本文關(guān)鍵詞：人源性抗FGF-2中和性抗體的篩選，表達(dá)及鑒定　出處：《南方醫(yī)科大學(xué)》2010年博士論文　論文類型：學(xué)位論文

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【摘要】： 目的和背景 FGF-2又稱為堿性成纖維細(xì)胞生長因子(basic fibroblast growth factor,FGF-2),是成纖維生長因子家族的一員,最早由Gospodarowicz (1974)從牛腦垂體中提取的一種對BALB／C 3T3細(xì)胞等有明顯促進(jìn)生長作用的細(xì)胞因子。FGF-2有五種分子量,從18kD到34kD不等。所有的FGF-2異構(gòu)體都缺乏信號肽,是通過一種現(xiàn)在還不十分清楚的機(jī)制分泌到細(xì)胞外的,其中18kD FGF-2在體內(nèi)分布廣泛,存在于中胚層及神經(jīng)外胚層來源的細(xì)胞及多種腫瘤細(xì)胞中,對這些細(xì)胞有促增殖分化功能,參與了胚胎發(fā)育、血管生成、損傷修復(fù)、神經(jīng)再生、腫瘤生長、組織纖維化等多項(xiàng)生理及病理過程。 FGF-2的生物學(xué)效應(yīng),除對成纖維細(xì)胞和血管內(nèi)皮細(xì)胞具有顯著的促增殖作用外,還參與腫瘤發(fā)生和發(fā)展,特別對腫瘤的浸潤和轉(zhuǎn)移具有重要作用。有相當(dāng)部分的腫瘤(如乳癌、結(jié)腸癌、甲狀腺癌、肺癌、膀胱癌、食道癌等)存在FGF-2的高表達(dá)(表2)。已知其作用環(huán)節(jié)主要有(1)FGF-2能促進(jìn)表皮、內(nèi)皮細(xì)胞再生,促進(jìn)血管內(nèi)皮細(xì)胞分裂,誘導(dǎo)其從基膜中分離出來,以刺激內(nèi)皮細(xì)胞向腫瘤組織趨化運(yùn)動,并形成管狀結(jié)構(gòu),還提高組織中血纖維蛋白溶解酶原激活因子類及誘導(dǎo)內(nèi)皮細(xì)胞產(chǎn)生其他蛋白酶,從而促進(jìn)毛細(xì)血管的形成；(2)FGF-2能促進(jìn)多種生長因子的分泌,如VEGF、HGF、FGF-1等,這些因子相互調(diào)節(jié),共同促進(jìn)腫瘤的生長；(3)腫瘤細(xì)胞存在FGF-2受體的高表達(dá),通常比正常細(xì)胞上FGF-2R高10倍以上,許多腫瘤細(xì)胞(如神經(jīng)膠質(zhì)瘤、橫紋肌肉瘤、白血病、肺瘤、黑色素瘤、肝癌等)既表達(dá)FGF2又表達(dá)FGFRs,因此FGF2可直接作用于腫瘤細(xì)胞,使腫瘤織的各種蛋白酶及膠原酸分泌增加,從而加速腫瘤細(xì)胞的轉(zhuǎn)移過程；(4)FGF-2還與腫瘤的耐藥性有關(guān),增強(qiáng)腫瘤對藥物誘導(dǎo)的細(xì)胞凋亡的抵抗能力[20]。故FGF-2與腫瘤生長、轉(zhuǎn)移、預(yù)后有著非常密切的關(guān)系。 FGF-2與腫瘤腫瘤生長、轉(zhuǎn)移、耐藥有關(guān),用抗體阻斷FGF-2的活性被認(rèn)為是一種治療腫瘤的有效方法。已有多株抗FGF-2抗體體內(nèi)外抑制腫瘤試驗(yàn)的報道。在多種動物體內(nèi),一株中和性的的單抗(GD2)能抑制FGF-2或腫瘤組織誘導(dǎo)的血管新生。在大鼠體內(nèi)內(nèi)GD2能抑制骨肉瘤的生長。另一株單抗,3H3,能抑制十二指腸潰瘍的血管新生,從而延遲潰瘍的愈合,同時它還能抑制一種轉(zhuǎn)染的致瘤性細(xì)胞在裸鼠體內(nèi)的生長。Aonuma等制備了兩株中和性單抗,2G11和1E6,前者識別的是FGF-2的肝素結(jié)合位點(diǎn),后者識別的是FGF-2與受體結(jié)合的位點(diǎn),體內(nèi)實(shí)驗(yàn)證實(shí)1E6能抑制腫瘤生長,而2G11不具備抗瘤作用。國內(nèi),謝慶祥等[7]通過建立裸鼠皮下移植膀胱癌動物模型進(jìn)行研究,發(fā)現(xiàn)FGF-2抗體治療組皮下瘤體積和重量比對照組均顯著減少,瘤組織中增殖細(xì)胞核抗原(PCNA)指數(shù)和微血管密度(MVD)也顯著降低,表明FGF-2抗體對膀胱癌的生長具有明顯抑制作用,其主要通過抑制膀胱癌細(xì)胞增殖和減少腫瘤血管形成的途徑而在荷瘤裸鼠體內(nèi)發(fā)揮抗腫瘤作用。