Generation of truncated recombinant form of tumor necrosis factor receptor-1 to produce cancer vaccine

Purpose: To produce truncated recombinant form of tumor necrosis factor receptor 1 (TNFR1), cysteine-rich domain 2 (CRD2) and CRD3 regions of the receptor were generated using pET28a and E. coli/BL21.Methods: DNA coding sequence of CRD2 and CRD3 was cloned into pET28a vector and the corresponding protein was expressed under induction of isopropyl β-D-1-thiogalactopyranoside (IPTG) as 6×His tagged using E.coli BL21 (DE3) expression system. The protein was then purified by Ni-NTA affinity chromatography. The fragment insertion, expression of recombinant protein and the yield of expression were evaluated.Results: Protein expression was achieved by identifying a band with molecular weight of 1488.3 Da. The recombinant protein of CRD2 and CRD3 was most efficiently expressed in 0.5 mM IPTG and 3 h of incubation at 37 °C with high yield equal to 0.3 μg/μl. Also, the highest concentration of imidazole for purification of the recombinant protein was 250 mM.Conclusion: A truncated form of TNFR-1 has been successfully expressed in a bacterial expression system and purified on affinity column. The purified protein can be used in in vivo experiments to prepare specified agonist antibodies for TNFR-1.Keywords: Tumor necrosis factor receptor 1 (TNFR-1), Cysteine rich domain 2 (CRD2), Cysteine rich domain (CRD3), Apoptosis, Cancer vaccine, Antibodies, Recombinant protein, pET28

Isolation of a Penicillin Acylase Producing E.coli and Kinetic Characterization of the Whole Cell Enzyme Activity

ABSTRACT Penicillin acylase (EC 3.5.1.11) has been a target of study for a long time because of its pivotal role in the deacylation of the penicillin into the 6-aminopenicillanic acid (6-APA) and the side-chain organic acids. This property of penicillin acylase has been exploited commercially for large scale production of 6-APA, which is the key intermediate in the manufacture of semi-synthetic penicillins. Due to the worldwide demand for semi-synthetic penicillins, production of 6-APA has been increased up to 7000 tons in recent years. In this study, Sixty-five strains of E. coli were investigated for penicillin acylase activity using fluorescamine method. The 6-aminopenicillanic acid formed in the reaction mixture was developed on thin layer chromatography. One-minute beta-lactamase test was carried out to follow any trace of penicillinase activity. Only one sample designated as E.coli PPA78 was found to be penicillin acylase producer. The optimal pH and temperature of penicillin acylase activity of the whole cells were determined to be 8.0 and 57°C, respectively. Km value and activation energy of the enzymatic hydrolysis reaction of penicillin G by intracellular enzyme were estimated as 0.004 mmol and 6.2 Kcal/mol, respectively. Iran. Biomed. J. 6 (2 & 3): 93-96, 200

Fusion protein consisting of hemagglutinin small subunit and truncated nucleoprotein as a universal influenza vaccine candidate: Starting in-silico evaluation toward In Vitro expression

Background: Influenza virus is a respiratory pathogen, which causes high degree of mortality and morbidity during seasonal epidemics and sporadic pandemics. By selecting conserved antigenic proteins, for example, hemagglutinin small subunit (HA2) and nucleoprotein (NP), we aimed to develop a vaccine based on a fusion protein leading to both cellular and humoral responses that are the most challenging aspects in designing a universal vaccine. Materials and Methods: The bioinformatics analysis was performed for HA2-NP structure and function prediction. Primers for the antigenic part of NP were designed using bioinformatics tools. The desired product was amplified via polymerase chain reaction using the designed primers, which was then penetrated into T vector, followed by insertion into pET28a vector in order to construct pET28a/NP. The pET28a/HA2, previously generated in our lab, was digested with the same restriction enzymes as pET28a/NP (HindIII/Xhol). Then, NP was inserted to the downstream region of HA2 to construct pET28a/HA2. Results: The generated pET28a/HA2-NP was transformed into Escherichia coli BL21 (DE3). The expression was induced by isopropyl β-d-l-thiogalactopyranoside. The results showed that the antigenic segment of NP was successfully cloned into pET28a/ HA2. The protein band of HA2-NP was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis, confirmed by Western blotting and purified with Ni-NTA purification system (QIAGEN, Germany). Conclusion: As currently available vaccines can cause some allergic reactions, using a chimer protein based on the bioinformatics analysis is continual, safe, and affordable, thus stimulating both cellular and humoral immunity systems. Our construct could potentially provide a basis for a universal vaccine candidate

