“…We conjectured that EV71S1 might recognize a protein on RD cell which is important for EV71 infection, such as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1, which molecular weight without transmembrane domain is approximately 45 kDa, is the receptor on cells for EV71 infection [ 52 , 53 ]. The EV71S1 might bind to the PSGL-1 to inhibit EV71 virus infection.…”
Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.
“…We conjectured that EV71S1 might recognize a protein on RD cell which is important for EV71 infection, such as P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1, which molecular weight without transmembrane domain is approximately 45 kDa, is the receptor on cells for EV71 infection [ 52 , 53 ]. The EV71S1 might bind to the PSGL-1 to inhibit EV71 virus infection.…”
Enterovirus 71 (EV71) is the major etiological agent contributing to the development of hand-foot-mouth disease (HFMD). There are not any global available vaccines or antibody drugs against EV71 released yet. In this study, we perform the virus immunization in a cost-effective and convenient approach by preparing virus particles from size exclusion and immunization of chicken. Polyclonal yolk-immunoglobulin (IgY) was simply purified from egg yolk and monoclonal single-chain variable fragments (scFv) were selected via phage display technology with two scFv libraries containing 6.0 × 106 and 1.3 × 107 transformants. Specific clones were enriched after 5 rounds of bio-panning and four identical genes were classified after the sequence analysis. Moreover, the higher mutation rates were revealed in the CDR regions, especially in the CDR3. IgY showed specific binding activities to both EV71-infected and Coxsackievirus 16-infected cell lysates and high infectivity inhibitory activity of EV71. However, while IgY detected a 37 kDa protein, the selected scFv seemingly detected higher size proteins which could be cell protein instead of EV71 proteins. Despite the highly effective chicken antibody generation, the purity of virus particles prepared by size exclusion is the limitation of this study, and further characterization should be carried out rigorously.
“…PCR products and pGEX-5X1 plasmid were treated with double enzymes to create compatible sticky ends and then ligated using T4 ligase (Thermo Scientific). After transforming the ligated products into competent E. coli DH5α cells via heat-shock method, bacterial transformations were spread onto a LB agar plate containing 100 μg/ml ampicillin [ 20 ]. The target plasmids were confirmed by PCR cloning and sequencing.…”
Section: Methodsmentioning
confidence: 99%
“…Hsp60 gene was cut by Xba I and Xho I (Thermo Scientific) and then cloned into predigested pET22b plasmid with compatible sticky ends. The ligation was also delivered into competent E. coli DH5α cells via chemical transformation and screened by selective medium, LB agar containing 100 ng/μl ampicillin [ 20 ]. The growth colonies were confirmed via PCR colony and sequencing.…”
The distinctive morphology characteristics of microfold cells (M cells) allow the vaccine antigen not only to interact with immune cells directly, but also to effectively stimulate mucosal immune responses via receptors on its apical surface. Human prion protein, a transmembrane receptor for
Brucella abortus
Hsp60, is highly expressed on the M cell surface. Nonetheless, this protein tends to express in inclusion body in prokaryotic hosts. In this study, the shorter interacting regions of human prion protein were identified via computational methods such as docking and molecular dynamics simulations to minimize its aggregation tendency. The computational calculations revealed three novel human prion protein-interacting regions, namely PrP125, PrP174, and PrP180. In accordance with in silico prediction, the biologically synthesized peptides fusing with GST tag demonstrated their specific binding to Hsp60 protein via pull-down assay. Hence, this finding laid the groundwork for M-cell targeting candidate validation through these newly identified interacting regions.
“…Vector pET22b-scrb2-IIIx3 sau khi cấu trúc thành công được thu nhận và biến nạp vào tế bào E. coli BL21(DE3) để biểu hiện theo quy trình của Seidmoradei với một số điều chỉnh (Vo-Nguyen et al, 2021). Các khuẩn lạc E. coli BL21(DE3) sau khi được xác nhận mang vector, được hoạt hóa trong môi trường LB-amp lỏng và nuôi cấy lắc ở 37 o C trong 16 giờ.…”
Section: Biểu Hiện Protein Scrb2-iiix3unclassified
“…Việc tái gấp cuộn protein SCRB2-IIIx3 được thực hiện như mô tả với một số điều chỉnh (Vo-Nguyen et al, 2021). Dịch môi trường nuôi vi khuẩn E. coli BL21(DE3) mang vector pET22b-scrb2-IIIx3 sau khi được cảm ứng biểu hiện, tiến hành phá bằng sóng siêu âm ở điều kiện lạnh trong dung dịch đệm ly giải (20 mM Tris -HCl pH 8,0, 1 M NaCl, 5 mM EDTA) với tỉ lệ sinh khối thu từ 50 ml dịch canh khuẩn hòa với 20 ml dịch ly giải.…”
Section: Tái Gấp Cuộn Và Tinh Sạch Protein Scrb2-iiix3unclassified
Enterovirus A71 (EV-A71) là tác nhân chính gây nên biến chứng nguy hiểm của bệnh Tay Chân Miệng, có thể dẫn đến thương tật, và tử vong ở trẻ nhỏ. Hiện nay, vaccine và hợp chất điều trị EV-A71 còn nhiều hạn chế, chưa được áp dụng rộng rãi. Vì thế, nhiều nghiên cứu tìm giải pháp thay thế hoặc bổ trợ đang được tiến hành, trong đó có bẫy virus. Với chức năng chính là bắt, cố định virus, ngăn sự xâm nhiễm, thể bám của bẫy cấu trúc từ thụ thể liên kết EV-A71, cụ thể là SCARB2. Trong nghiên cứu này, tiểu phần thụ thể tái tổ hợp SCARB2 dung hợp foldon peptide được dòng hóa, biểu hiện, và tinh sạch nhằm tạo thể bám ở dạng trimer hóa có ái lực cao với virus. Vector pET22b-scrb2-IIIx3 được cấu trúc trong E. coli DH5α, protein được biểu hiện trong E. coli BL21(DE3), và chỉ thu được cấu hình monomer, phần lớn ở dạng thể vùi. Việc tái gấp cuộn và tinh sạch protein được tiến hành nhằm đưa protein trở về dạng tan có hoạt tính sinh học. Những kết quả trên cung cấp thêm nhiều thông...
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