抗人L-PGDS基因工程抗體的構(gòu)建及表達
發(fā)布時間：2018-10-25 12:45
【摘要】：Lipocalin型前列腺素D合成酶(Lipocalin-type Prostagladin D Synthase,L-PGDS)亦稱β-trace蛋白,是機體組織屏障的主要成分,屬Lipocal in家族。L-PGDS主要分布于哺乳動物的中樞神經(jīng)系統(tǒng)和雄性生殖器官,并分泌至腦脊液、血清、精液、尿液、羊水等體液中。L-PGDS是一種雙功能蛋白,除催化PGD_2產(chǎn)生外,亦可結(jié)合和運輸親脂/疏水分子。該蛋白具有廣泛的生理學作用,它不僅與睡眠誘導、體溫調(diào)節(jié)、感受傷害、氣味應(yīng)答等有關(guān),亦是一種生育相關(guān)蛋白,可能參與精子的發(fā)生和成熟。研究表明,L-PGDS與許多疾病密切相關(guān),如L-PGDS不同程度地表達于卵巢癌、乳腺癌等組織,病毒性腦膜炎、腦脊膜瘤、椎管狹窄、腦栓塞等病人的CSF中L-PGDS明顯升高。 為了建立L-PGDS的免疫學檢測方法并探討其在男性生殖系統(tǒng)中的具體功能、代謝過程、作用機制以及與男性不育的關(guān)系等,本實驗室以畢赤氏酵母表達了人睪丸L-PGDS抗原,成功制備了兔抗人L-PGDS多克隆抗體和鼠抗人L-PGDS單克隆抗體。兔源多抗實際上是多種抗體的混合物,具有不均一性,無論是對抗體分子結(jié)構(gòu)與功能的研究還是臨床應(yīng)用都受到很大限制;而特異性鼠單抗具有異源性,應(yīng)用于人體研究會引發(fā)人抗鼠抗體的毒性反應(yīng),并使抗體在體內(nèi)消除加快等。因此,應(yīng)用于臨床的理想抗體應(yīng)是人源的,但人一人雜交瘤技術(shù)目前尚未突破,且還存在人-人雜交瘤體外傳代不穩(wěn)定、抗體親合力低及產(chǎn)量不高等問題。目前解決此問題的較好辦法之一是研制基因工程抗體,代替鼠源單抗應(yīng)用于臨床。本研究試利用基因工程技術(shù),構(gòu)建和表達目前研究較多也較成熟的單鏈抗體、人—鼠嵌合抗體,為L-PGDS的功能性研究以及臨床相關(guān)疾病的診斷、治療奠定基礎(chǔ)。 本研究第一部分首先采用RT-PCR技術(shù),以一組自前導序列的簡并引物,從1株穩(wěn)定分泌抗人L-PGDS單抗的鼠雜交瘤細胞中擴增出抗體的輕鏈可變區(qū)基因2個、重鏈可變區(qū)基因1個:然后分別克隆至pGEM-T載體,并測序。測序結(jié)果表明,其中1個V k為鼠骨髓瘤細胞系中固有的無功能基因:另一V k和VH經(jīng)KabatIg比對分析,與其同源性較高的序列均為鼠免疫球蛋白可變區(qū)基因,且堿基序列符合鼠抗體可變區(qū)特征。 本研究第二部分利用一柔性連接短肽[(Gly)_4Set]_3,SOE PCR法將上述重、
[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.
【學位授予單位】：南京師范大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392.1
本文編號：2293730
[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.
【學位授予單位】：南京師范大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392.1
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