林衛(wèi)等觀察了FGF-2抗體對人卵巢癌細(xì)胞的增殖、卵巢癌腹腔移植瘤裸鼠的生存率和人卵巢癌移植瘤血管生成和腫瘤生長的影響。研究結(jié)果顯示,FGF-2抗體能明顯抑制卵巢癌細(xì)胞的增殖,卵巢癌的生長和血管生成,并呈濃度依賴性,提示FGF-2單抗有望成為卵巢癌生物治療的新方法。本實(shí)驗(yàn)室前期制備了19株鼠抗FGF-2單抗,通過體外實(shí)驗(yàn)證實(shí),其中三株抗體能夠誘導(dǎo)B16細(xì)胞凋亡,近期的體內(nèi)試驗(yàn)發(fā)現(xiàn)其中一株抗體能夠明顯抑制黑色素瘤的生長和轉(zhuǎn)移,延長荷瘤小鼠的生存期,同時與化療藥物順鉑、紫杉醇等聯(lián)合使用能夠明顯抑制黑色素癌的生長,而對于那些對化療藥物耐藥的腫瘤抗FGF-2單抗能逆轉(zhuǎn)其耐藥性。 這些報道證實(shí)在多種試驗(yàn)條件下,抗FGF-2抗體能阻斷血管新生和抑制腫瘤生長。但這些抗體都是動物來源的抗體,用于人類疾病的治療存在半衰期短、血清病發(fā)生率高、易在人體內(nèi)產(chǎn)生抗體導(dǎo)致不能重復(fù)使用等弊。制備全人源性的抗FGF-2抗體,是克服上述弊端有效方法。 人源性抗體的制備技術(shù)主要有三種,第一種是通過表面展示技術(shù)從人源性抗體庫中篩選人源性抗體,第二種技術(shù)是通過免疫轉(zhuǎn)入人免疫球蛋白基因的轉(zhuǎn)基因鼠來獲得針對特異抗原的人源性抗體,第三種技術(shù)是通過細(xì)胞分選技術(shù),從某些病人骨髓細(xì)胞或外周血細(xì)胞中篩選特異性針對某種抗原的B細(xì)胞或記憶性B細(xì)胞,通過某些技術(shù)如與人骨髓瘤融合或用EB病毒使其永生化,使得這些B細(xì)胞能在體外無限繁殖,從而分泌人源性抗體。這三種技術(shù)中第二種技術(shù)需要購買或構(gòu)建轉(zhuǎn)基因小鼠,需要耗費(fèi)大量財(cái)力和物力。第三種技術(shù)需要特殊病人的血細(xì)胞,并且需要獲得人源性雜交瘤或刺激B細(xì)胞的永生化,這些技術(shù)目前還不成熟。表面展示技術(shù)具有極強(qiáng)的篩選功能,將序列互不相同的一組多樣化的抗體表達(dá)到細(xì)菌,酵母,病毒表面,得到抗體展示庫因此,利用其表型與基因型的統(tǒng)一以及易于擴(kuò)增的特性,可很容易的從庫中篩選出針對某種特異抗原的抗體。其中以噬菌體展示技術(shù)應(yīng)用最為廣泛。 從噬菌體抗體庫中直接篩選獲得高親和力抗體,其主要影響因素是抗體庫容量大小和抗體基因的成熟度,其多樣性要能保證有這種抗體存,并且在經(jīng)過擴(kuò)增后具有被篩選出來所要求的拷貝數(shù),因此增加噬菌體抗體庫的庫容量尤其重要。根據(jù)已有報道,從大于109的大容量天然抗體庫中,往往可以得到高親和力的抗體,無需進(jìn)行抗體親和力成熟。本研究所用的抗體庫為經(jīng)過單載體細(xì)胞內(nèi)重組法(Lxop-cre定位重組系統(tǒng))兩次重組配對后的人源性大容量抗體庫,重組后庫容約為6×1010,沉淀濃縮后滴度約為1013cfu／ml,具有良好的多樣性,可以充分滿足篩選的需要,在獲得高親和力抗體以及進(jìn)行抗體性能改造方面具有較強(qiáng)的優(yōu)勢。 在抗體的進(jìn)化過程中,基因突變并不是隨機(jī)發(fā)生于可變區(qū)內(nèi)的,而是傾向性的集中在CDR區(qū)的某些位置,即突變熱點(diǎn),有多種類型,其中研究較詳細(xì)的熱點(diǎn)類型有AGY／RGYW和TAY(R=A／G,Y=C／T,W=A／T)。這些位點(diǎn)也常常是位于和抗原直接結(jié)合的區(qū)域中。針對這類位點(diǎn)引入突變,相較于這類位點(diǎn)之外的區(qū)域?qū)⒏赡艿玫接H和力提高的基因工程抗體,而且可以通過構(gòu)建小型的突變庫來實(shí)現(xiàn)。