Study on β-Chitosan against the binding of SARS-CoV-2S-RBD/ACE2

SARS-CoV-2 invades human respiratory epithelial cells via an interaction between its spike RBD protein (SARS-CoV-2 S-RBD) and the host cell receptor angiotensin converting enzyme II (ACE2). Blocking this interaction provides a potent approach to preventing and controlling SARS-CoV-2 infection. In this work, the ability of {beta}-chitosan to block the binding interaction between SARS-CoV-2 S-RBD and ACE2 was investigated. The inhibitory effect of {beta}-chitosan on inflammation induced by the SARS-CoV-2 S-RBD was also studied. Native-PAGE analysis indicated that {beta}-chitosan could bind with ACE2 and the SARS-CoV-2 S-RBD and a conjugate of {beta}-chitosan and ACE2 could no longer bind with the SARS-CoV-2 S-RBD. HPLC analysis suggested that a conjugate of {beta}-chitosan and the SARS-CoV-2 S-RBD displayed high binding affinity without dissociation under high pressure (40 MPa) compared with that of {beta}-chitosan and ACE2. Furthermore, immunofluorescent staining of Vero E6 cells and lungs from hACE2 mice showed that the presence of {beta}-chitosan prevented SARS-CoV-2 S-RBD from binding to ACE2. Meanwhile, {beta}-chitosan could dramatically suppress the inflammation caused by the presence of the SARS-CoV-2 S-RBD both in vitro and vivo. Moreover, the decreased expression of ACE2 caused by {beta}-chitosan treatment was restored by addition of TAPI-1, an inhibitor of the transmembrane protease ADAM17. Our findings demonstrated that {beta}-chitosan displays an antibody-like function capable of neutralizing the SARS-CoV-2 S-RBD and effectively preventing the binding of the SARS-CoV-2 S-RBD to ACE2. Moreover, ADAM17 activation induced by {beta}-chitosan treatment can enhance the cleavage of the extracellular domain of ACE2, releasing the active ectodomain into the extracellular environment, which can prevent the binding, internalization, and degradation of ACE2 bound to the SARS-CoV-2 S-RBD and thus diminish inflammation. Our study provides an alternative avenue for preventing SARS-CoV-2 infection using {beta}-chitosan.

Construction of the Expression Vector of Viruslike Particles Containing FMDV IRES RNA / 中国生物工程杂志

The Coat protein and Maturase gene of E.coli bacteriophage MS2 was amplified by PCR,then the gene was cloned into pET32a to construct the intermediate vector pET32aCP.The conservative sequence of FMDV internal ribosome entry site(IRES) was cloned into the downstream of pET32aCP bacteriophage gene to construct the prokaryotic expression vector pCPES.The recombinant plasmid pCPES transformed into E.coli strain BL21(DE3) was induced to express with 1mmol/L IPTG.The expression products were purified by sucrose density gradient centrifugation.The expression products observed by TEM were circular viruslike particles,and the diameter of these particles was about 26nm.The stability of viruslike particles was detected,and the viruslike particles was identified by RTPCR.The results showed that the viruslike particles contain the FMDV IRES RNA and have good stability.The viruslike particles have great prospect as the standard and quality control in the area of RNA virus detection.

Cloning and Expression of Vibrio cholerae CTB Gene and the Recombinant CTB Protein Activation Assay / 中国生物工程杂志

CTB protein possessed mucosal adjuvant immunoactivity. The CTB gene was amplified by PCR method from a strain V. cholerae. The nucleotide sequence of CTB gene was 375 bp and shared 96.0%~99.2% homology with other 6 CTB genes. The recombinant plasmid pTWIN1-CTB transformed E. coli strain BL21(DE3) expressed with 0.8 mmol/L IPTG. The molecular weight of expression products was identical with expectative weight by SDS-PAGE electrophoresis. The CTB fusion proteins mainly assembled inclusion bodies and the outputs of proteins were approximately 20% of the total bacterial proteins. The CTB proteins possessed mucosal immunoactivity by GM1-ELISA assay.