磷酰化组氨酸特异性抗体的制备与应用

doi:10.7503/cjcu20200078

摘要/Abstract

摘要：

以4-碘吡唑和亚磷酸二乙酯为初始原料, 经3步反应合成了新型的磷酰化组氨酸结构类似物, 并以其为半抗原制备了磷酰化组氨酸特异性抗体(Anti-pHis). 采用酶联免疫吸附(ELISA)、斑点印迹(Dot blot)和免疫印迹(Western blot)实验等方法对Anti-pHis的特异性进行了检测. 结果表明, Anti-pHis对组氨酸磷酰化蛋白表现出优异的亲和力和选择性识别能力, 并可应用于微生物细胞裂解液中组氨酸磷酰化蛋白的检测.

关键词: 半抗原, 磷酰化组氨酸抗体, 特异性, 亲和力

Abstract:

A novel phosphorylated histidine analogue was synthesized using 4-iodopyrazole and diethyl phosphite as raw materials and characterized by nuclear magnetic resonance spectroscopy(NMR). It was used as hapten to prepare phosphohistidine antibody(Anti-pHis). Enzyme-linked immunosorbent assay, dot blot and western blot methods were used to detect the specificity and sensitivity of the Anti-pHis. The results showed that the Anti-pHis had excellent avidity and sensitivity for histidine phosphorylated protein, and the Anti-pHis was successfully applied to the detection of histidine phosphorylated protein in the lysate of microbial cells.

Key words: Hapten, Phosphohistidine antibody, Specificity, Avidity

中图分类号:

O621.4

TrendMD:

李容庆,刘佳璐,范若辰,白鹏阳,张丽影,权春善. 磷酰化组氨酸特异性抗体的制备与应用. 高等学校化学学报, 2020, 41(7): 1552.

LI Rongqing,LIU Jialu,FAN Ruochen,BAI Pengyang,ZHANG Liying,QUAN Chunshan. Preparation and Application of Specificity Phosphohistidine Antibody^†. Chem. J. Chinese Universities, 2020, 41(7): 1552.

图/表 9

Scheme 1 Synthetic routes of Hap

Table 1 Appearance, yields, melting points, elemental analysis, MS and IR data for compounds 1, 2 and Hap

Compd.	Appearance	Yield^*(%)	m. p./℃	Elemental analysis(%, calcd.)			MS, m/z [M+H]⁺	IR(KBr), $ν ˙$ /cm^-1
Compd.	Appearance	Yield^*(%)	m. p./℃	C	H	N	MS, m/z [M+H]⁺	IR(KBr), $ν ˙$ /cm^-1
1	White solid	89	69.7—70.3	41.09(41.18)	6.37(6.42)	13.77(13.72)	204.95	2962, 2923, 2852, 1658, 1619, 1439
2	White solid	92	227.4—228.1	45.19(45.14)	6.88(6.94)	13.22(13.16)	320.10	3356, 3119, 3053, 2978, 2932, 2871, 1704, 1511, 1430
Hap	White solid	86	179.2—179.9	38.42(38.36)	6.38(6.44)	19.13(19.17)	234.00	3250, 3090, 2976, 2904, 2811, 1643, 1602, 1483

Table 1 Appearance, yields, melting points, elemental analysis, MS and IR data for compounds 1, 2 and Hap

Compd.	Appearance	Yield^*(%)	m. p./℃	Elemental analysis(%, calcd.)			MS, m/z [M+H]⁺	IR(KBr), $ν ˙$ /cm^-1
Compd.	Appearance	Yield^*(%)	m. p./℃	C	H	N	MS, m/z [M+H]⁺	IR(KBr), $ν ˙$ /cm^-1
1	White solid	89	69.7—70.3	41.09(41.18)	6.37(6.42)	13.77(13.72)	204.95	2962, 2923, 2852, 1658, 1619, 1439
2	White solid	92	227.4—228.1	45.19(45.14)	6.88(6.94)	13.22(13.16)	320.10	3356, 3119, 3053, 2978, 2932, 2871, 1704, 1511, 1430
Hap	White solid	86	179.2—179.9	38.42(38.36)	6.38(6.44)	19.13(19.17)	234.00	3250, 3090, 2976, 2904, 2811, 1643, 1602, 1483

Table 2 1H NMR and 13C NMR data for compounds 1, 2 and Hap

Compd.	¹H NMR(400 MHz), δ^a	¹³C NMR(100 MHz), δ^b
1	7.54(s, 1H, Pyrazole CH), 7.46(s, 1H, Pyrazole CH), 3.54(s, 1H, NH), 1.25(s, 3H, Alkyl CH₃), 0.88(s, 2H, Alkyl CH₂)	138.21, 106.28, 77.09, 16.32
2	7.59(s, 1H,Pyrazole CH), 7.50(s, 1H, Pyrazole CH), 4.48(s, 1H, NH), 3.23(t, J=3.8 Hz, 2H, Alkyl CH₂), 3.14(t, J=4.0 Hz, 2H, Alkyl CH₂), 1.83(q, J=7.0 Hz, 2H, Alkyl CH₂), 1.60(q, J=6.8 Hz, 2H, Alkyl CH₂), 1.43[s, 9H, Alkyl (CH₃)₃], 1.29(q, J=6.8 Hz, 2H, Alkyl CH₂)
Hap	7.78(s, 1H,Pyrazole CH), 7.63(s, 1H, Pyrazole CH), 4.19(t, J=6.7 Hz, 2H, Alkyl CH₂), 2.95(t, J=7.6 Hz, 2H, Alkyl CH₂), 1.85(p, J=6.9 Hz, 2H, Alkyl CH₂), 1.64(p, J=7.7 Hz, 2H, Alkyl CH₂), 1.28(p, J=7.9 Hz, 2H, Alkyl CH₂)	144.22, 136.15, 55.87, 51.93, 39.53, 29.10, 26.43, 22.82

Fig.1 1H NMR(A) and 13C NMR(B) spectra of Hap

Fig.2 Absorption spectra of complete antigen BSA-Hap(A) and coating antigen OVA-Hap(B)

Fig.3 SDS PAGE results of complete antigen BSA-Hap and coating antigen OVA-Hap M: marker; lane 1: BSA; lane 2: BSA-Hap; lane 3: OVA; lane 4: OVA-Hap.

Fig.4 ELISA results of antiserum λ=450 nm, negative: preimmune serum, blank: buffer solution. a. Negative; b. 1:2000; c. 1:4000; d. 1:8000; e. 15000; f. 1:30000; g. 1:60000; h. 1:70000; i. 1:80000; j. 1:90000; k. 1:100000; l. 1:120000; m. 1:140000; n. blank.

Fig.5 Detection of histidine phosphorylated proteins by Anti-pHis

Fig.6 Changes of histidine phosphorylation protein of Bacillus amyloliquefaciens cultured at different pH conditions

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