Ho等研究發(fā)現(xiàn),CDR區(qū)的突變熱點(diǎn)分為胚系熱點(diǎn)和非胚系熱點(diǎn),只有突變胚系熱點(diǎn)才能提高抗體的親和力。因此對抗體胚系熱點(diǎn)進(jìn)行突變將是本文采取的方法。 方法 1.利用固相篩選技術(shù)從大容量噬菌體抗體庫(6×1010)中篩選能特異性結(jié)FGF-2的克隆,經(jīng)過三到四輪的洗脫篩選,將得到的陽性克隆通過酶切鑒定,可變區(qū)多樣性分析,基因測序和序列比對,確定能特異性結(jié)合FGF-2的序列正確的陽性克隆。 2.將測序正確的陽性克隆的基因插入到原核表達(dá)載體pComb3XSS中,在低溫培養(yǎng)條件下,利用IPTG誘導(dǎo)scFv基因的可溶性表達(dá),離心后收集菌體,PBS重懸后超聲破碎,收集破碎上清,通過SDS-PAGE, Western-blot, ELISA鑒定上清中抗體的特異性,通過競爭ELISA檢測者幾株scFv能否抑制FGF-2與其高親和力受體FGFR1的結(jié)合。 3.將能夠抑制FGF-2與其高親和力受體FGFR1的結(jié)合的44克隆通過重疊延伸PCR構(gòu)建成全長的人源性抗FGF-2抗體,插入到真核表達(dá)載體pIGG中,構(gòu)建成真和表達(dá)載體pIGG-44,利用轉(zhuǎn)染試劑FuGene HD轉(zhuǎn)染HEK293T細(xì)胞,進(jìn)行真核可溶性表達(dá),收集細(xì)胞培養(yǎng)上清,通過SDS-PAGE, Western-blot,ELISA鑒定上清中抗體的特異性,通過硫酸銨沉淀和蛋白G純化抗體,通過夾心ELISA測定抗體的濃度。 4.表達(dá)的全長的人源性抗體通過體外細(xì)胞實(shí)驗(yàn)驗(yàn)證抗體的中和FGF-2的活性,通過血管內(nèi)皮細(xì)胞促增值實(shí)驗(yàn),遷移實(shí)驗(yàn)和成管實(shí)驗(yàn)驗(yàn)證抗體對血管內(nèi)皮細(xì)胞的作用,通過腫瘤細(xì)胞增殖抑制試驗(yàn)驗(yàn)證抗體對腫瘤細(xì)胞的增值抑制作用,通過Hoechst 33258染色觀察抗體作用后腫瘤細(xì)胞細(xì)胞核的變化情況,來觀察細(xì)胞是否發(fā)上了凋亡 5.對具有中和活性的44號克隆進(jìn)行親和力成熟,利用基因比對從Genebank中搜索出與44號克隆序列最接近的抗體胚系基因序列,然后通過IMTG中的抗體可變區(qū)基因在線分析工具V-QUEST,找出重鏈胚系基因中CDR區(qū)的突變熱點(diǎn),并與44號克隆進(jìn)行比較,確定這些熱點(diǎn)在44號克隆重鏈CDR區(qū)中的位置,隨后我們通過定點(diǎn)突變對重鏈可變區(qū)CDR1區(qū)中的三個胚系熱點(diǎn)進(jìn)行定點(diǎn)隨機(jī)突變,構(gòu)建噬菌體抗體突變庫,通過三輪洗脫篩選,每輪逐漸增加洗脫次數(shù)和減少包被的FGF-2的量來得到親和力提高的抗體突變株,將高親和力突變株進(jìn)行可溶性表達(dá)后,通過異硫氰酸銨洗脫法測定抗體的相對親和力。計(jì)算抗體親和力提高倍數(shù)。同時通過競爭ELISA法檢測突變株是否還能抑制FGF-2與其高親和力受體FGFR1的結(jié)合。 結(jié)果 1.經(jīng)過3到4輪篩選從104個隨機(jī)挑選的克隆中,篩得39株抗FGF-2的抗體,通過對抗體可變區(qū)基因多樣性分析,發(fā)現(xiàn)有8種不同抗體可變區(qū)基因型,挑選其中8株phage ELISA顯色最高的克隆進(jìn)行DNA酶切和測序鑒定,發(fā)現(xiàn)其中7株為正確的抗體基因序列,通過大腸桿菌HB2151成功的進(jìn)行了scFv的可溶性表達(dá),通過競爭ELISA發(fā)現(xiàn)其中三株scFv能夠抑制FGF-2與其高親和力受體FGFR1βⅢC的結(jié)合,其中44號克隆的抑制效果最好。 2.通過對轉(zhuǎn)染試劑,轉(zhuǎn)染方法,培養(yǎng)溫度,轉(zhuǎn)染試劑與細(xì)胞的孵育時間,組蛋白抑制劑的加入等條件進(jìn)行優(yōu)化使人源性抗FGF-2抗體在HEK293T細(xì)胞中的表達(dá)量從1.5mg／L提高到了15.1mg／L。