中和性rhbFGF单克隆抗体抗原表位的研究与VBP3复合肽疫苗的安全性评价

发布时间：2018-02-16 15:18

  本文关键词： 碱性成纤维细胞生长因子 单克隆抗体 抗原表位 基质辅助激光解吸电离飞行时间质谱 VPB3 复合肽疫苗 安全性评价　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景和目的： (1)本室前期筛选出三株具有中和活性的单克隆抗MabF7, MabF10和MabF12,三株单抗MabF7, MabF10和MabF12均能抑制bFGF与FGFR1的结合,细胞实验显示三株单抗可抑制bFGF促血管内皮细胞增殖活性,并且三株单抗均可抑制黑色素瘤的生长,并诱导其发生凋亡。而且,MabF7具有显著的抑制肿瘤生长的作用。抗体识别表位与抗体的体内治疗、亲和力及药代动力学均有密切关系。因此本研究的目的就是确定三株rhbFGF单克隆抗体的抗原表位。 (2)另外用本室构建的VBP3基因工程菌表达的VBP3复合肽疫苗免疫C57BL/6J小鼠获得高效价的抗bFGF血清和抗VEGF血清,接种B16黑色素瘤后发现VBP3复合肽疫苗可诱导小鼠产生高滴度抗VEGF和抗bFGF抗体,可抑制体内异种移植黑色素瘤的生长。本研究的目的是初步评价复合肽疫苗的安全性和稳定性。方法： 单克隆抗体抗原表位的分析：ELISA法初步检测这三株bFGF单抗抗原表位的类型；采用免疫亲和质谱的方法确定三株单抗的抗原表位序列。(a)针对线性表位：水解bFGF成肽段并用单克隆抗体与表位肽结合,除去未与抗体结合的肽段,然后分离出与抗体结合的表位肽进行分析；(b)针对构象表位：水解抗原抗体复合物并除去未与抗体结合的肽段,然后分离出与抗体结合的表位肽进行分析。 复合肽疫苗VBP3的安全性评价：通过注射VBP3复合肽疫苗分别观察家兔的皮下注射的局部刺激反应和豚鼠的全身过敏状况,初步评价该复合肽疫苗的安全性。 复合肽疫苗VBP3的稳定性评价：VBP3复合肽疫苗分别于不同温度条件保存两个月后免疫Balb/c小鼠,通过检测抗bFGF血清的效价评估其稳定性。 结果： 单克隆抗体抗原表位的分析：ELISA法初步分析出MabF7识别bFGF的连续线性表位,单抗MabF10和MabF12识别bFGF的构象表位；亲和质谱分析出MabF7表位序列为110-120, MabF10的作用氨基酸位于53-60,34-44,99-108,111-119和136-145这5个肽段上。 复合肽疫苗VBP3的安全性评价：家兔局部无充血,出血,水肿等改变,注射部位有少量炎性细胞浸润,未发现水肿、坏死等组织学改变。豚鼠接种VBP3复合肽疫苗组仅3只出现弱阳性过敏反应,且很快缓解,其余豚鼠未见过敏症状。 复合肽疫苗VBP3的稳定性评价：复合肽在-20℃下保存两个月后免疫原性下降了一倍左右,在4℃条件下放置复合肽VBP3,效果很差,两个月后其免疫原性只有原来的十分之一,而在室温(25℃)条件下保存,复合肽的免疫原性基本消失。 结论： 亲和质谱的方法分析出了单抗MabF7和MabF10的抗原表位,证明采用亲和质谱的方法分析单抗的抗原表位是可行的。 通过家兔的局部刺激反应和豚鼠的全身过敏性分析,证明复合肽疫苗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，

本文编号：1515817