Cloning and Expression of HBV Infection Related Novel Gene C12orf49

Abstract Objective To clone, express and purify C12orf49 recombinant protein. To prepare rabbit anti-C12orf49 protein polyclonal antibody in order to further elucidate its biological function. Methods PCR was used to amplify the gene C12orf49 in vitro. pET-32a (+)-C12orf49, the recombinant protein prokaryotic expression vector, was transformed into E. coli. IPTG was used as the inductive agent to obtain C12orf49 recombinant protein, and the recombinant protein was analyzed with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Specific polyclonal antibody was derived from rabbits that immunized by recombinant protein. ELISA and Western blot were used to test its titer and specificity, respectively. MTT cell proliferation experiment was carried out to observe effect of the protein on proliferation of HepG2 cells. Results The C12orf49 recombinant protein was expressed in a large quantity. Data of ELISA indicated that the titer of polyclonal antibody was higher than 1:1 280 000. And the antibody also had a good specificity, confirmed by Western blot. C12orf49 recombinant protein may had a advanced effect on the proliferation of HepG2 cells. Conclusions Using C12orf49 recombinant protein, we can obtain the polyclonal antibody with great titer and good specificity. Human novel gene C12orf49 encoded protein could promote the proliferation of HepG2 cells.

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Identification of Human Hepatocyte Proliferation Related Gene C2orf69

Infection International ◽

10.1515/ii-2017-0066 ◽

2014 ◽

Vol 3 (1) ◽

pp. 1-9

Author(s):

Ren-wen Zhang ◽

Yong Qiao ◽

Xiao-hua Hao ◽

Hong-min Li ◽

Hui Ren ◽

...

Keyword(s):

Cell Proliferation ◽

Recombinant Protein ◽

Western Blot ◽

Polyclonal Antibody ◽

Prokaryotic Expression ◽

Liver Cells ◽

Hepatocyte Proliferation ◽

Enzyme Linked Immunosorbent Assay ◽

Gene Fragment

Abstract Objective To construct the prokaryotic expression vector pET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained. Methods Gene fragment of C2orf69 was amplified by PCR and then prokaryotic expression plasmid pET-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identified by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and specificity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully amplified, results of gene sequencing were consistent with the sequence in GenBank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

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Gluthathione S-transferase-resuscitation-promoting factor B recombinant protein of Mycobacterium tuberculosis induces the production of interferon-γ and interleukin-12 in mice splenocytes

Medical Journal of Indonesia ◽

10.13181/mji.v28i3.2444 ◽

2019 ◽

Vol 28 (3) ◽

pp. 234-40

Author(s):

Andriansjah Rukmana ◽

Burhanuddin Rasyid ◽

Fitriyah Sjatha

Keyword(s):

Mycobacterium Tuberculosis ◽

Recombinant Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Interferon Γ ◽

Interleukin 12 ◽

Potential Candidate ◽

Factor B ◽

Latent Tb

BACKGROUND As the only TB vaccine available, Bacillus Calmette-Guérin shows variable efficacy in adults and does not provide protection against the resuscitation of latent TB infections. Resuscitation-promoting factor B (RpfB) is a protein produced by Mycobacterium tuberculosis during the resuscitation phase and is promising as a novel TB vaccine. This study was aimed to analyze the immunogenicity of the gluthathione S-transferase (GST)-RpfB recombinant protein on mice splenocytes in vitro. METHODS After induction with isopropyl β-D-1-thiogalactopyranoside, the protein was extracted by sonication followed by solubilization in 8 M urea buffer. Protein was then re-natured and purified with a GST chromatography column. The isolated protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot using anti-GST antibodies, and its concentration was determined using the Bradford method. Each group of splenocytes was treated with 25 μg/ ml of the recombinant protein (GST-RpfB), GST, and phytohemagglutinin. Antigen induction was repeated twice at 24 and 72 hours. The supernatant was collected at 96 hours and interferon gamma (IFNγ), interleukin (IL-12, IL-4, and IL-10) levels were measured with enzyme-linked immunosorbent assays. RESULTS GST-RpfB recombinant proteins were expressed in the form of inclusion bodies with a molecular weight of approximately 66 kDa. Based on the independent t-test, GST-RpfB stimulated IFNγ and IL-12 production but not IL-4 and IL-10. CONCLUSIONS The GST-RpfB protein has been immunogenically proven and is a potential candidate as a novel subunit TB vaccine.