表達(dá)產(chǎn)物經(jīng)硫酸銨沉淀,透析后上蛋白G親和層析柱純化,純化后的產(chǎn)物經(jīng)超濾管超濾除鹽,最后經(jīng)SDS-PAGE鑒定,獲得純度為90%的電泳純抗體,雙抗夾心法測定抗體含量,顯示產(chǎn)量可以達(dá)到每升細(xì)胞培養(yǎng)上清12mg抗體。 3.通過一系列體外實(shí)驗(yàn)證實(shí)44號克隆能夠抑制人臍靜脈血管內(nèi)皮細(xì)胞的增殖,遷移和成管,同時還能抑制神經(jīng)膠質(zhì)瘤細(xì)胞株U87MG的增殖,Hoechst 33258染色后發(fā)現(xiàn)抗FGF-2抗體作用能夠誘導(dǎo)U87MG的凋亡 4.通過熱點(diǎn)隨機(jī)突變構(gòu)建44號克隆的單鏈抗體噬菌體突變庫,通過三輪洗脫篩選,篩選到了4株親和力提高的抗體突變株,經(jīng)過可溶性表達(dá)后,通過異硫氰酸銨洗脫法測定抗體的相對親和力,顯示其中最高的一株親和力比44號克隆的親和力提高了4倍。 結(jié)論 1.通過對人源性噬菌體抗體庫的篩選得到了七株不同的人源性抗FGF-2抗體,經(jīng)過體外實(shí)驗(yàn)證實(shí)其中一株抗體能夠中和FGF-2的多種生物學(xué)作用,如促血管內(nèi)皮增殖作用,促細(xì)胞遷移作用,內(nèi)皮細(xì)胞成管作用等,更為重要的此株抗體還能誘導(dǎo)神經(jīng)膠質(zhì)瘤細(xì)胞株U87MG的凋亡。 2.通過胚系熱點(diǎn)突變使此株中和性抗體的親和力提高了4倍,與此同時也提高了抗體的中和活性,從而證實(shí)了胚系熱點(diǎn)突變提高抗體親和力的可行性。 本研究的創(chuàng)新處： 1.成功篩選到三株人源性中和性抗FGF-2抗體,其中一株的抑制FGF-2與其高親和力受體FGFR1結(jié)合的效果最好,通過細(xì)胞實(shí)驗(yàn)證實(shí)此株抗體能夠中和FGF-2的多種生物學(xué)作用,為FGF-2抗體藥物研究奠定基礎(chǔ)。 2.成功利用CDR區(qū)胚系熱點(diǎn)突變提高了一株中和性抗體的親和力,同時也使得抗體的中和活性增強(qiáng),證實(shí)了此方法可在不改變抗原識別位點(diǎn)的情況下提高抗體的親和力。
[Abstract]:Purpose and background
FGF-2 is also called the basic fibroblast growth factor (basic fibroblast, growth factor, FGF-2), is a member of the fibroblast growth factor family, first by Gospodarowicz (1974) extracted from bovine pituitary in a cell growth factor.FGF-2 had significant promoting effect of the five kinds of molecular weight of BALB / C 3T3 the cell, from 18kD to 34kD FGF-2 range. All of the isoforms are lack of signal peptide, is through a now is not clear the mechanism of secretion to the extracellular 18kD FGF-2, which is widely distributed in the body, cells and many tumor cells exist in the mesoderm and neuroectoderm source, can promote the proliferation and differentiation of function these cells are involved in embryonic development, angiogenesis, wound repair, nerve regeneration, tumor growth, tissue fibrosis and other physiological and pathological processes.
