本文關鍵詞： 堿性成纖維細胞生長因子 單克隆抗體 抗原表位 基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜 VPB3 復合肽疫苗 安全性評價　出處：《暨南大學》2011年碩士論文　論文類型：學位論文

【摘要】：背景和目的： (1)本室前期篩選出三株具有中和活性的單克隆抗MabF7, MabF10和MabF12,三株單抗MabF7, MabF10和MabF12均能抑制bFGF與FGFR1的結(jié)合,細胞實驗顯示三株單抗可抑制bFGF促血管內(nèi)皮細胞增殖活性,并且三株單抗均可抑制黑色素瘤的生長,并誘導其發(fā)生凋亡。而且,MabF7具有顯著的抑制腫瘤生長的作用�？贵w識別表位與抗體的體內(nèi)治療、親和力及藥代動力學均有密切關系。因此本研究的目的就是確定三株rhbFGF單克隆抗體的抗原表位。 (2)另外用本室構(gòu)建的VBP3基因工程菌表達的VBP3復合肽疫苗免疫C57BL/6J小鼠獲得高效價的抗bFGF血清和抗VEGF血清,接種B16黑色素瘤后發(fā)現(xiàn)VBP3復合肽疫苗可誘導小鼠產(chǎn)生高滴度抗VEGF和抗bFGF抗體,可抑制體內(nèi)異種移植黑色素瘤的生長。本研究的目的是初步評價復合肽疫苗的安全性和穩(wěn)定性。方法： 單克隆抗體抗原表位的分析：ELISA法初步檢測這三株bFGF單抗抗原表位的類型；采用免疫親和質(zhì)譜的方法確定三株單抗的抗原表位序列。(a)針對線性表位：水解bFGF成肽段并用單克隆抗體與表位肽結(jié)合,除去未與抗體結(jié)合的肽段,然后分離出與抗體結(jié)合的表位肽進行分析；(b)針對構(gòu)象表位：水解抗原抗體復合物并除去未與抗體結(jié)合的肽段,然后分離出與抗體結(jié)合的表位肽進行分析。 復合肽疫苗VBP3的安全性評價：通過注射VBP3復合肽疫苗分別觀察家兔的皮下注射的局部刺激反應和豚鼠的全身過敏狀況,初步評價該復合肽疫苗的安全性。 復合肽疫苗VBP3的穩(wěn)定性評價：VBP3復合肽疫苗分別于不同溫度條件保存兩個月后免疫Balb/c小鼠,通過檢測抗bFGF血清的效價評估其穩(wěn)定性。 結(jié)果： 單克隆抗體抗原表位的分析：ELISA法初步分析出MabF7識別bFGF的連續(xù)線性表位,單抗MabF10和MabF12識別bFGF的構(gòu)象表位；親和質(zhì)譜分析出MabF7表位序列為110-120, MabF10的作用氨基酸位于53-60,34-44,99-108,111-119和136-145這5個肽段上。 復合肽疫苗VBP3的安全性評價：家兔局部無充血,出血,水腫等改變,注射部位有少量炎性細胞浸潤,未發(fā)現(xiàn)水腫、壞死等組織學改變。豚鼠接種VBP3復合肽疫苗組僅3只出現(xiàn)弱陽性過敏反應,且很快緩解,其余豚鼠未見過敏癥狀。 復合肽疫苗VBP3的穩(wěn)定性評價：復合肽在-20℃下保存兩個月后免疫原性下降了一倍左右,在4℃條件下放置復合肽VBP3,效果很差,兩個月后其免疫原性只有原來的十分之一,而在室溫(25℃)條件下保存,復合肽的免疫原性基本消失。 結(jié)論： 親和質(zhì)譜的方法分析出了單抗MabF7和MabF10的抗原表位,證明采用親和質(zhì)譜的方法分析單抗的抗原表位是可行的。 通過家兔的局部刺激反應和豚鼠的全身過敏性分析,證明復合肽疫苗VBP3的安全性良好。
[Abstract]:Background and purpose:. Three monoclonal antibodies MabF7, MabF10 and MabF12, three McAbs MabF7, MabF10 and MabF12 could inhibit the binding of bFGF to FGFR1. Cell experiments showed that three McAbs could inhibit the proliferation activity of bFGF induced vascular endothelial cells, and MabF7, MabF12, MabF7, MabF7, MabF7, MabF7, MabF7, MabF7 and MabF12, respectively. Moreover, three monoclonal antibodies can inhibit the growth of melanoma and induce its apoptosis. MabF7 has a significant inhibitory effect on tumor growth. The affinity and pharmacokinetics are closely related, so the aim of this study is to identify the epitopes of three rhbFGF monoclonal antibodies. In addition, C57BL / 6J mice were immunized with VBP3 complex peptide vaccine expressed by VBP3 genetically engineered bacteria in our laboratory to obtain high titer anti-#en2# serum and anti-#en3# serum. After inoculation with B16 melanoma, it was found that VBP3 complex peptide vaccine could induce mice to produce high titer anti VEGF and anti bFGF antibodies. It can inhibit the growth of xenotransplantation melanoma in vivo. The aim of this study was to evaluate the safety and stability of compound peptide vaccine. Analysis of Monoclonal Antibody epitopes the types of epitopes of the three bFGF McAbs were preliminarily detected by Elisa. The epitope sequence of three McAbs was determined by immuno-affinity mass spectrometry. The epitope was targeted at linear epitopes: hydrolysis of bFGF peptide segment and binding of monoclonal antibody to epitope peptide, removal of peptides not bound to antibody. Then the epitope peptides which bind to antibody were isolated and analyzed. The epitopes were hydrolyzed and the peptides which were not bound to the antibody were removed and then the peptides which were bound to the antibody were separated and analyzed for the purpose of analyzing the conformational epitope: the antigen-antibody complex was hydrolyzed and the peptides that were not bound to the antibody were removed for analysis. Safety evaluation of compound peptide vaccine VBP3: the safety of the compound peptide vaccine was preliminarily evaluated by observing the local stimuli of rabbit subcutaneous injection and the systemic allergy of guinea pigs by injecting VBP3 compound peptide vaccine. Evaluation of the stability of VBP3 vaccine the stability of Balb/c mice was evaluated by detecting the titer of anti-VBP3 complex peptide vaccine after two months of preservation at different temperature. Results:. Analysis of Monoclonal Antibody epitopes the conformational epitopes of bFGF were identified by MabF7, MabF10 and MabF12, respectively. The sequence of MabF7 epitope was 110-120, and the amino acid of MabF10 was located at 53-6034-44-108C 111-119 and 136-145 by affinity mass spectrometry. Safety evaluation of VBP3: there were no changes in local hyperemia, hemorrhage and edema, a small amount of inflammatory cells infiltrated at injection site, and no edema was found. Only 3 guinea pigs received VBP3 compound peptide vaccine had weak positive allergic reaction, and the other guinea pigs had no allergic symptoms. Stability evaluation of compound peptide vaccine VBP3: the immunogenicity of compound peptide was reduced by about twice after two months of preservation at -20 鈩，

本文編號：1515817