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In Vitro Antihepatoma Activity of Novel 3D-Copper Cyanide Supramolecular Coordination Polymers

Journal of AOAC International ◽

10.5740/jaoacint.16-0141 ◽

2016 ◽

Vol 99 (5) ◽

pp. 1240-1246

Author(s):

Noura M Darwish ◽

Ahmed S Sultan ◽

Ahmed M Malki ◽

Hossam Khamis ◽

Mohamed El-Ziady

Keyword(s):

Protein Expression ◽

Western Blot ◽

Coordination Polymers ◽

Hepg2 Cells ◽

Inhibitory Effect ◽

Dependent Manner ◽

Invasive Potential ◽

Dna Ladder ◽

Supramolecular Coordination

Abstract This study aimed to investigate the inhibitory effect of novel 3D-organocopper supramolecular coordination polymers (SCPs) on the invasive potential of HepG2 cells. Chemoprevention could represent an important means to inhibit the process of hepatocarcinogenesis. The inhibitory effect of an SCP compound on the proliferation of HepG2 hepatoma cells was evaluated by cell vibility assay. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of the SCP compound on phosphorylated ERK1/2, Bcl-2, and β-catenin protein expression of HepG2 cells was analyzed by Western blot. The SCP compound exerted an inhibitory effect on HepG2 cell proliferation in a dose-dependent manner. This inhibitory effect was confirmed by examination of cell morphology and DNA fragmentation. Furthermore, Western blot analysis revealed that phosphorylated ERK1/2 and β-catenin protein expression was inhibited after 24 h of treatment with the SCP compound, and that this event was associated with decreased Bcl-2 expression. We concluded that SCP can effectively inhibit the invasive potential of the ERK signaling pathway in HepG2 cells by altering apoptosis and by inhibiting Bcl-2 and β-catenin, which may play a significant role in this process.

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Effect of Tumor Suppressor MiR-34a Loaded on ZSM-5 Nanozeolite in Hepatocellular Carcinoma: In Vitro and In Vivo Approach

Current Gene Therapy ◽

10.2174/1566523219666191108103739 ◽

2019 ◽

Vol 19 (5) ◽

pp. 342-354 ◽

Cited By ~ 3

Author(s):

Zeinab Salah ◽

Eman M. Abd El Azeem ◽

Hanan F. Youssef ◽

Amira M. Gamal-Eldeen ◽

Abdel R. Farrag ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Tumor Suppressor ◽