The biological effects of FGF-2, besides of fibroblasts and vascular endothelial cells proliferation significantly, is also involved in the occurrence and development of malignant tumor, especially in tumor invasion and metastasis plays an important role. There is a considerable part of the tumor (such as breast cancer, colon cancer, thyroid cancer, lung cancer, bladder cancer, esophageal cancer, etc.) the high expression of FGF-2 (Table 2). The known part of its role mainly (1) FGF-2 can promote the regeneration of epidermis, endothelial cells, promote vascular endothelial cell division and induce the separated from the basement, to stimulate endothelial cells to tumor tissue chemotaxis, and the formation of tubular structure, but also improve the blood of fibrous tissue protein plasminogen activators and other proteases induced by endothelial cells, thereby promote capillary formation; (2) FGF-2 can promote the secretion of various growth factors, such as VEGF, HGF, FGF-1 and so on, these factors regulate each other and jointly promote Progressive tumor growth; (3) the high expression of FGF-2 receptor in tumor cells than in normal cells, usually FGF-2R 10 times higher than that of many tumor cells (such as glioma, rhabdomyosarcoma, leukemia, lung, melanoma, liver cancer) expressed both FGF2 and FGFRs, so the FGF2 can be directly applied to the tumor the tumor cells, proteases and collagen fabric acid secretion increased, thus speeding up the transfer process of tumor cells; (4) FGF-2 is associated with tumor resistance, enhance the ability to resist [20]. on the apoptosis of the tumor FGF-2 and tumor growth, metastasis, prognosis has a very close relationship.
