT-PCR的方法從人外周血PHA刺激活化的T細胞中克隆擴增了人IL-22去信號肽基因片斷。應(yīng)用BamHI單酶切酶切載體和目的基因片斷,膠回收酶切產(chǎn)物后以T4連接酶連接。連接產(chǎn)物轉(zhuǎn)化大腸桿菌Top10,經(jīng)酶切和煮菌PCR驗證陽性克隆。測序正確后,抽提質(zhì)粒重新轉(zhuǎn)化至宿主工程菌株M15。在LB細菌培養(yǎng)基中大量擴增M15工程表達菌,經(jīng)IPTG誘導(dǎo)表達產(chǎn)生hIL-22/His重組蛋白。經(jīng)過SDS-PAGE電泳鑒定重組蛋白以包涵體的形式存在。包涵體經(jīng)過超聲波裂解和離心,沉淀變性和復(fù)性后經(jīng)親和層析柱純化。實驗結(jié)果表明,純化后的IL-22/His融合蛋白純度可達94%以上。應(yīng)用SDS-PAGE和Western Blotting對融合蛋白進行鑒定,在相對分子量約為18kd的位置為人IL-22特異性條帶。 2.人重組IL-22蛋白生物學(xué)特性的初步分析 根據(jù)NCBI數(shù)據(jù)庫中人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)基因序列設(shè)計合成Real-Time PCR引物。對人肝癌細胞株HepG2、人結(jié)腸癌細胞株SW480分別加入終濃度為100ng/ml的人重組IL-22及商品化人IL-22蛋白進行體外激發(fā)。于規(guī)定時間點收集細胞并使用Trizol抽提細胞Total RNA進行逆轉(zhuǎn)錄。以相當(dāng)于100ng Total RNA逆轉(zhuǎn)錄的cDNA為模板進行PCR檢測在上述細胞株上Gadd45b、Gadd45g、Serpin A3、SAA2 mRNA的表達。結(jié)果顯示通過原核表達并純化的人重組IL-22與商品化人IL-22蛋白均能刺激上述細胞中相關(guān)基因的表達上調(diào)。由此表明,本實驗所研制的人重組IL-22具有良好的生物學(xué)活性。 綜上所述,本項研究我們成功地應(yīng)用原核表達系統(tǒng)表達出了人IL-22重組蛋白,并進行了生物學(xué)活性的初步研究。所表達的蛋白具有良好的生物學(xué)活性,為進一步研究人IL-22分子在腫瘤中的作用提供了有價值的生物材料,具有潛在運用價值。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前2條

1 Svetlana Radaeva;;Hydrodynamic Gene Delivery of Interleukin-22 Protects the Mouse Liver from Concanavalin A-, Carbon Tetrachloride-,and Fas Ligand-Induced Injury via Activation of STAT3[J];Cellular & Molecular Immunology;2004年01期

2 趙欽一;蛋白質(zhì)折迭的不可逆熱力學(xué)理論及蛋白質(zhì)動態(tài)熱力學(xué)結(jié)構(gòu)(英文)[J];生物化學(xué)與生物物理進展;2001年03期

，

本文編號：1413448