Hepg2 Cells ◽

Cloning And Expression ◽

Great Promise ◽

Control Group ◽

Qrt Pcr ◽

Nano Formulation

Background: MicroRNA modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC), however Efficient tissue-specific and safe delivery remains a major challenge. Objective: We sought to develop an inorganic-organic hybrid vehicle for the systemic delivery of the tumor suppressor miR-34a, and to investigate the efficiency of the delivered miR-34a in the treatment of HCC in vitro and in vivo. Methods: In the present study, pEGP-miR cloning and expression vector, expressing miR-34a, was electrostatically bound to polyethyleneimine (PEI), and then loaded onto ZSM-5 zeolite nanoparticles (ZNP). Qualitative and quantitative assessment of the transfection efficiency of miR-34a construct in HepG2 cells was applied by GFP screening and qRT-PCR, respectively. The expression of miR-34a target genes was investigated by qRT-PCR in vitro and in vivo. Results: ZNP/PEI/miR-34a nano-formulation could efficiently deliver into HepG2 cells with low cytotoxicity, indicating good biocompatibility of generated nanozeolite. Furthermore, five injected doses of ZNP/PEI/miR-34a nano-formulation in HCC induced male Balb-c mice, significantly inhibited tumor growth, and demonstrated improved cell structure, in addition to a significant decrease in alphafetoprotein level and liver enzymes activities, as compared to the positive control group. Moreover, injected ZNP/PEI/miR-34a nano-formulation led to a noticeable decrease in the CD44 and c-Myc levels. Results also showed that ZNP/PEI/miR-34a nano-formulation inhibited several target oncogenes including AEG-1, and SOX-9, in vitro and in vivo. Conclusion: Our results suggested that miR-34a is a powerful candidate in HCC treatment and that AEG-1 and SOX-9 are novel oncotargets of miR-34a in HCC. Results also demonstrated that our nano-formulation may serve as a candidate approach for miR-34a restoration for HCC therapy, and generally for safe gene delivery.

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Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

Chinese Journal of Agricultural Biotechnology ◽

10.1079/cjb200565 ◽

2005 ◽

Vol 2 (2) ◽

pp. 113-117

Author(s):

Pan Li-Jing ◽

Li Zhao-Fei ◽

Yin Chong ◽

Lv Lei ◽

Pang Yi

Keyword(s):

Fusion Protein ◽

Prokaryotic Expression ◽

Recombinant Plasmid ◽

Nitrilotriacetic Acid ◽

Cloning And Expression ◽

Reading Frame ◽

Pcr Product ◽

Prokaryotic Expression Vector ◽

Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

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Production of Polyclonal Antibody against Interleukin-33 and Assessment of Its Distribution in Murine Liver and Lung

Journal of Biomedicine and Biotechnology ◽

10.1155/2009/729197 ◽

2009 ◽

Vol 2009 ◽

pp. 1-6 ◽

Cited By ~ 4

Author(s):

Xiaojin Liu ◽

Yan Wu ◽

Mingcai Li ◽

Sui Chen ◽

Yanchun Zhou

Keyword(s):

Recombinant Protein ◽

Affinity Chromatography ◽

Polyclonal Antibody ◽

Metal Ion ◽

High Titer ◽

Enzyme Linked Immunosorbent Assay ◽

Coding Region ◽

Good Tool ◽

Antibody Preparation ◽

Prokaryotic Expression Vector

Interleukin (IL)-33 is the latest member of IL-1 cytokine family. In this study, the cloning, expression, purification, and polyclonal antibody preparation of mouse IL-33 were described. The coding region of IL-33 mature protein was cloned into the prokaryotic expression vector pET-44. The recombinant protein, IL-33 containing a hexahistidine tag in the C-terminal, was expressed inEscherichia coli. The expressed soluble protein was purified by immobilized metal-ion affinity chromatography usingNi2+-nitrilotriacetic acid agarose. The rabbits were immunized with the purified recombinant protein. The obtained antiserum was precipitated by saturated ammonium sulfate and then purified by Protein A affinity chromatography. The sensitivity and specificity of the antibodies were evaluated by enzyme-linked immunosorbent assay and immunohistochemistry. The high titer (1:32000) polyclonal antibodies with high specificity were obtained by immunizing rabbits with the purified recombinant protein. Significant expression of IL-33 was seen in mouse liver and lung tissues determined with the anti-IL-33. The production of the polyclonal antibody against IL-33 provides a good tool for studying the biofunctions of IL-33.

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CD137 Signaling Promotes Endothelial Apoptosis by Inhibiting Nrf2 Pathway, and Upregulating NF-κB Pathway

Mediators of Inflammation ◽

10.1155/2020/4321912 ◽

2020 ◽

Vol 2020 ◽

pp. 1-15

Author(s):

Tianxin Geng ◽

Yang Yan ◽

Yue Zhang ◽

Liangjie Xu ◽

Guangyao Zang ◽

...