FGF-2 and tumor growth, metastasis, drug resistance, antibody blocking FGF-2 activity was found to be an effective method for treating tumors. Tumor inhibition test has been reported in several strains of anti FGF-2 antibodies in vitro and in vivo. In a variety of animal, a neutralizing monoclonal antibody (GD2) could inhibit FGF-2 induced tumor angiogenesis can inhibit osteosarcoma. GD2 in vivo in rat growth. Another monoclonal antibody, 3H3, can inhibit the angiogenesis of duodenal ulcer, and delayed healing of the ulcer, and it can inhibit a transfection of tumorigenic cells by two strains of neutralizing monoclonal antibody in nude mice and the growth of.Aonuma so, 2G11 and 1E6, the former is the identification of the heparin binding site of FGF-2, which is a combination of FGF-2 and receptor recognition sites in vivo experiments confirmed that 1E6 can inhibit the growth of tumor, and 2G11 does not have antitumor effect. At home, Xie Qingxiang [7]. Study to establish the animal model of bladder carcinoma transplanted subcutaneously in nude mice, found FGF-2 antibody treatment than in the control group were significantly reduced in group subcutaneous tumor volume and weight of tumor tissues, proliferating cell nuclear antigen (PCNA) index and microvessel density (MVD) were significantly decreased, indicating that the growth of FGF-2 antibody on bladder cancer has obvious inhibitory effect, the mainly through the inhibition of tumor angiogenesis and ways to reduce the proliferation of bladder cancer cells and the antitumor effect in nude mice. Lin Wei observed the proliferation of FGF-2 antibody against human ovarian cancer cells, and affect the survival of human ovarian cancer xenograft tumor angiogenesis and tumor growth in nude mice to study. The results showed that FGF-2 antibody can inhibit ovarian cancer cell proliferation, growth and angiogenesis of ovarian cancer, and in a concentration dependent manner, suggesting that FGF-2 is expected to become the biological therapy of ovarian carcinoma monoclonal antibody The new method. The laboratory has prepared 19 strains of mouse anti FGF-2 monoclonal antibody, in vitro experiments confirmed that the three antibodies can induce B16 cell apoptosis in vivo, recently found that one strain antibody can significantly inhibit melanoma growth and metastasis, prolong the survival time of the mice, at the same time with the chemotherapy drug the combined use of cisplatin, paclitaxel can inhibit the growth of melanoma, and to those of the chemotherapy drug resistance tumor anti FGF-2 monoclonal antibody can reverse the drug resistance.
These reports have confirmed that in a variety of experimental conditions, the anti FGF-2 antibody can inhibit angiogenesis and tumor growth inhibition. But these antibodies are antibodies of animal origin, for the treatment of human disease has short half-life, high rate of occurrence of serum sickness, easy to produce antibody can not cause repeatition harm in the human body. Preparation of the whole people the source of anti FGF-2 antibody, is the effective method to overcome these disadvantages.
There are three main types of human antibody preparation technology, the first is through the surface screening of human antibodies from human antibody library, second transgenic mice by immune technology is transferred to Human Immunoglobulin gene to obtain human antibodies against specific antigen, third kinds of technology through cell sorting technique screening, specific to antigens of B cells and memory B cells in some patients with bone marrow cells or peripheral blood cells, and by some techniques such as human myeloma or fusion with EB virus immortalized B cells can proliferate, the infinite in vitro, and the secretion of human antibodies. Three second kinds of technology technology need to purchase or construction of transgenic mice, requires a lot of financial and material resources. Third techniques require special blood cells, and need to get human hybridoma or stimulation of B cells The immortal, the technology is not mature. The surface display technology has the function of screening, a group of diverse expression of antibody to bacteria, yeast, virus surface sequences are different from each other, so the obtained antibody display library, using the unified phenotype and genotype and easy amplification characteristics, can be very easy screening from the library for a specific antigen antibody. The phage display technology is most widely used.
Screening from phage antibody library directly to obtain high affinity antibodies, the main factor is the antibody library size and antibody gene maturity, its diversity to ensure the antibody, and after amplification has been screened out copy number required, thus increasing the storage capacity of particular phage antibody library important. According to the existing reports, from more than 109 large capacity natural antibody library, often can get high affinity antibody, without antibody affinity maturation. Antibody library used in this study is through a single cell carrier recombination method (Lxop-cre recombination system) of human large phage antibody library two recombinant pairing after the reorganization, the volume is 6 x 1010, precipitation concentration was approximately 1013cfu / ml, with good diversity, can fully meet the need in the screening, to obtain high affinity antibodies and anti The improvement of body performance has strong advantages.
