一组导向融合蛋白的基因工程研究
摘要
很多高免疫反应的疾病中都发现有白细胞介-2受体(IL-2R)阳性细胞的浸涧和聚集，如器官移植排斥、自身免疫性甲状腺病、Ⅰ型糖尿病、类风湿性关节炎等。成人T淋巴细胞白血病细胞和爱滋病病毒HIV感染的细胞表面也含有丰富的IL-2受体。IL2受体阳性淋巴细胞在这些疾病的病理过程中起着关键性作用。因此可以通过IL-2与毒素蛋白的基因融合，表达出融合毒素用以对IL-2受体异常丰富的靶细胞进行特异杀伤，从而达到治疗疾病的目的。然而到目前为止，要将此类融合毒素导向药物推向实际临床应用却存在一个很大的障碍：大分子异源蛋白在体内的免疫原性问题。
本论文分为四个部分。第一部分：融合蛋白IL-2(60)-PEZN，IL-2(60)-PEZNK，IL-2-PEZNM，IL-2-PEZNKM，IL-2-K-PEZNM的基因构建与诱导表达。第二部分：5种融合蛋白的快速纯化、生物活性测定及融合毒素抗血清的制备。第三部分：融合蛋白IL-2-K-PEZNM的纯化制备及药理活性的初步研究。前三部分的研究对象是IL-2与绿脓毒素的融合，主要目的是利用基因工程和蛋白质工程的方法，对本室曾经构建的融合毒素IL-2(60)-PE40进行一系列的基因突变与改造，探讨融合毒素结构与功能的关系，通过对突变体的活性比较，找到最适的构建突变融合基因的方式。第四部分：新型融合蛋白IL-2-TNFαM的纯化与初步药理实验。目的是探索一条构建无免疫原性融合毒素的新路
在本文第一部分中，我们通过PCR反应、插入突变及缺失突变等方法，构建了5种融合蛋白IL-2(60)-PEZN，IL-2(60)-PEZNK，IL-2-PEZNM，IL-2-PEZNKM，IL-2-K-PEZNM的基因，绿脓毒素突变体PEZN是我们利用本实验室克隆到的PE40基因的特异性所得到的一种突变体形式以求创新，将经典的PE40删除其360-380位氨基酸而得到；突变体PEZNK是将PE40中360-380位氨基酸删除并以—段扩增于人IgG4基因的富含Lys编码的基因片段取代而得到；突变体PEZNM是在突变体PEZN的基础上删除其C末端最后一个Lys而得到。其中IL-2(60)-PEZN，IL-2(60)-PEZNK的基因分别克隆于表达质粒pT7-7的T7启动子下游，与质粒pGP1-2共转化大肠杆菌K38，温控表达，表达水平分别为18.1％，18.5％；IL-2-PE38M，IL-2-PE38KM，IL-2-K-PE38M的基因分别克隆于本室所构建的高效表达质粒pLSD13的P_L启动子下游，转化大肠杆菌DH5α，温控表达，表达水平分别为19.6％，19.8％，
Activated Interleukin 2 (IL2) receptor-positive lymphocytes have been found to accumulate as cellular infiltrates in many diseases, such as autoimmune thyroid disease, type I diabetes, allograft rejection, rheumatoid arthritis, etc. The abnormal T-lymphocytes of adult T-cell leukemia (ATL) and AIDS patients also express aberrant IL2 receptors. It is considered that IL-2 receptor-positive lymphocytes play a crucial role in the pathogenesis of such kind of diseases. Therefore, DL-2 receptor-bearing cells are important targets for immunosuppression. It leads to a therapeutical idea on these autoimmune diseases. So, many kind of fusion immutoxin targeting to IL-2 receptor bvearing cells were constructed. However there is an obstacle for using such kind of fusion proteins as a drug in clinical treatment. That is the immunogenicity of the foreign protein in vivo.
The research was divided into four parts.
Part I: Construction and induced expression of the fusion protein genes :
IL-2(60)-PEZN, IL-2(60)-PEZNK, IL-2-PEZNM, IL-2-PEZNKM, IL-2-K-
PEZNM.
Part II: Rapid purification of the fusion proteins, bioactivity assay in vitro and
preparation of the antisera against chimeric proteins.
Part III: Purification of the fusion protein IL-2-PEZNKM and Fundamental
investigation on pharmacological activities.
Part IV: Purification of a new fusion protein IL-2-TNFαM and Fundamental
investigation on pharmacological activities.