Keyword(s):

Endothelial Cells ◽

Recombinant Protein ◽

Western Blot ◽

Nuclear Translocation ◽

Umbilical Vein ◽

In Vivo Studies ◽

Control Group ◽

Nrf2 Pathway

Background. Endothelial dysfunction and apoptosis resulting from oxidative stress can lead to the development of atherosclerosis. Our group has previously showed that CD137 signaling contributes to the progression of atherosclerosis and the vulnerability of plaques. The aim of this study is to investigate the effects of CD137 signaling in atherosclerosis on endothelial cells (ECs) apoptosis and to explore the underlying mechanisms. Methods. Serum samples were collected from 11 patients with acute myocardial infarction and 4 controls. Peritoneal injection of agonist-CD137 recombinant protein in ApoE−/− mice was used to determine whether CD137 signaling can promote apoptosis in vivo, and human umbilical vein endothelial cells treated with agonist-CD137 recombinant protein, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) were used to explore the effect of Nrf2 and NF-κB pathway in CD137 signaling-induced ECs apoptosis in vitro. Results. ELISA showed that Bcl-2 in the serum of AMI patients was lower than that of the control group, while TNF-α and sCD137 were higher than that of the control group. Confocal microscopy and Western blot analysis showed that the nuclear translocation of Nrf2 in the agonist-CD137 group was significantly inhibited, and the expression of its downstream antioxidant enzymes was also decreased when compared with control. Immunofluorescence and Western blot results showed that the nuclear translocation of NF-κB in the agonist-CD137 group was enhanced, and ELISA results showed that the secretion of proinflammatory cytokines in the agonist-CD137 group was increased. Immunofluorescence results revealed that ROS production in the agonist-CD137 group was higher than that in control, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) groups. In vitro studies using HUVECs and in vivo studies using high-fat-fed ApoE−/− mice showed that the number of apoptotic endothelial cells was the highest in the agonist-CD137 group. By contrast, both M5580 and CAPE treatments were able to reduce CD137 induced ECs apoptosis. Conclusions. Our results showed that CD137 signaling promotes ECs apoptosis through prooxidative and proinflammatory mechanisms, mediated by Nrf2 and NF-κB pathways, respectively.

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The Effect of Protein FAM172A on Proliferation in HepG2 Cells and Investigation of the Possible Molecular Mechanism

Analytical Cellular Pathology ◽

10.1155/2019/5901083 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8

Author(s):

Hong Zhao ◽

Yujie Wang ◽

Yufeng Liu ◽

Xiaohua Hao ◽

Hongshan Wei ◽

...

Keyword(s):

Cell Proliferation ◽

Mass Spectrum ◽

Protein Kinase ◽

Membrane Proteins ◽

Recombinant Protein ◽

Western Blot ◽

Hepg2 Cell ◽

Mtt Assay ◽

Hepg2 Cells ◽

Molecular Mechanisms

Background. In our previous study, we found that the FAM172A recombinant protein could promote proliferation of L02 cells. However, the underlying mechanisms are still unknown. The present study was aimed at investigating the effect of FAM172A on proliferation of HepG2 cells and exploring the possible molecular mechanisms and its role in hepatocellular carcinoma (HCC). Methods. Cell proliferation was measured by MTT assay. Western blot test was carried out to investigate the mechanism. Rabbit antibodies against FAM172A and membrane proteins isolated from lysate of HepG2 cell were coprecipitated and the resultant precipitates were analyzed by mass spectrum. Results. The MTT assay showed that recombinant protein FAM172A isoform 1 (FAM172A-1) could induce HepG2 cell proliferation at the concentration of 10-100 ng/mL, while protein FAM172A isoform 3 (FAM172A-3) was at the concentration of 80-100 ng/mL. Western blot demonstrated that both FAM172A-1 and FAM172A-3 could activate the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) pathway and the phosphatidylinositol 3-kinase/threonine-protein kinase (PI3K/Akt) pathway. Mass spectrum analysis suggested that there were some membrane proteins interacting with FAM172A. Several candidate interacting proteins might mediate proliferation signals induced by FAM172A recombinant protein, including seven membrane proteins. Conclusion. In conclusion, FAM172A recombinant protein could induce proliferation of HepG2 cells, in which the MAPK/ERK and PI3K/Akt signaling pathways might be involved. The role of FAM172A in HepG2 cell proliferation also indicated its possible involvement in HCC. The receptor of FAM172A on cells still needs to be exploited.

