白细胞介素2受体α基因结构和功能的研究
摘要
由白细胞介素2(IL-2)及其受体(IL-2R)为核心的自分泌体系在T淋巴细胞的正常生长、增殖和免疫功能的发挥中起着关键作用。高亲和力的白细胞介素2受体至少由α、β、γ三种亚基组成，其中只有α亚基呈诱导表达，而且是淋巴细胞中结合IL-2的唯一特异成分，因此，IL-2α亚基在机体对IL-2作用的免疫效应中起着重要作用，与其相一致，IL-2Rα亚基的表达受到严格的调控。
基因定向整合技术是新近发展起来可用于细胞基因组定向修饰的新方法。为了研究IL-2Rα基因在T淋巴细胞的功能，并建立体细胞基因敲除的模式系统，我们对在Jurkat细胞中敲除IL-2Rα基因进行了探索，并对IL-2Rα基因第一内含子的转录调控活性进行了初步研究。
一、悬浮细胞甲基纤维素半固体克隆化培养方法的建立与Jurkat Tet-on细胞系的建立。我们借鉴在杂交瘤细胞中用于克隆化培养的甲基纤维素半固体培养方法，用于悬浮细胞Jurkat细胞的克隆化培养，成功地建立了Jurkat Tet-on细胞系-rtTA③。通过Southern杂交显示，在rtTA③细胞中有rtTA基因的整合，Luciferase报告基因的转染结果表明，rtTA③细胞系在Dox 1μg／ml浓度下诱导近30倍的Luciferase的表达。
二，IL-2Rα基因第三内含子的克隆与核苷酸序列测定。应用PCR方法，扩增了IL-2Rα基因第三内含子2.5Kb片段，并成功克隆到pBS-SK载体中。构建了一系列亚克隆并测定了2.5Kb全核苷酸序列，其中2228bp碱基序列为国内外首次测定，此核苷酸序列已被Genbank接受，接受号为U56389。通过计算机分析发现IL-2Rα基因第三内含子具有典型的G↓GU……Py rich(～15bp)NCAG↓GU结构特点，
Interleukin 2 (IL-2), a key regulator of the immune response, is secreted by activated T cells upon antigenic stimulation. Binding of IL-2 to its high-affinity receptor (IL-2R ) induces proliferation as well as functional differentiation of T and B lymphocytes and natural killer activation. The high affinity IL-2 receptor is comprised of three component chains, IL-2Rα, IL-2Rβ, and IL-2Rγ. IL-2Rβ and IL-2Rγ are present constitutively in resting lymphocytes. In contrast, IL-2Rα is expressed only following activation and it is the unique subunit which strictly bind with IL-2. Therefore, the interleukin 2 receptor α-chain plays a critical role in IL-2 responsiveness, thus explaining why expression of this chain is highly regulated.
Homologous recombination is a powerful tool which has only recently become available for correcting or mutating the desired chromosomal locus. To establish the model of gene targeting in somatic cells and study the function of IL-2Rα gene in lymphocytes, we perform gene targeting of IL-2Rα in Jurkat cells and investigate the transcription regulation activity of the 1st intron of IL-2Rα gene.
I. Establishment of cell cloning in semi-solid medium in suspension cell and the construction of Jurkat Tet-on Cell line.
We successfully constructed Jurkat Tet-on cell line-rtTA(3) according to the protocol of hybridoma cloning culture using semi-solid medium of methyl cellulose. rtTA gene integrated into the chromosome of rtTA(3) cell line . These positive rtTA(3) cell line identified by Southern blot could express 30 folds luciferase highly while induced by 1 μg/ml of Dox.
基因定向整合技术是新近发展起来可用于细胞基因组定向修饰的新方法。为了研究IL-2Rα基因在T淋巴细胞的功能，并建立体细胞基因敲除的模式系统，我们对在Jurkat细胞中敲除IL-2Rα基因进行了探索，并对IL-2Rα基因第一内含子的转录调控活性进行了初步研究。
一、悬浮细胞甲基纤维素半固体克隆化培养方法的建立与Jurkat Tet-on细胞系的建立。我们借鉴在杂交瘤细胞中用于克隆化培养的甲基纤维素半固体培养方法，用于悬浮细胞Jurkat细胞的克隆化培养，成功地建立了Jurkat Tet-on细胞系-rtTA③。通过Southern杂交显示，在rtTA③细胞中有rtTA基因的整合，Luciferase报告基因的转染结果表明，rtTA③细胞系在Dox 1μg／ml浓度下诱导近30倍的Luciferase的表达。
二，IL-2Rα基因第三内含子的克隆与核苷酸序列测定。应用PCR方法，扩增了IL-2Rα基因第三内含子2.5Kb片段，并成功克隆到pBS-SK载体中。构建了一系列亚克隆并测定了2.5Kb全核苷酸序列，其中2228bp碱基序列为国内外首次测定，此核苷酸序列已被Genbank接受，接受号为U56389。通过计算机分析发现IL-2Rα基因第三内含子具有典型的G↓GU……Py rich(～15bp)NCAG↓GU结构特点，
Interleukin 2 (IL-2), a key regulator of the immune response, is secreted by activated T cells upon antigenic stimulation. Binding of IL-2 to its high-affinity receptor (IL-2R ) induces proliferation as well as functional differentiation of T and B lymphocytes and natural killer activation. The high affinity IL-2 receptor is comprised of three component chains, IL-2Rα, IL-2Rβ, and IL-2Rγ. IL-2Rβ and IL-2Rγ are present constitutively in resting lymphocytes. In contrast, IL-2Rα is expressed only following activation and it is the unique subunit which strictly bind with IL-2. Therefore, the interleukin 2 receptor α-chain plays a critical role in IL-2 responsiveness, thus explaining why expression of this chain is highly regulated.
Homologous recombination is a powerful tool which has only recently become available for correcting or mutating the desired chromosomal locus. To establish the model of gene targeting in somatic cells and study the function of IL-2Rα gene in lymphocytes, we perform gene targeting of IL-2Rα in Jurkat cells and investigate the transcription regulation activity of the 1st intron of IL-2Rα gene.
I. Establishment of cell cloning in semi-solid medium in suspension cell and the construction of Jurkat Tet-on Cell line.
We successfully constructed Jurkat Tet-on cell line-rtTA(3) according to the protocol of hybridoma cloning culture using semi-solid medium of methyl cellulose. rtTA gene integrated into the chromosome of rtTA(3) cell line . These positive rtTA(3) cell line identified by Southern blot could express 30 folds luciferase highly while induced by 1 μg/ml of Dox.
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