Prokaryotic Expression of the Influenza A (H5N1) Neuraminidase

Background and purpose: Highly Pathogenic Influenza A(H5N1) viruses cause vast economic losses throughout the world. They may circulate in animals and be able to spread from human to human. Therefore, launching diagnostic tests are highly essential to control the influenza infection. Studies on the amino acid sequence of the neuraminidase (NA) protein of influenza viruses revealed that NA is the most immunogenic protein in naïve animals which can simply stimulate the humoral immune system well. Materials and methods: Influenza virus NA gene (A/Indonesia/5/2005(H5N1) was cloned into pET21a and expressed in host E. coli (BL21) strains. Then, the expression level of NA protein was optimized for different IPTG inductor concentrations and times. Results: Findings showed the integrity of pET21a-NA construct. Emerging bands with the expected molecular weight (38KDal) on SDS-PAGE and WB analysis confirmed the successful expression of target protein in E.coli BL21 strain. In silico analysis showed integrity of major epitopes in the structure of fused version of NA produced in this work. Conclusion: The new recombinant NA has the potential to be used directly in serological tests. It could be also used in polyclonal antibody preparation which is employed as an essential material in western blot analyses and other immunological and serological studies, such as ELISA, immunocytochemistry, and immunohistochemistry

Developing of SARS-CoV-2 fusion protein expressed in E. coli Shuffle T7 for enhanced ELISA detection sensitivity – an integrated experimental and bioinformatic approach

In the recent COVID-19 pandemic, developing effective diagnostic assays is crucial for controlling the spread of the SARS-CoV-2 virus. Multi-domain fusion proteins are a promising approach to detecting SARS-CoV-2 antibodies. In this study, we designed an antigen named CoV2-Pro, containing two RBD domains from SARS-CoV-2 Omicron and Delta variants and one CTD domain of the nucleoprotein in the order of RBD-RBD-N, linked by a super flexible glycine linker. We evaluated the suitability of E. coli Shuffle T7 and BL21 (DE3) strain for expressing CoV2-Pro. Moreover, Bioinformatic studies were conducted first to analyze the tertiary structure of CoV2-Pro. The CoV2-Pro sequences were cloned into a pET-32b (+) vector for expression in E. coli Shuffle T7 and BL21 (DE3). SDS-PAGE and western blot confirmed the protein expression and folding structure. The CoV2-Pro-TRX was purified by Ni-NTA affinity chromatography. Dot blot analysis was performed to evaluate the antigenic characterization of the CoV2-Pro. A molecular docking simulation was conducted to assess the binding affinity of CoV2-Pro with LY-COV555 (Bamlanivimab) monoclonal antibody. A molecular dynamic was performed to analyze the stability of the structure. Bioinformatic and experimental studies revealed a stable conformational 3D structure of the CoV2-Pro. The CoV2-Pro interacted with SARS-CoV-2 antibodies, confirming the correct antigenic structure. We assert with confidence that CoV2-Pro is ideal for developing an ELISA assay for precise diagnosis and rigorous vaccine evaluation during the COVID-19 prevalence. Communicated by Ramaswamy H. Sarma</p

Phylogenetic analysis and docking study of neuraminidase gene of influenza A/H1N1 viruses circulating in Iran from 2010 to 2019

