恩拉霉素人工抗原的合成与鉴定
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摘要
建立酶联免疫方法以检测食品中恩拉霉素的残留,采用戊二醛法,偶联半抗原恩拉霉素(Er)和载体牛血清白蛋白(BSA)或卵清白蛋白(OVA),制备人工免疫抗原和人工包被抗原,分别为Er-BSA和Er-OVA。经紫外光谱鉴定,在紫外270～280 nm波长处得到Er-BSA和Er-OVA特征性偶联吸收峰。试验结果表明人工合成的2个人工抗原均偶联成功。人工免疫抗原和人工包被抗原的偶联比均达到26∶1,为进一步制备抗血清和酶联检测方法的建立奠定了基础。
In order to establish an enzyme-linked immunosorbent assay(ELISA) for examing residual Enramycin(Er) in food.The antibody with high titer and specificity must be firstly prepared. So it was necessary for antibody production to conjugate the hapten(Er) with a carrier protein to synthesize immunogent(antigen). Enramycin was coupled with carrier protein bovine serum albumin(BSA) to obtain immunogent(Er-BSA) by glutaraldehyde(GA) method and with ovalnumin(OVA) to obtain coating antigen(Er-OVA) by GA respectively. The successful linkage of Er-BSA and Er-OVA were identified by UV spectrophotometry. The UV spectral characterization demonstrated that Er-BSA and Er-OVA were successfully synthesized at 270～280 nm. The coupling ratios between Er and BSA or OVA were determined to be 26∶1, respectively.
In order to establish an enzyme-linked immunosorbent assay(ELISA) for examing residual Enramycin(Er) in food.The antibody with high titer and specificity must be firstly prepared. So it was necessary for antibody production to conjugate the hapten(Er) with a carrier protein to synthesize immunogent(antigen). Enramycin was coupled with carrier protein bovine serum albumin(BSA) to obtain immunogent(Er-BSA) by glutaraldehyde(GA) method and with ovalnumin(OVA) to obtain coating antigen(Er-OVA) by GA respectively. The successful linkage of Er-BSA and Er-OVA were identified by UV spectrophotometry. The UV spectral characterization demonstrated that Er-BSA and Er-OVA were successfully synthesized at 270～280 nm. The coupling ratios between Er and BSA or OVA were determined to be 26∶1, respectively.
引文
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[9]张天杰,李岩松,孟星宇,等.荧蒽完全抗原的合成及其免疫效果评价[J].吉林农业大学学报,2015,37(4):424-429.
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[19]张岩蔚,金艳丹,栗慧,等.氨苄西林人工抗原的合成及鼠源多克隆抗体的制备[J].黑龙江畜牧兽医,2018(1):227-229.
[20]张春辉,邢广旭,胡骁飞,等.氟苯尼考琥珀酸钠抗原的制备与鉴定[J].中兽医医药杂志,2017,36(6):17-20.
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[22]徐志康,朱凌燕,徐又一.基质辅助激光解吸与离子化时间飞行质谱在高分子研究中的应用[J].分析化学,1999,27(2):224-229.
[23]刘益华,徐俊福.基质辅助激光解吸飞行时间质谱在生物大分子中的应用[J].中国生化药物杂志,2004,25(3):192-193.
[24]胡骁飞,邓歌,柴书军,等.玉米赤霉醇完全抗原的制备及质量鉴定[J].西北农业学报,2016,25(5):637-645.
[25]孙凤霞,康立超,庞芳琴,等.克百威人工抗原合成及其免疫应用[J].西北农业学报,2018,27(1):131-137.
[2]ASAI M,MUROI M,SUGITA N,et al.Enduracidin,a new antibiotic II.Isolation and characterization[J].Antibiotics,1968,21(2):138-146.
[3]张子臣,张翠芬,刘宪军,等.恩拉霉素的最新研究进展[J].中国抗生素杂志,2013,38(3):185-189.
[4]高艳江,张勇.多肽类饲料添加剂——恩拉霉素[J].饲料博览,2010(4):32-35.
[5]国家进出口商品检验局科研组.农药标准物质的提纯与定值[M].北京:中国对外经济贸易出版社,1994.
[6]袁而森,陈子红,林安清.出口肉及肉制品中持久霉素残留量检验方法的研究[J].口岸卫生控制,1999(2):5-8.
[7]苏敏,李继安,邵雷,等.恩拉霉素与雷莫拉宁生物合成研究进展[J].中国抗生素杂志,2011,36(6):401-405
[8]汝晓飞,张伟,王兴,等.马波沙星人工抗原的合成及其免疫原性的鉴定[J].畜牧与兽医,2018,50(4):66-71.
[9]张天杰,李岩松,孟星宇,等.荧蒽完全抗原的合成及其免疫效果评价[J].吉林农业大学学报,2015,37(4):424-429.
[10]陈宇熹,方振霞,雷美玲,等.黄曲霉毒素B1人工抗原的制备、鉴定与免疫特性研究[J].武夷科学,2017,33(1):80-87.
[11]王亚楠,王晓斐,牛琳琳,等.G族黄曲霉毒素半抗原分子设计、抗原合成及抗体特性[J].西北农业学报,2018,27(5):626-633.
[12]王重庆.分子免疫学基础[M].北京:北京大学出版社,1997.
[13]洪孝庄,孙曼霁.蛋白质连接技术[M].北京:中国医药科学技术出版社,1993.
[14]刘媛媛,陈奇娜,徐光辉,等.邻苯二甲酸酯类半抗原的设计合成与抗原制备[J].精细化工,2018,35(3):534-540.
[15]陈继明,龚晓明,陆承平.T细胞表位研究进展[J].中华微生物学与免疫学杂志,1998,18(1):78-82.
[16]GB/T 22262-2008,饲料中氯羟吡啶的测定高效液相色谱法[S].
[17]赵朋玲,郑海涛,姜盼盼,等.两种方法制备氟苯尼考人工抗原及其鉴定[J].食品科学,2010,31(15):225-230.
[18]郉云瑞,姚静静,邢广旭,等.环丙氨嗪完全抗原的合成及免疫原性鉴定[J].食品安全质量检测学报,2018,9(1):68-73.
[19]张岩蔚,金艳丹,栗慧,等.氨苄西林人工抗原的合成及鼠源多克隆抗体的制备[J].黑龙江畜牧兽医,2018(1):227-229.
[20]张春辉,邢广旭,胡骁飞,等.氟苯尼考琥珀酸钠抗原的制备与鉴定[J].中兽医医药杂志,2017,36(6):17-20.
[21]EILANGE B F.The preparation of antigenic hapten-carrier conjugates:a survey[J].Methods in enzymology,1980,70(A):85-104.
[22]徐志康,朱凌燕,徐又一.基质辅助激光解吸与离子化时间飞行质谱在高分子研究中的应用[J].分析化学,1999,27(2):224-229.
[23]刘益华,徐俊福.基质辅助激光解吸飞行时间质谱在生物大分子中的应用[J].中国生化药物杂志,2004,25(3):192-193.
[24]胡骁飞,邓歌,柴书军,等.玉米赤霉醇完全抗原的制备及质量鉴定[J].西北农业学报,2016,25(5):637-645.
[25]孙凤霞,康立超,庞芳琴,等.克百威人工抗原合成及其免疫应用[J].西北农业学报,2018,27(1):131-137.