抗T-2毒素单链抗体的制备及该毒素对细胞毒性影响的初步研究

发布时间：2018-08-29 13:04

【摘要】： T-2毒素是由多种真菌产生的单端孢霉烯族(trichothecenes,TS)毒素中最毒的毒素之一。该毒素在自然界中分布极为广泛,是危险的食品污染物,对人畜健康危害十分严重。为此,在本研究中,我们构建了抗T-2毒素的噬菌体单链抗体库,从中筛选亲和力较高的单链抗体,从而为T-2毒素的检测奠定基础。此外,并对其引起的细胞毒性进行了研究。根据T-2毒素是小分子半抗原的特性,我们首先将T-2毒素与牛血清白蛋白BSA通过琥珀酸酐法进行交联使之变成完全抗原。然后用交联物T-2HS-BSA免疫Balb/c雌性小鼠。达到一定的效价后,从小鼠的脾脏中提取总RNA,反转录得到cDNA第一链,通过PCR扩增得到重链和轻链可变区VH、VL基因,通过重叠延伸PCR(SOE-PCR)组装成scFv并克隆到噬菌体载体pCANTAB5E中,构建噬菌体抗体库。通过噬菌体展示技术经过四轮淘选得到一株有亲和力的单链抗体T1。为研究T-2毒素对骨髓瘤细胞SP2/0的毒性,我们通过显微镜观察细胞形态的变化,MTT法检测其对骨髓瘤细胞增殖抑制用,流式细胞仪检测细胞的周期,实时荧光定量PCR检测Caspase-3基因的表达情况。结果表明,T-2毒素使骨髓瘤细胞凋亡,且呈时间和剂量依赖关系。实时定量PCR表明T-2使Caspase-3基因mRNA表达发生上调。这些结果表明,Caspase-3可能在T-2毒素诱导的SP2/0细胞凋亡信号通路中起重要作用。
[Abstract]:T-2 toxin is one of the most toxic toxins in monosporin (trichothecenes,TS) toxin produced by many fungi. The toxin is widely distributed in nature and is a dangerous food pollutant, which is very harmful to human and animal health. Therefore, in this study, we constructed a phage scFv library against T-2 toxin, and screened scFv with high affinity from it, thus laying a foundation for the detection of T-2 toxin. In addition, the cytotoxicity was studied. According to the characteristic that T-2 toxin is a small molecule hapten, T-2 toxin and bovine serum albumin (BSA) BSA are crosslinked to complete antigen by succinic anhydride method. Then Balb/c female mice were immunized with T-2HS-BSA. After reaching a certain titer, the first strand of cDNA was obtained by reverse transcription of total RNA, from mouse spleen, and the VH,VL gene of heavy chain and light chain variable region was obtained by PCR amplification. ScFv was assembled by overlapping extension of PCR (SOE-PCR) and cloned into phage vector pCANTAB5E. The phage antibody library was constructed. A single chain antibody T 1 with affinity was obtained by phage display after four rounds of panning. In order to study the cytotoxicity of T-2 toxin to myeloma cell line SP2/0, the cell morphology was observed by microscope and the cell cycle was detected by flow cytometry. The expression of Caspase-3 gene was detected by real-time fluorescence quantitative PCR. The results showed that T-2 toxin induced apoptosis of myeloma cells in a time and dose dependent manner. Real-time quantitative PCR showed that T-2 up-regulated the mRNA expression of Caspase-3 gene. These results suggest that Caspase-3 may play an important role in T-2 toxin induced apoptosis signaling pathway in SP2/0 cells.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】