Influenza A viruses (H1N1) have been consistently one of the most evolving viruses that escape from vaccine-induced immunity. Although there has been a rapid rise in human influenza virus knowledge since the 2009 pandemic, the molecular information about Iranian strains is still inadequate. The aim of this study was to analyze the neuraminidase (NA) segment of the Iranian isolates in terms of phylogenetic, antiviral resistance, and vaccine efficiency. Ninety-three NA sequences collected among 1758 nasopharyngeal swab samples during the 2015–2016 influenza season were sequenced and submitted to NCBI. Moreover, all the submitted Iranian influenza H1N1 NA sequences since 2010 till 2019 were included in the study.Software including MEGA-X, MODELLER, UCSF ChimeraX, Auto-Dock 4.2, and other online tools were used to analyze the phylogenetic relationship, vaccine efficiency, and binding affinity to sialic acid of the selected NA proteins. Moreover, the information about antiviral drug resistance mutations of NA were gathered and compared to the Iranian NA segments to check the presence of antiviral drug-resistant strains.The phylogenetic study showed that most Iranian NA sequences (between 2015 and 2016) were located in a single clade and following years were located in its subclade by 3 major mutations (G77R/K, V81A, and J188T). Resistant mutations in drug targets of NA including I117M, D151E, I223V, and S247N were ascertained in 10 isolates during the 2015–2016 flu seasons.Investigation of vaccination effect revealed that Iranian isolates in 2017 and 2018 were best matched to A/Brisbane/02/2018 (H1N1), and in 2019 to A/Guangdong-Maonan/SWL1536/2019 (H1N1).Furthermore, we performed an in-silico analysis of NA enzymatic activity of all Iranian sequences by assessment of enzyme stability, ligand affinity, and active site availability. Overall, the enzyme activity of four Iranian strains (AUG84119, AUG84157, AUG84095, and AUG84100) was assumed as the maximum enzyme activity. This study highlighted the evolutionary trend of influenza A virus/H1N1 circulating in Iran, which provides a preliminary viewpoint for a better comprehension of new emerging strains’ virulence and thus, more appropriate monitoring of influenza virus A/H1N1 during each outbreak season

Immunological Assessment of Three Tandem Repeat of Influenza Virus M2 Extracellular Domain with Adjuvant in Balb/c Mice Model

Abstract Background: Influenza A viruses are globally important respiratory pathogens which cause a high degree of morbidity and mortality during annual epidemics. M2 protein which expressed on the viral surface facilitates virus entry to the host cells. The extracellular domain of M2 protein (M2e) consists of N-terminal 24 residue which shows remarkable conservation among all subtypes of influenza A viruses. In this study, we evaluated the immunogenicity of three tandem repeats of M2e along with different adjuvants in BALB/C mice model. Materials and Methods: Recombinant protein (3M2e) was expressed in Escherichia coli and purified. Six weeks old BALB/c mice were immunized interdermally with three doses of 3M2e alone or supplemented with Alum/CpG motif as adjuvant. Control group was injected with PBS. Two weeks after the last immunization, specific anti-M2 was measured using ELISA method and finally mice were challenged with one lethal dose (LD90) of PR8 virus. Results: The results showed that 3M2e can induce specific antibody alone. However, 3M2e protein supplemented with Alum-CpG induced higher level of specific antibodies, so that, there was a significant difference with 3M2e group (p<0.05). Anti-M2 antibodies mostly consisted of IgG2a subclass which considered as activity index of TH1 Cells. Moreover, this group showed enhanced protection against wild-type virus (survival rate=60%). Conclusion: Applying Alum-CpG as a complex adjuvant may play a crucial role in integrating innate and acquisitive immunity. We increased density of M2e in combination with complex adjuvant and showed that this vaccine induced power immune responses and semi-protected mice against lethal challenge

Purification of Influenza virus A (H1N1) recombinant Hemagglutinin (HA1) and polyclonal antibody production

Background: Each year, Human influenza A (H1N1) virus causes moderate to severe infections with a high prevalence throughout the world. Accordingly, the rapid, sensitive and cost-effective laboratory diagnosis based on viral antigen detection is important. Moreover, the generation of specific antibodies directed against Influenza antigens is essential to the success of both basic and applied research programs. Hemagglutinin (HA) is the major surface envelope glycoprotein of influenza virus, which is subsequently cleaved into two subunits, HA1 and HA2. Since most antigenic sites are in the HA1 domain of HA, HA1 domain of influenza virus was studied as antigen to produce polyclonal antibody. Methods: In this experimental study we expressed and purified the recombinant HA1 protein in the second half of 2015 at department of influenza and other respiratory viruses, Pasteur Institute of Iran and then prepared the polyclonal rabbit antibody against it. The vector of pET28aHA1 expressing HA1-His tagged protein of H1N1 influenza A/PR/8/34 virus was used for large scale production of HA1 into E. Coli (BL21). By changing expression conditions such as IPTG (Isopropyl &beta;-D-1-thiogalactopyranoside) concentration, time and temperature of incubation, the expression conditions for HA1 were optimized. The total cell protein harvested and purified by nickel affinity chromatography. All above mentioned experiments monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The efficiency of HA1 recombinant protein was high, equal to 400-600 mg/ml of cell lysate. The polyclonal antibody was prepared by immunizing the rabbits using recombinant HA1 with Freund&rsquo;s adjuvant according to standard protocols. Efficiency of the antiserum evaluated by enzyme linked immunosorbent assay (ELISA). Determination of antibody level in the collected antiserum using serum-based ELISA showed that the specific antibody has risen well through the immunization schedule. Conclusion: Our data shows that this polyclonal antibody has potential to be produced in rabbit. It will also be used in the future in influenza diagnosis as well as in other immunological applications such as western blot analyses, immunocytochemistry, and immunohistochemistry. &nbsp