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Immunization with a Recombinant Protein of Trichinella britovi 14-3-3 Triggers an Immune Response but No Protection in Mice

Vaccines ◽

10.3390/vaccines8030515 ◽

2020 ◽

Vol 8 (3) ◽

pp. 515

Author(s):

Anna Stachyra ◽

Sylwia Grzelak ◽

Katarzyna Basałaj ◽

Anna Zawistowska-Deniziak ◽

Justyna Bień-Kalinowska

Keyword(s):

Recombinant Protein ◽

Meat Products ◽

Cloning And Expression ◽

Adapter Proteins ◽

Trichinella Britovi ◽

Protein Immunization ◽

Eukaryotic Organisms ◽

Protection Against Infection ◽

Cellular Compartments

14-3-3 proteins are present in all eukaryotic organisms and are ubiquitously expressed in a broad range of tissues and cellular compartments. They are regulatory adapter proteins that play key roles in a variety of signaling pathways, and have been proposed as suitable targets for the control and detection of certain parasites. Trichinella britovi is a widely-distributed parasitic nematode, transmitted through ingestion of meat products containing invasive larvae. The present study describes the cloning and expression of Tb14-3-3, and investigates the immunological and protective potential of the recombinant protein. Immunization of mice with rTb14-3-3 triggered an IgG response, and significant differences, in the profiles of secreted cytokines observed in vitro, between experimental groups. Nonetheless, neither specific antibodies, nor increased secretion of IFNγ, IL-4, and IL-10 cytokines, conferred greater protection against infection. No reduction in larval burden was observed during recovery at 48 dpi. Additionally, rTb14-3-3 was not recognized by sera from the infected control mice, except for one, suggesting some mismatch between native and recombinant Tb14-3-3 antigenic sites. Therefore, before 14-3-3 can be considered a potential tool for Trichinella detection and vaccination, more research regarding its target proteins, and actual specific function, is needed.

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Silencing the Expression of Cyclin G1 Enhances the Radiosensitivity of Hepatocellular Carcinoma in vitro and in vivo by Inducing Apoptosis

10.21203/rs.3.rs-40051/v1 ◽

2020 ◽

Author(s):

Gang Xu ◽

Shanshan Bu ◽

Xiushen Wang ◽

Hong Ge

Keyword(s):

Hepatocellular Carcinoma ◽

Western Blot ◽

Blot Analysis ◽

Hepg2 Cells ◽

Western Blot Analysis ◽

Cyclin G1 ◽

Related Proteins ◽

Hcc Cells

Abstract Background: Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 was a novel member of the cyclin family, and it is abnormally expressed in HCC. The aim of this study was to investigate the role of cyclin G1 in the radiotherapy of HCC cells. Methods: The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and western blot analysis. The proliferation was analysed by MTT assay, and the radiosensitivity of HCC cells was detected by using a colony formation assay and a xenograft tumour model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by western blot analysis. Results: The expression of cyclin G1 mRNA and protein in HepG2-cyclin G1-siRNA cells is significantly decreased compared with that in HepG2 cells. Silencing the expression of cyclin G1 inhibits the proliferation of HCC cells and enhances the radiosensitivity of HepG2 cells in vitro and in vivo. HepG2-cyclin G1-siRNA significantly decreases Bcl-2 expression and increases Bax expression in cells.Conclusion: Silencing the expression of cyclin G1 enhances the radiosensitivity of HCC cells in vitro and in vivo, and the mechanism is probably related to the regulation of apoptosis-related proteins.

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