Preparation and Application of Specificity Phosphohistidine Antibody†

doi:10.7503/cjcu20200078

Abstract

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

CLC Number:

O621.4

TrendMD:

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

Figures/Tables 9

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

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

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

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