脫氧雪腐鐮刀菌烯醇的環(huán)七肽模擬表位篩選
發(fā)布時(shí)間：2019-06-14 12:04
【摘要】：利用噬菌體隨機(jī)環(huán)七肽庫(kù)篩選脫氧雪腐鐮刀菌烯醇(Deoxynivalenol,DON)模擬表位。以DON單抗為靶分子,四輪固相淘選噬菌體隨機(jī)環(huán)七肽庫(kù),篩選出陽(yáng)性噬菌體,并通過ELISA實(shí)驗(yàn)檢測(cè)其特異性。對(duì)陽(yáng)性克隆的DNA進(jìn)行測(cè)序,取序列不同的陽(yáng)性克隆,分別建立陽(yáng)性噬菌體與DON標(biāo)準(zhǔn)品的競(jìng)爭(zhēng)抑制曲線。對(duì)30個(gè)噬菌體單克隆進(jìn)行活性測(cè)定,結(jié)果顯示能與DON單抗結(jié)合的噬菌體克隆有28株,其中的22株能被DON標(biāo)準(zhǔn)品阻斷與DON單抗結(jié)合。DNA測(cè)序結(jié)果顯示,21株陽(yáng)性噬菌體的序列是PFPNHPY,另一株的序列是TPWTQHL。其相應(yīng)噬菌體與DON毒素標(biāo)準(zhǔn)品的競(jìng)爭(zhēng)曲線結(jié)果顯示,8號(hào)噬菌體建立的競(jìng)爭(zhēng)抑制曲線線性范圍是0.0292~0.636μg/mL,IC_(50)為0.169μg/mL;4號(hào)噬菌體建立的競(jìng)爭(zhēng)抑制曲線線性范圍是0.0124~0.271μg/mL,IC_(50)為0.058μg/mL。在隨機(jī)環(huán)七肽庫(kù)中篩選到TPWTQHL和PFPNHPY兩個(gè)DON模擬表位,可替代DON毒素標(biāo)準(zhǔn)品,建立DON的免疫學(xué)無毒檢測(cè)技術(shù)。
[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.
【作者單位】： 暨南大學(xué)生命科學(xué)技術(shù)學(xué)院生物工程學(xué)系;
【基金】：“十一五”國(guó)家科技支撐計(jì)劃項(xiàng)目(2008BAK42B05)
【分類號(hào)】：S816.17;TS210.7
本文編號(hào)：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.
【作者單位】： 暨南大學(xué)生命科學(xué)技術(shù)學(xué)院生物工程學(xué)系;
【基金】：“十一五”國(guó)家科技支撐計(jì)劃項(xiàng)目(2008BAK42B05)
【分類號(hào)】：S816.17;TS210.7
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