抗人L-PGDS基因工程抗体的构建及表达

发布时间：2018-10-25 12:45

【摘要】：Lipocalin型前列腺素D合成酶(Lipocalin-type Prostagladin D Synthase,L-PGDS)亦称β-trace蛋白,是机体组织屏障的主要成分,属Lipocal in家族。L-PGDS主要分布于哺乳动物的中枢神经系统和雄性生殖器官,并分泌至脑脊液、血清、精液、尿液、羊水等体液中。L-PGDS是一种双功能蛋白,除催化PGD_2产生外,亦可结合和运输亲脂/疏水分子。该蛋白具有广泛的生理学作用,它不仅与睡眠诱导、体温调节、感受伤害、气味应答等有关,亦是一种生育相关蛋白,可能参与精子的发生和成熟。研究表明,L-PGDS与许多疾病密切相关,如L-PGDS不同程度地表达于卵巢癌、乳腺癌等组织,病毒性脑膜炎、脑脊膜瘤、椎管狭窄、脑栓塞等病人的CSF中L-PGDS明显升高。为了建立L-PGDS的免疫学检测方法并探讨其在男性生殖系统中的具体功能、代谢过程、作用机制以及与男性不育的关系等,本实验室以毕赤氏酵母表达了人睾丸L-PGDS抗原,成功制备了兔抗人L-PGDS多克隆抗体和鼠抗人L-PGDS单克隆抗体。兔源多抗实际上是多种抗体的混合物,具有不均一性,无论是对抗体分子结构与功能的研究还是临床应用都受到很大限制;而特异性鼠单抗具有异源性,应用于人体研究会引发人抗鼠抗体的毒性反应,并使抗体在体内消除加快等。因此,应用于临床的理想抗体应是人源的,但人一人杂交瘤技术目前尚未突破,且还存在人-人杂交瘤体外传代不稳定、抗体亲合力低及产量不高等问题。目前解决此问题的较好办法之一是研制基因工程抗体,代替鼠源单抗应用于临床。本研究试利用基因工程技术,构建和表达目前研究较多也较成熟的单链抗体、人—鼠嵌合抗体,为L-PGDS的功能性研究以及临床相关疾病的诊断、治疗奠定基础。本研究第一部分首先采用RT-PCR技术,以一组自前导序列的简并引物,从1株稳定分泌抗人L-PGDS单抗的鼠杂交瘤细胞中扩增出抗体的轻链可变区基因2个、重链可变区基因1个:然后分别克隆至pGEM-T载体,并测序。测序结果表明,其中1个V k为鼠骨髓瘤细胞系中固有的无功能基因:另一V k和VH经KabatIg比对分析,与其同源性较高的序列均为鼠免疫球蛋白可变区基因,且碱基序列符合鼠抗体可变区特征。本研究第二部分利用一柔性连接短肽[(Gly)_4Set]_3,SOE PCR法将上述重、
[Abstract]:Lipocalin type prostaglandin D synthase (Lipocalin-type Prostagladin D Synthase,L-PGDS), also known as 尾 trace protein, is a major component of the body's tissue barrier and belongs to the Lipocal in family. L-PGDS is mainly distributed in the central nervous system and male reproductive organs of mammals and secreted to cerebrospinal fluid, serum and semen. In urine and amniotic fluid, L-PGDS is a bifunctional protein, which not only catalyzes the production of PGD_2, but also binds and transports lipophilic / hydrophobic molecules. This protein has a wide range of physiological functions, it is not only related to sleep induction, thermoregulation, perception of injury, odor response, but also a procreation related protein, which may be involved in spermatogenesis and maturation. Studies have shown that L-PGDS is closely related to many diseases, such as L-PGDS expression in ovarian cancer, breast cancer and other tissues, viral meningitis, meningioma, spinal stenosis, cerebral embolism and other patients in CSF increased significantly. In order to establish an immunological method for the detection of L-PGDS and to explore its specific function, metabolic process, mechanism and relationship with male infertility in male reproductive system, the human testis L-PGDS antigen was expressed by Pichia pastoris in our laboratory. Rabbit anti-human L-PGDS polyclonal antibody and mouse anti-human L-PGDS monoclonal antibody were successfully prepared. Rabbit polyantibodies are actually a mixture of many kinds of antibodies and have heterogeneity, both in the study of the molecular structure and function of antibodies and in clinical application, but the specific mouse monoclonal antibodies are heterogenous. Application in human body can induce the toxic reaction of human anti-mouse antibody and accelerate the elimination of anti-mouse antibody in vivo. Therefore, the ideal antibody for clinical application should be human, but the technology of one-person hybridoma has not been broken through, and there are still some problems such as unstable passage of human hybridoma in vitro, low affinity and low yield of antibody. One of the better ways to solve this problem is to develop genetic engineering antibody instead of mouse monoclonal antibody. In this study, genetic engineering techniques were used to construct and express more mature single-chain antibodies, human-mouse chimeric antibodies, which laid a foundation for the functional study of L-PGDS and the diagnosis and treatment of clinically-related diseases. In the first part of this study, using RT-PCR technique and a group of degenerate primers of leading sequence, two genes of light chain variable region of antibody were amplified from a mouse hybridoma cell stably secreting anti-human L-PGDS monoclonal antibody. Heavy chain variable region gene 1: then cloned into pGEM-T vector and sequenced. Sequencing results showed that one of the VK genes was an inherent nonfunctional gene in the mouse myeloma cell line, and the other V k and VH were all mouse immunoglobulin variable region genes by KabatIg alignment analysis. The nucleotide sequence was in accordance with the variable region of mouse antibody. In the second part of this study, we use a flexible binding short peptide [(Gly) _ 4Set] _ 3SOE PCR method to weight the above.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1

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