scholarly journals A Dual Target-Directed Single Domain-Based Fusion Protein Against Interleukin-6 Receptor Decelerate Experimental Arthritis Progression Via Modulating JNK Expression

2021 ◽  
Author(s):  
Xiaole Chen ◽  
Yize Bian ◽  
Yongqing Xie ◽  
Ningning Zheng ◽  
Kaimei Nie ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Interleukin 6 ◽  
Expression System ◽  
Single Domain ◽  
No Production ◽  
Dependent Manner ◽  
Drug Study ◽  
Prokaryotic Expression System ◽  
Interleukin 6 Receptor ◽  
Dual Target

Abstract The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signalling that cause collateral damage to protective signalling cascades. The single domain chain firstly discovered in Camelidae displays fully functional ability in antigen-binding against variable targets, which has been seemed as attractive candidates for the next-generation biologic drug study. In this study, we established a simple prokaryotic expression system for a dual target-directed single domain-based Fusion Protein against the interleukin-6 receptor and human serum, albumin, the recombinant anti-IL-6R fusion protein (VHH-0031). VHH-0031 exhibited potent anti-inflammatory effects produced by LPS on cell RAW264.7, where the major cytokines and NO production were down-regulated after 24 hr incubation with VHH-0031 in a dose-dependent manner. In vivo, VHH-0031 presented significant effects on the degree reduction of joint swelling in the adjuvant-induced arthritis (AIA) rat, having a healthier appearance compared to the dexamethasone. The expression level of JNK protein in the VHH-0031 group was significantly decreased, demonstrating that VHH-0031 provides a low-cost and desirable effect in the treatment of more widely patients.

scholarly journals The recombinant anti-TNF-α fusion protein ameliorates rheumatoid arthritis by the protective role of autophagy

2020 ◽  
Vol 40 (9) ◽  
Author(s):  
Xiaole Chen ◽  
Kaimei Nie ◽  
Xin Zhang ◽  
Shuangyu Tan ◽  
Qingmei Zheng ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Fusion Protein ◽  
Prokaryotic Expression ◽  
High Efficiency ◽  
Expression System ◽  
Inflammatory Factors ◽  
Cytokine Signaling ◽  
Drug Study ◽  
Tnf Α ◽  
Prokaryotic Expression System

Abstract The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signaling that cause collateral damage to protective signaling cascades carrying the potential for unwanted side effects. The variable domains of heavy-chain only antibodies (HCAbs) discovered in Camelidae are stable and display to be fully functional in antigen-binding against variable targets, which seem to be attractive candidates for the next-generation biologic drug study. The purpose of our study was to establish a simple prokaryotic expression system for large-scale expression, purification, and refolding of the recombinant anti-tumor necrosis factor α (TNF-α) fusion protein (FVH1-1) from inclusion bodies. Over 95% purity of the recombinant anti-TNF-α fusion proteins was obtained by just one purification step in our developed prokaryotic expression system, while the results of surface plasmon resonance (SPR) established the high-efficiency potent binding ability of FVH1-1 to human TNF-α. The counteraction of TNF-α cytotoxic effect experiment on the mouse fibroblast fibrosarcoma cell line (L929) confirmed that the expressed FVH1-1 were able to selectively and highly combine with human recombinant TNF-α (hTNF-α) in vitro. Western blot results showed that FVH1-1 can inhibit the activation of caspase-9 and PARP, which are the apoptotic signaling pathway proteins activated by hTNF-α. Meanwhile, lysosome autophagy signaling pathways stimulated by hTNF-α were inhibited by FVH1-1, which down-regulated the expression of LC3II/LC3I and up-regulated the expression of P62, indicating that the autophagy linked with TNF-α-induced apoptosis in response to rheumatoid arthritis. The results of the AIA rat model experiment presented that FVH1-1 can reduce the degree of joint swelling and inflammatory factors to a certain extent in vivo.