In the evolutionary process of antibody, gene mutation and random variable is not occurred in the area, but the tendency of certain locations concentrated in the CDR area, namely hot spot mutation, there are many types, including detailed research hotspot types are AGY / RGYW and TAY (R=A / G, Y=C / T, W=A / T). These sites are often located and direct antigen binding region. For this kind of site mutation, compared to the outside of this kind of site will be more likely to improve the affinity of gene engineering antibody, but also through the construction of mutant library to achieve small.Ho research found that mutation hotspot region CDR points for germline hot and non germline mutations in the germ line hot hot, only to improve the affinity of the antibody. So the mutation will be the approach of germline antibody hotspot.
Method
1. using the solid-phase screening technology from a large phage antibody library (6 * 1010) in screening the specific binding of FGF-2 clones, after three to four rounds of panning, the positive clones were obtained by enzyme digestion, diversity analysis of variable region gene sequencing and sequence alignment, can determine the sequence of positive clone FGF-2 the correct binding specificity.
2. sequencing positive clones the gene was inserted into prokaryotic expression vector pComb3XSS, in low temperature conditions, the soluble expression of scFv gene induced by IPTG, after centrifugation, collecting thalli, ultrasonic suspended in PBS, collect the broken supernatant, through SDS-PAGE, Western-blot, ELISA and identification of specific antibodies in the supernatant by. With the competitive ELISA detection of several strains of scFv can inhibit FGF-2 and its high affinity receptor FGFR1.
3. will be able to inhibit the binding of FGF-2 to its high affinity receptor FGFR1 44 cloned by overlap extension PCR to construct a full-length humanized anti FGF-2 antibody, inserted into eukaryotic expression vector pIGG, construct and true expression vector pIGG-44 using transfection reagent FuGene HD transfected HEK293T cells, eukaryotic expression of soluble, collecting cells the culture supernatant by SDS-PAGE, Western-blot, ELISA and identification of specific antibodies in the supernatant, purified by ammonium sulfate precipitation and protein G, the antibody concentration was determined by sandwich ELISA.
4. activity and FGF-2 expression of human antibodies against full-length antibody by in vitro experiments, the endothelial cell proliferation experiment, migration and tube formation experiment experimental verification antibody on vascular endothelial cells, the inhibition of tumor cell proliferation test antibody inhibition value of tumor cells, changes by Hoechst 33258 antibody staining of tumor cells after the role of the nucleus, to observe the hair on the cell apoptosis
5. of the 44 clones with neutralizing activity of affinity maturation, using gene ratio from Genebank to search the closest and clone 44 sequence antibody germline gene sequence, then the antibody variable genes in IMTG online analysis tool V-QUEST, find out the heavy chain germline gene mutation hotspot in CDR region, and compared with the 44 clones, determine the location of these hot spots in clone 44 heavy chain in the CDR area, then we by point mutation in CDR1 region of heavy chain variable region in three germline hot point mutation, mutant phage antibody library, through three rounds of panning, each round gradually increased and washing times reduce the amount of FGF-2 package is to improve the affinity of antibody mutant, the high affinity mutant soluble expression, by ISO thiocyanate elution method for the determination of the relative affinity of antibody antibody affinity calculation. At the same time, a competitive ELISA method was used to detect whether the mutant strain could inhibit the combination of FGF-2 and its high affinity receptor FGFR1.
Result
1. to 4 after 3 rounds of screening from 104 clones randomly selected, screened anti FGF-2 antibody of 39 strains, the gene of antibody variable region diversity analysis, found that 8 genes of different antibody variable type, selected among 8 strains of phage ELISA were cloned by DNA color of the highest enzyme digestion and DNA sequencing. 7 of them were found, antibodies to the correct gene sequence of Escherichia coli HB2151, through the success of the soluble expression of scFv, ELISA found through competition among the three scFv strains could inhibit FGF-2 to its high affinity receptor FGFR1 beta III binding to C, one of the best inhibitory effect of clone 44.
2. through the transfection reagent, transfection method, transfection reagent and cell culture temperature, incubation time, adding conditions such as inhibitors of histone deacetylase optimize expression of anti FGF-2 antibody in HEK293T cells from 1.5mg / L to 15.1mg / L. expression products were purified by ammonium sulfate precipitation, dialysis protein G affinity chromatography, purified by ultrafiltration ultrafiltration

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

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