本文關(guān)鍵詞：一種鼠源抗CTLA4單鏈抗體mS的構(gòu)建及制備　出處：《四川大學(xué)》2006年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： CTLA-4 scFv 包涵體 復(fù)性 ELISA

【摘要】：免疫抑制劑在臨床上的廣泛應(yīng)用是二十世紀(jì)器官移植領(lǐng)域取得的重大突破性進(jìn)展之一。免疫抑制劑的廣泛應(yīng)用在大規(guī)模降低急性排斥反應(yīng)發(fā)生率的同時(shí)，也引起了長(zhǎng)期蓄積的毒副作用，從而導(dǎo)致移植器官的損傷，制約了病人或供器官長(zhǎng)期存活率的進(jìn)一步提高。已有研究表明，活化T細(xì)胞是介導(dǎo)移植排斥的重要細(xì)胞，選擇性清除活化T細(xì)胞而保留靜息T細(xì)胞，能控制移植排斥反應(yīng)發(fā)生，減少藥物的毒副作用。 活化的T細(xì)胞表面會(huì)誘導(dǎo)表達(dá)細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4(CTLA4)分子，，基于識(shí)別CTLA4分子而殺傷活化T細(xì)胞的免疫毒素可以特異性殺傷活化T細(xì)胞而保留靜息T細(xì)胞。作為免疫毒素關(guān)鍵的導(dǎo)向分子決定著目標(biāo)分子的識(shí)別，詳細(xì)探討導(dǎo)向分子的功能對(duì)構(gòu)建免疫毒素非常必要。基于此，我們?cè)O(shè)計(jì)鼠源anti-CTLA-4單鏈抗體(scFv)(簡(jiǎn)稱(chēng)mS)，用大腸桿菌表達(dá)系統(tǒng)表達(dá)該單鏈抗體，初步探索其功能，結(jié)果如下： 以裝載了鼠源Anti-CTLA-4-scFv基因的PBMN質(zhì)粒為模板，根據(jù)鼠源Anti-CTLA-4-scFv的基因序列設(shè)計(jì)引物，在引物設(shè)計(jì)時(shí)引入相關(guān)雙酶切位點(diǎn)(BamH Ⅰ／Kpn Ⅰ)，PCR擴(kuò)增獲得單鏈抗體基因，再克隆到pQE30載體，提取pQE30-mS質(zhì)粒，轉(zhuǎn)入大腸桿菌中表達(dá)，提取表達(dá)產(chǎn)物發(fā)現(xiàn)蛋白mS絕大部分以包涵體形式表達(dá)，占細(xì)菌總蛋白量30％，極少量以
[Abstract]:The wide application of immunosuppressants in clinical practice is one of the most important breakthroughs in organ transplantation in 20th century. The extensive application of immunosuppressants can reduce the incidence of acute rejection on a large scale. It also causes long-term accumulation of toxic side effects, resulting in organ transplantation damage, restricting the patient or donor organ long-term survival rate of further improvement. Activated T cells are important cells in mediating transplantation rejection. Selective clearance of activated T cells and retention of resting T cells can control the occurrence of transplant rejection and reduce the toxic and side effects of drugs. Activated T cells can induce the expression of cytotoxic T lymphocyte associated antigen (CTLA4) molecules. Immunotoxins that kill activated T cells based on the recognition of CTLA4 molecules can specifically kill activated T cells and retain resting T cells. As the key guiding molecules of immunotoxins, the recognition of target molecules is determined. A detailed study of the function of the directed molecules is necessary for the construction of immunotoxins. Based on this, we designed murine anti-CTLA-4 scFvv (short for mSs). The scFv was expressed in E. coli expression system and its function was preliminarily explored. The results are as follows: Using the PBMN plasmid loaded with mouse Anti-CTLA-4-scFv gene as template, primers were designed according to the gene sequence of mouse Anti-CTLA-4-scFv. The single chain antibody (scFv) gene was amplified by polymerase chain reaction with BamH 鈪

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