Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application

Zhou, Yaping; Cao, Limin; Ding, Cuijun; Ji, Yun; Jiang, Qinbo; Liu, Xiping; Li, Xiang; Hou, Xueling; Peng, Hongjun; Shi, Weifeng

doi:10.1590/s1517-83822014005000016

Cited by 7 publications

(4 citation statements)

References 34 publications

(36 reference statements)

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“…However, the negative serum of monkey had weak cross-reaction with the recombinant protein ( Figure 6 C). In the negative control, empty vector pET-32a (+) could not be induced to express the gD protein; it did not have a specific band in the western blot analysis ( 34 ).…”

Section: Resultsmentioning

confidence: 99%

Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli, and Production of Its Monoclonal Antibodies

Jin

Sun

Xia

et al. 2016

Jundishapur J Microbiol

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BackgroundHerpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus.ObjectivesThis study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research.Materials and MethodsThe gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid.ResultsThe optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass.ConclusionsThe cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.

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Section: Resultsmentioning

confidence: 99%

Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli, and Production of Its Monoclonal Antibodies

Jin

Sun

Xia

et al. 2016

Jundishapur J Microbiol

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“…As presented in Fig. 2, VP1-overexpressing mouse Schwann cells exhibited characteristic features of autophagy, including swelling mitochondria, dilation and degranulation of rough ER and vesicle-like dilation of Golgi (40). By contrast, the organelles in untransfected and empty vector-transfected control mouse Schwann cells were morphologically normal (Fig.…”

Section: Overexpression Of Vp1 Activates Mouse Schwann Cell Autophagymentioning

confidence: 88%

Enterovirus 71 VP1 promotes mouse Schwann cell autophagy via ER stress‑mediated PMP22 upregulation

Yang

et al. 2019

Int J Mol Med

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Enterovirus 71 (EV71) accounts for the majority of hand, foot and mouth disease-related deaths due to fatal neurological complications. EV71 structural viral protein 1 (VP1) promotes viral replication by inducing autophagy in neuron cells, but the effect of VP1 on myelin cells is unclear. The present study aimed to investigate the role and mechanism of VP1 in autophagy of mouse Schwann cells. An EV71 VP1-expressing vector (pEGFP-c3-VP1) was generated and transfected into mouse Schwann cells. Transmission electron microscopy and western blot analysis for microtubule-associated protein 1 light chain 3 α (Lc3) II (an autophagy marker) were used to assess autophagy. Reverse transcription-quantitative PcR and immunofluorescence were performed to determine the expression of peripheral myelin protein 22 (PMP22). Small interfering RNA against PMP22 was used to investigate the role of PMP22 in mouse Schwann cell autophagy. Salubrinal [a selective endoplasmic reticulum (ER) stress inhibitor] was used to determine whether PMP22 expression was affected by ER stress. The present results indicated that VP1 promoted mouse Schwann cell autophagy. Overexpression of VP1 upregulated PMP22. PMP22 deficiency downregulated LC3II and thus inhibited autophagy. Furthermore, PMP22 expression was significantly suppressed by salubrinal. In conclusion, VP1 promoted mouse Schwann cell autophagy through upregulation of ER stress-mediated PMP22 expression. Therefore, the VP1/ER stress/PMP22 autophagy axis may be a potential therapeutic target for EV71 infection-induced fatal neuronal damage.

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“…EV71 carries four structural proteins: VP1, VP2, VP3, and VP4. VP1 homodimers are the main component of the icosahedral viral capsid, which contributes to the pathogenicity and stability of EV71 and allows it to survive in the gastrointestinal tract (27,28). Our previous study found that VP1 overexpression induced autophagy in Schwann cells in mice.…”

Section: Discussionmentioning

confidence: 99%

Enterovirus 71 structural viral protein 1 promotes autophagy by inducing an m6A-mediated PMP22 overexpression in mouse Schwann cells

Zhu

et al. 2021

Preprint

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Objective Enterovirus 71 (EV71), one of the enteroviruses responsible for the hand, foot and mouth disease (HFMD), can cause severe neurologic diseases such as brainstem encephalitis and demyelination. The molecular mechanism of demyelination is still not fully understood. This study aims to investigate the mechanism of how the EV71 structural viral protein 1, VP1 can act on host cellular pathways in mouse Schwann cells. Methods An EV71 VP1-expressing vector was generated and transfected into mouse Schwann cells (MSCs). Selective mRNA methylation inhibitor (DAA) was employed to identify key members of m6A pathway that are targeted by VP1. To investigate the role of METTL14 and YTHDF1 in PMP22 expression, small interfering RNA against METTL14 and YTHDF1 was employed to knockdown the METTL14 and YTHDF1 expression in MSCs. Real-time PCR and Western blot analysis were performed to determine the expression of PMP22 and m6A modification-associated proteins. Results Our results demonstrated VP1 upregulated m6A pathway by targeting METTL14 and YTHDF1. The expression of PMP22 was decreased by inhibiting the expression of METTL14 and YTHDF1. Conclusions VP1 upregulates m6A modification, which in turn causes the hypermethylation of PMP22 in MSCs, resulting in a higher level of PMP22. Ultimately, this VP1-induced PMP22 overexpression leads to MSC autophagy.

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Expression of enterovirus 71 capsid protein VP1 in Escherichia coli and its clinical application

Cited by 7 publications

References 34 publications

Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli, and Production of Its Monoclonal Antibodies

Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli, and Production of Its Monoclonal Antibodies

Enterovirus 71 VP1 promotes mouse Schwann cell autophagy via ER stress‑mediated PMP22 upregulation

Enterovirus 71 structural viral protein 1 promotes autophagy by inducing an m6A-mediated PMP22 overexpression in mouse Schwann cells

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