Fusion protein of interleukin-6 and interleukin-6 receptor without a polypeptide linker

2003 ◽  
Vol 96 (1) ◽  
pp. 38-46 ◽  
Author(s):  
Kiyoshi Yasukawa ◽  
Shigeo Tsuchiya ◽  
Teiji Ekida ◽  
Hiroshi Iida ◽  
Teruhiko Ide ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Interleukin 6 ◽  
Interleukin 6 Receptor

Expression of Recombinant Human Cytomegalovirus Fusion Proteins pp150-pp65 Fragments and its Application

2013 ◽  
Vol 634-638 ◽  
pp. 1313-1318
Author(s):  
Yu Fen Jin ◽  
Yan Lei Li ◽  
Yan Hua ◽  
Xiao Gang Zhang ◽  
Ting Yu
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Human Cytomegalovirus ◽  
Colloidal Gold ◽  
Fusion Proteins ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Serum Samples ◽  
Igg Antibody ◽  
Prokaryotic Expression System

Objective To evaluate the effectiveness of prokaryotic expression of fusion proteins pp150-pp65 of human cytomegalovirus (hCMV) for its application as antigen, the fusion protein of pp150-pp65 were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column for preparing the colloidal gold kit. Methods Using DNA from HCMV strain as template, the genes encoding pp150 and pp65 protein fragment were amplified by PCR technique, respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pET28a-pp150-pp65 was transformed into E.Coil.BL21(DE3) and induced to express with IPTG. The expressed fusion protein was characterized by SDS-PAGE and western blot after purified, then we used the purified fusion protein to develop combined detection kit of IgM/IgG antibody against HCMV (colloidal gold method) with Beijing Innovita Bio-tech Co., Ltd for detecting the samples, compared with the imported kits (ELISA). Results The gene of fusion fragment pp150-pp65 was correctly amplified and the recombinant vector was successfully constructed. The purified protein with the molecular weight of 45KD had good antigenicity by western blot. The protein was subjected to assay with an ELI SA capture kit in its specific and sensitive assay based on colloidal gold nanoparticles, testing of 600 serum samples indicated that this kit had a sensitivity of 92.7%;, specificity of 83.1%, crude consistency o f 90.2%, compared to the imported HCMV-IgG kit; the sensitivity of the kit was 88.1%, specificity was 89.2%, coarse consistency was 88.5%, compared to the imported HCMV-IgM kit; Conclusion In this experiment, the HCMV antigen with high purity and specificity (pp150-pp65 recombinant protein) was prepared effectively through genetic engineering technology. Compared to imported reagents, the colloidal gold kit consisting of fusion protein in a capture assay had high sensitivity and specificity. Preliminary clinical use warrants further development and use of this kit. Furthermore, it provides a technological basis for detection of HCMV in different stages of clinical infection.

scholarly journals Soluble interleukin-6 receptor triggers NO production in mouse macrophage lines induced by interleukin-6

1997 ◽  
Vol 75 ◽  
pp. 85
Author(s):  
Kuniaki Takagi ◽  
Hidetoshi Murao ◽  
Sanae Ikegaya ◽  
Junko Mine ◽  
Yasunobu Suketa
Keyword(s):  
Interleukin 6 ◽  
No Production ◽  
Mouse Macrophage ◽  
Interleukin 6 Receptor

scholarly journals Soluble interleukin-6 receptor activates the human papillomavirus type 18 long control region in SW756 cervical carcinoma cells in a STAT3-dependent manner

2001 ◽  
Vol 82 (10) ◽  
pp. 2335-2339 ◽  
Author(s):  
Sigrun Smola-Hess ◽  
Ute Sandaradura de Silva ◽  
Dirk Hadaschik ◽  
Herbert J. Pfister
Keyword(s):  
Human Papillomavirus ◽  
Control Region ◽  
Cervical Carcinoma ◽  
Interleukin 6 ◽  
Carcinoma Cells ◽  
Dominant Negative ◽  
Soluble Form ◽  
Long Control Region ◽  
Dependent Manner ◽  
Interleukin 6 Receptor

