脱氧雪腐镰刀菌烯醇的环七肽模拟表位筛选
发布时间：2019-06-14 12:04
【摘要】：利用噬菌体随机环七肽库筛选脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)模拟表位。以DON单抗为靶分子,四轮固相淘选噬菌体随机环七肽库,筛选出阳性噬菌体,并通过ELISA实验检测其特异性。对阳性克隆的DNA进行测序,取序列不同的阳性克隆,分别建立阳性噬菌体与DON标准品的竞争抑制曲线。对30个噬菌体单克隆进行活性测定,结果显示能与DON单抗结合的噬菌体克隆有28株,其中的22株能被DON标准品阻断与DON单抗结合。DNA测序结果显示,21株阳性噬菌体的序列是PFPNHPY,另一株的序列是TPWTQHL。其相应噬菌体与DON毒素标准品的竞争曲线结果显示,8号噬菌体建立的竞争抑制曲线线性范围是0.0292~0.636μg/mL,IC_(50)为0.169μg/mL;4号噬菌体建立的竞争抑制曲线线性范围是0.0124~0.271μg/mL,IC_(50)为0.058μg/mL。在随机环七肽库中筛选到TPWTQHL和PFPNHPY两个DON模拟表位,可替代DON毒素标准品,建立DON的免疫学无毒检测技术。
[Abstract]:The simulated epitopes of deoxynivalenol (Deoxynivalenol,DON) were screened by bacteriophage random cyclic seven-peptide library. Using DON monoclonal antibody as the target molecule, the random cyclic seven peptide library of bacteriophage was selected by four rounds of solid phase scouring, and the positive bacteriophage was screened out, and its specificity was detected by ELISA assay. The DNA of the positive clone was sequenced, and the competitive inhibition curves of the positive bacteriophage and the DON standard were established by taking the positive clones with different sequences. The activity of 30 bacteriophage monoclonal antibodies was determined. the results showed that there were 28 bacteriophage clones that could bind to DON monoclonal antibodies, 22 of which could be blocked by DON standard to bind to DON monoclonal antibodies. DNA sequencing results showed that the sequence of 21 positive bacteriophages was the sequence of the other strain of PFPNHPY, was TPWTQHL.. The results of the competition curve between the corresponding bacteriophage and DON toxin standard showed that the linear range of competitive inhibition curve established by bacteriophage 8 was 0.0292 ~ 0.636 渭 g / mL,IC_ (50). The linear range of competitive inhibition curve established by bacteriophage 0.169 渭 g / mL;4 was 0.0124 ~ 0.271 渭 g / mL,IC_ (50). 0.058 渭 g / mL.. Two DON mimic epitopes, TPWTQHL and PFPNHPY, were screened from the random cyclic seven peptide library, which can replace the standard of DON toxin and establish the immunological nontoxic detection technique of DON.
【作者单位】： 暨南大学生命科学技术学院生物工程学系;
【基金】：“十一五”国家科技支撑计划项目(2008BAK42B05)
【分类号】：S816.17;TS210.7
本文编号：2499368
[Abstract]:The simulated epitopes of deoxynivalenol (Deoxynivalenol,DON) were screened by bacteriophage random cyclic seven-peptide library. Using DON monoclonal antibody as the target molecule, the random cyclic seven peptide library of bacteriophage was selected by four rounds of solid phase scouring, and the positive bacteriophage was screened out, and its specificity was detected by ELISA assay. The DNA of the positive clone was sequenced, and the competitive inhibition curves of the positive bacteriophage and the DON standard were established by taking the positive clones with different sequences. The activity of 30 bacteriophage monoclonal antibodies was determined. the results showed that there were 28 bacteriophage clones that could bind to DON monoclonal antibodies, 22 of which could be blocked by DON standard to bind to DON monoclonal antibodies. DNA sequencing results showed that the sequence of 21 positive bacteriophages was the sequence of the other strain of PFPNHPY, was TPWTQHL.. The results of the competition curve between the corresponding bacteriophage and DON toxin standard showed that the linear range of competitive inhibition curve established by bacteriophage 8 was 0.0292 ~ 0.636 渭 g / mL,IC_ (50). The linear range of competitive inhibition curve established by bacteriophage 0.169 渭 g / mL;4 was 0.0124 ~ 0.271 渭 g / mL,IC_ (50). 0.058 渭 g / mL.. Two DON mimic epitopes, TPWTQHL and PFPNHPY, were screened from the random cyclic seven peptide library, which can replace the standard of DON toxin and establish the immunological nontoxic detection technique of DON.
【作者单位】： 暨南大学生命科学技术学院生物工程学系;
【基金】：“十一五”国家科技支撑计划项目(2008BAK42B05)
【分类号】：S816.17;TS210.7
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