The Prokaryotic Expression and Activity Analysis of the Fifth Domain of β2GPⅠ and Its Mutants or Short Peptide Fragments

doi:10.13523/j.cb.20180801

China Biotechnology

2018, Vol. 38

Issue (8): 1-9 DOI: 10.13523/j.cb.20180801

The Prokaryotic Expression and Activity Analysis of the Fifth Domain of β2GPⅠ and Its Mutants or Short Peptide Fragments

Ming-ying LI,Ren-jun WANG,Fun ZHANG,Yan CHI(

)

School of Life Science and Technology, Dalian University,Key Laboratory of Glycolipid Metabolism, Dalian 116622, China

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Abstract

Beta 2-glycoprotein Ⅰ (β2GPⅠ)is the main antigen of antiphospholipid antibody (aPL) in serum of antiphospholipid syndrome (APS). β2GPⅠ binding to oxLDL via its the fifth domian and then subsequently recognized by aPL is a key event in the development of APS arterial thrombosis. In this study, a prokaryotic expression vector encoding β2GPⅠ fifth domain (β2GPⅠ-DⅤ), β2GPⅠ-DⅤ mutant and the Phe280-Ala320 fragment of β2GPⅠ-DⅤ were constructed, their induced expression and purification were performed, and the molecular mechanism of the binding of β2GPⅠ-DⅤ to negative phospholipids was analyzed. Results showed that the spatial configuration of the Cys281-Cys288 and Ser311-Lys317 segments in β2GPⅠ-DⅤ, which are maintained by the two disulfide bonds in C245-C296 and C288-C326, is a necessary precursor condition for the binding to CL. On this basis, the binding activity of rDⅤ to oxLDL and oxLDL in the serum of APS were further examined, indicating that rDⅤ has biological activity consistent with that of natural β2GPⅠ. The obtainment of rβ2GPⅠ-DⅤ and the establishment of the method in rβ2GPⅠ-DⅤ binding to oxLDL in this study lay a foundation for the early laboratory diagnosis of APS.

Key words： Antiphospholipid syndrome Beta 2-glycoprotein Ⅰ Cardiolipin Oxidized low density protein Prokaryotic expression

Received: 23 March 2018 Published: 11 September 2018

ZTFLH:

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Corresponding Authors: Yan CHI E-mail: chi_yan@126.com

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Ming-ying LI,Ren-jun WANG,Fun ZHANG,Yan CHI. The Prokaryotic Expression and Activity Analysis of the Fifth Domain of β2GPⅠ and Its Mutants or Short Peptide Fragments. China Biotechnology, 2018, 38(8): 1-9.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180801 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I8/1

Fig.1 PCR amplification of DⅤ、mDⅤ and cDⅤ (a) PCR product of DⅤ.M:DNA Marker;1:PCR product of DⅤ (287bp) (b) PCR product of mDⅤ.M:DNA Marker;1:PCR product of mDⅤ(287bp) (c) PCR product of cDⅤ.M:DNA Marker;1:PCR product of cDⅤ(120bp)

Fig.2 Identification of recombinant vectors by restriction enzyme digestion (a) Identification of pET32a-DⅤ by restriction enzyme digestion.M:DNA Marker; 1: pET32a-DⅤ digested with EcoR I and KpnI (b) Identification of pET32a-mDⅤ by restriction enzyme digestion.M:DNA Marker; 1:pET32a-mDⅤ digested with NcoI and BamHI (c) Identification of pET32a-cDⅤ by restriction enzyme digestion.M:DNA Marker; 1: pET32a-cDⅤ digested with NcoI and BamHI

Fig.3 Expression of rDⅤ, rmDⅤ and rcDⅤ (a) Expression of rDⅤ. M:marker;1:without induction;2:induction (b) Expression of rmDⅤ. M:marker;1:control; 2:without induction;3:induction (c) Expression of rcDⅤ. M:marker;1:without induction;2:induction

Fig.4 Purification of rDⅤ, mDⅤ and cDⅤ (a) Purification of rDⅤ. M:marker;1:Purification of rDⅤ (b) Purification of rmDⅤ. M:marker;1:Purification of rmDⅤ (c) Purification of rcDⅤ. M:marker;1:total protein; 2:Purification of rcDⅤ

Fig.5 Analysis of rDⅤ, rmDⅤ and rcDⅤ by Western blot (a) Analysis of rDⅤ by Western blot (b) Analysis of rmDⅤ by Western blot (c) Analysis of rcDⅤ by Western blot

Fig.6 Analysis of rDⅤ,rmDⅤ and rcDⅤ binding with CL by HPTLC and ELISA (a) CL (50 μg each plate) was spotted on HPTLC plate, developed with solution of chloroform/methanol (8∶1, v/v), incubated with recombinant proteins and its related antibodies(WB-CAL-I for nβ2GPⅠ, anti-His Antibodies for rDⅤ, rmDⅤ and rcDⅤ) (b) CL (50 μg each plate) was coated, incubated with nβ2GPⅠ, rDⅤ, rmDⅤ or rcDⅤ. The plates were probed by WB-CAL-1(for nβ2GPⅠ) and anti-His antibodies (for rDⅤ, rmDⅤ and rcDⅤ), while blank is without the primary antibody. Observation wavelength in 492 nm

Table 1 Analysis of rDⅤ,rmDⅤ and rcDⅤ binding with CL by ELISA

Fig.7 The binding activity of rβ2GPⅠ-DⅤ to oxLDL and oxLDL in APS serum (a) Lane 1: incubated with natural nβ2GPⅠ and oxLDL oxidized by Cu²⁺; Lane 2: incubated with rβ2GPⅠ-DⅤ and oxLDL oxidized by Cu²⁺; Lane 3: incubated with rβ2GPⅠ-DⅤ and LDL;Lane 4: negative control (b) The rβ2GPⅠ-DⅤ/the nβ2GPⅠ(50 μg/ml,50 μl) were coated, incubated with the serum from the patients with APS (100-fold diluted). The plates were probed by HRP-labeled anti-apoB-100 Ab, while control is without the the serum from the patients ,Observation wavelength in 492 nm

Table 2 Analysis of rβ2GPⅠ-DⅤ binding with oxLDL by ELISA

Table 3 Analysis of rβ2GPⅠ-DⅤ binding with oxLDL in APS serum by ELISA


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