In the research of part I: The fusion genes of IL-2(60)-PEZN, IL-2(60)-PEZNK, IL-2-PEZNM, IL-2-PEZNKM, EL-2-K-PEZNM obtained by polymerase chain reaction and deletion mutagenesis were cloned into expression vector. The fusion genes of IL-2(60)-PEZN, IL-2(60)-PEZNK were cloned into plasmid pT7-7 under the T7 promoter and were expressed in E.coli K38 at high level respectively under thermal induction after co-tansformation with plasmid pGP1-2. Desitometric scanning on SDS-PAGE gel revealed that IL-2(60)-PEZN protein, IL-2(60)-
本论文分为四个部分。第一部分：融合蛋白IL-2(60)-PEZN，IL-2(60)-PEZNK，IL-2-PEZNM，IL-2-PEZNKM，IL-2-K-PEZNM的基因构建与诱导表达。第二部分：5种融合蛋白的快速纯化、生物活性测定及融合毒素抗血清的制备。第三部分：融合蛋白IL-2-K-PEZNM的纯化制备及药理活性的初步研究。前三部分的研究对象是IL-2与绿脓毒素的融合，主要目的是利用基因工程和蛋白质工程的方法，对本室曾经构建的融合毒素IL-2(60)-PE40进行一系列的基因突变与改造，探讨融合毒素结构与功能的关系，通过对突变体的活性比较，找到最适的构建突变融合基因的方式。第四部分：新型融合蛋白IL-2-TNFαM的纯化与初步药理实验。目的是探索一条构建无免疫原性融合毒素的新路
在本文第一部分中，我们通过PCR反应、插入突变及缺失突变等方法，构建了5种融合蛋白IL-2(60)-PEZN，IL-2(60)-PEZNK，IL-2-PEZNM，IL-2-PEZNKM，IL-2-K-PEZNM的基因，绿脓毒素突变体PEZN是我们利用本实验室克隆到的PE40基因的特异性所得到的一种突变体形式以求创新，将经典的PE40删除其360-380位氨基酸而得到；突变体PEZNK是将PE40中360-380位氨基酸删除并以—段扩增于人IgG4基因的富含Lys编码的基因片段取代而得到；突变体PEZNM是在突变体PEZN的基础上删除其C末端最后一个Lys而得到。其中IL-2(60)-PEZN，IL-2(60)-PEZNK的基因分别克隆于表达质粒pT7-7的T7启动子下游，与质粒pGP1-2共转化大肠杆菌K38，温控表达，表达水平分别为18.1％，18.5％；IL-2-PE38M，IL-2-PE38KM，IL-2-K-PE38M的基因分别克隆于本室所构建的高效表达质粒pLSD13的P_L启动子下游，转化大肠杆菌DH5α，温控表达，表达水平分别为19.6％，19.8％，
Activated Interleukin 2 (IL2) receptor-positive lymphocytes have been found to accumulate as cellular infiltrates in many diseases, such as autoimmune thyroid disease, type I diabetes, allograft rejection, rheumatoid arthritis, etc. The abnormal T-lymphocytes of adult T-cell leukemia (ATL) and AIDS patients also express aberrant IL2 receptors. It is considered that IL-2 receptor-positive lymphocytes play a crucial role in the pathogenesis of such kind of diseases. Therefore, DL-2 receptor-bearing cells are important targets for immunosuppression. It leads to a therapeutical idea on these autoimmune diseases. So, many kind of fusion immutoxin targeting to IL-2 receptor bvearing cells were constructed. However there is an obstacle for using such kind of fusion proteins as a drug in clinical treatment. That is the immunogenicity of the foreign protein in vivo.
The research was divided into four parts.
Part I: Construction and induced expression of the fusion protein genes :
IL-2(60)-PEZN, IL-2(60)-PEZNK, IL-2-PEZNM, IL-2-PEZNKM, IL-2-K-
PEZNM.
Part II: Rapid purification of the fusion proteins, bioactivity assay in vitro and
preparation of the antisera against chimeric proteins.
Part III: Purification of the fusion protein IL-2-PEZNKM and Fundamental
investigation on pharmacological activities.
Part IV: Purification of a new fusion protein IL-2-TNFαM and Fundamental
investigation on pharmacological activities.
In the research of part I: The fusion genes of IL-2(60)-PEZN, IL-2(60)-PEZNK, IL-2-PEZNM, IL-2-PEZNKM, EL-2-K-PEZNM obtained by polymerase chain reaction and deletion mutagenesis were cloned into expression vector. The fusion genes of IL-2(60)-PEZN, IL-2(60)-PEZNK were cloned into plasmid pT7-7 under the T7 promoter and were expressed in E.coli K38 at high level respectively under thermal induction after co-tansformation with plasmid pGP1-2. Desitometric scanning on SDS-PAGE gel revealed that IL-2(60)-PEZN protein, IL-2(60)-
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