Production of polyclonal antibody against Tehran strain influenza virus (A/H1N1/2009) hemagglutinin conserved domain (HA2): brief report

Background: The influenza virus is one of the most important factors for higher morbidity and mortality in the world. Recently, researchers have been focused on influenza conserved antigenic proteins such as hemagglutinin stalk domain (HA2) for vaccine production and serological studies. The HA2 plays a major role in the fusion of the virus with host cells membrane. The immunity system enables to produce antibody against HA2. The aim of this study is polyclonal antibody production against influenza HA2. Methods: This study was done in the Influenza Research Lab, Pasteur Institute of Iran, Tehran for one year from September 2013 to October 2014. In the present study, recombinant HA2 protein was produced in prokaryotic system and purified using Nickel affinity chromatography. The purified HA2 was mixed with Freund&rsquo;s adjuvant (complete and incomplete) and injected into two New Zealand white rabbits by intramuscularly and subcutaneously routes. Immunization was continued for several months with two weeks interval. Before each immunization, blood was drawn by venous puncture from the rabbit ear. Function of rabbit&#39;s sera was evaluated using radial immunodiffusion (RID) in both forms, Single RID (SRID) and Double RID (DRID). Finally, antiserum activity against HA2 was evaluated using western blotting as serological assay. Results: Sedimentary line and zone was observed in RID assays (SRID and DRID) represent interaction between HA2 protein and anti- HA2 antibody. As well as, western blotting results was positive for HA2 protein. Therefore, these results showed that polyclonal antibody produced against HA2 protein can identify HA2 protein antigenic sites. Conclusion: These findings show that humoral immune responses have properly been stimulated in rabbits and these antibodies can identify HA2 protein and may be suitable for other serological methods

Synthesis of Ni2+-functionalized polydopamine magnetic beads for facilitated purification of histidine-tagged proteins

Abstract Facilitated purification of proteins, at a low cost and a short time, is one of the key steps in the industrial production of recombinant proteins. In the current study, polydopamine nanoparticles (PDA-NPs) are considered in the synthesis of magnetic beads for purifying recombinant proteins due to advantages such as biocompatibility/ biodegradability, easy synthesis, as well as the ability to directly chelate metal ions. They were synthesized in Tris buffer (pH: 8:5), then chelated with Fe3+(20 mg) and Ni2+ ions at concentrations of 2, 3, 5, and 7 mg/ml. Prepared nanoparticles were characterized through scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV-vis), dynamic light scattering (DLS), Inductively Coupled Plasma (ICP), and vibrating sample magnetometer (VSM). The size distribution of the particles was reported in the narrow range of 120–140 nm and 200 to 220 nm by the SEM image and DLS analysis, respectively. The chelation of ions on the surface of the nanoparticle was confirmed by the ICP technique with a magnetization of 35.42 emu/g. The highest adsorption rate of Ni2+ ions to polydopamine was obtained at a ratio of 1.4. The SDS-PAGE and western blot analysis confirmed the purification of eGFP and Hsp40 by PDA/Fe3+/Ni2+ at 26 and 40 kDa compared to the commercial nickel column. Moreover, the concentration of purified eGFP by PDA/Fe3+/Ni2+ was reported 138.83 µg/ml by the fluorescent signals, which is almost equal to or more than the protein purified by commercial Ni-NTA column (108.28 µg/ ml). The stability of PDA/Fe3+/Ni2+ has also been evaluated by ICP-OES for 10 days, and the result suggested that PDA magnetic beads were stable. Therefore, it can be concluded that PDA/Fe3+/Ni2+ have the ability to purify recombinant proteins in one less step and shorter time