Cervical carcinoma cells producing high levels of interleukin-6 (IL-6) were shown to be unresponsive to the cytokine IL-6 due to the loss of their IL-6 receptor. Addition of IL-6 receptor in a soluble form restores IL-6 signalling in SW756 carcinoma cells. This leads to a rapid and strong activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). Nuclear factor IL-6 (NF-IL6, C/EBPβ) was induced only as a late event. While C/EBPβ significantly repressed the human papillomavirus type 18 long control region (HPV18-LCR), IL-6 signalling unexpectedly activated the HPV18-LCR in these cells. This IL-6 receptor-mediated induction could be completely reverted by transfection of a dominant-negative STAT3 but not STAT1 expression construct, indicating that STAT3 might play an important role in HPV18 oncogene promoter activation.

scholarly journals Recombinant Toxoplasma gondii Ribosomal Protein P2 Modulates the Functions of Murine Macrophages In Vitro and Provides Immunity against Acute Toxoplasmosis In Vivo

Vaccines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 357
Author(s):  
Zhengqing Yu ◽  
Yujia Lu ◽  
Zhaoyi Liu ◽  
Muhammad Tahir Aleem ◽  
Junlong Liu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Ribosomal Protein ◽  
Survival Time ◽  
Expression System ◽  
Prolonged Survival ◽  
No Production ◽  
Murine Macrophages ◽  
Prokaryotic Expression System ◽  
Acute Toxoplasmosis

Almost every warm-blooded animal can be an intermediate host for Toxoplasma gondii (T. gondii); there is still no efficient vaccine and medicine available for T. gondii infections. Detected on the surface of free tachyzoites of T. gondii, T. gondii ribosomal protein P2 (TgRPP2) has been identified as a target for protection against toxoplasmosis. In the present study, TgRPP2 was firstly expressed in a prokaryotic expression system, and the purified recombinant TgRPP2 (rTgRPP2) was characterized by its modulation effects on murine macrophages. Then, the purified rTgRPP2 was injected into mice to evaluate the immune protection of rTgRPP2. The results indicated that rTgRPP2 could bind to murine Ana-1 cells and showed good reactogenicity. After incubation with purified rTgRPP2, the proliferation, apoptosis, phagocytosis, nitric oxide (NO) production, and cytokines secreted by murine macrophages were modulated. Furthermore, the in vivo experiments indicated that animals immunized with rTgRPP2 could generate a significantly high level of antibodies, cytokines, and major histocompatibility complex (MHC) molecules, leading to a prolonged survival time. All of the results indicated that murine macrophages could be regulated by rTgRPP2 and are essential for the maintenance of tissue homeostasis. Immunization with rTgRPP2 triggered significant protection, with prolonged survival time in a mice model of acute toxoplasmosis. Our results lend credibility to the idea that rTgRPP2 could be a potential target for drug design and vaccine development.

Blockage of IL-6 Mediated JAK2/STAT3 Cell Signaling Pathway by sIL-6R Effectively Inhibited the Growth of Multiple Myeloma Cell.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5083-5083
Author(s):  
Ju-won Park ◽  
Kwang-Sung Ahn ◽  
Young-Ju Kim ◽  
Eunkyung Bae ◽  
Yong-Yeol Lee ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Interleukin 6 ◽  
Treatment Strategies ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Multiple Myeloma Cell ◽  
Survival Pathways ◽  
Comprehensive Knowledge ◽  
Interleukin 6 Receptor ◽  
Interfering Rna

Abstract Interleukin-6 (IL-6) is a key growth factor and thus plays a pivotal role in the pathogenesis of multiple myeloma. IL-6 is absolutely necessary for the cellular signalling cascade via JAK/STAT and RAS/MAPK pathways involved in proliferation and viability. We found interleukin (IL)-6 induced JAK-2 phosphorylation and induced proliferation of multiple myeloma cell line. Also phosphorylation of Stat1 and Stat3 was increased by IL-6 stimulation. Silencing JAK2 expression by small interfering RNA (siRNA) abrogated IL-6 induced phosphorylation of STAT3. In addition, soluble interleukin-6 receptor (sIL-6R) antagonized the function of hIL-6 and efficiently induced the growth arrest and apoptosis of U266 cells in a dose-dependent manner. Increasing levels of sIL-6R suppressed the phosphorylation of JAK-2 and Stat3. This suggests a comprehensive knowledge of the signaling and survival pathways could help find additional molecular targets and lead to the development of novel and more effective treatment strategies in multiple myeloma.

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