BackgroundHepatitis C virus (HCV) infection represents a worldwide health threat that still needs efficient protective vaccine and/or effective drug. The traditional medicine, such as camel milk, is heavily used by the large sector of HCV patients to control the infection due to the high cost of the available standard therapy. Camel milk contains lactoferrin, which plays an important and multifunctional role in innate immunity and specific host defense against microbial infection. Continuing the analysis of the effectiveness of camel lactoferrin against HCV, the current study aimed to separate and purify the native N- and C-lobes from the proteolytically cleaved camel lactoferrin (cLF) and to compare their in vitro activities against the HCV infection in Huh7.5 cells in order to determine the most active domain.MethodsLactoferrin and its digested N- and C-lobes were purified by Mono S 5/50 GL column and Superdex 200 5/150 column. The purified proteins were assessed through three venues: 1. To inhibit intracellular replication, HCV infected cells were treated with the proteins at different concentrations and time intervals; 2. The proteins were directly incubated with the viral particles (neutralization) and then such neutralized viruses were used to infect cells; 3. The cells were protected with proteins before exposure to the virus. The antiviral potentials of the cLf and its lobes were determined using three techniques: 1. RT-nested PCR, 2. Real-time PCR, and 3. Flow cytometry.ResultsN- and C-lobes were purified in two consecutive steps; using Mono-S and Superdex 200 columns. The molecular mass of N- and C-lobes was about 40 kDa. cLF and its lobes could prevent HCV entry into Huh 7.5 cells with activity reached 100% through direct interaction with the virus. The inhibition of intracellular viral replication by N-lobe is 2-fold and 3-fold more effective than that of the cLF and C-lobe, respectively.ConclusionGenerated native N- and C-lobes from camel lactoferrin demonstrated a range of noticeably different potentials against HCV cellular infectivity. The anti-HCV activities were sorted as N-lobe > cLf > C-lobe.

Hepatitis C virus (HCV) infection represents a world health problem and no protective vaccine or effective drug currently exists. For economic reasons, many patients use traditional medicines to control the infection. In Egypt, camel milk is one of the traditional medicines widely consumed by patients infected with HCV. The present study aimed to evaluate the efficacy of camel milk in the treatment of patients infected with HCV. Whole camel milk from a local farm was administered to patients for 4 months (250 ml/day/patient). Patient sera were collected prior to and following camel milk drinking, and three markers were set-up for sera-evaluation. The three markers indicating the effect of camel milk on HCV infection were: Liver function assays [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)]; a viral load assay; and anti-HCV antibodies profile and isotyping against synthetic HCV epitopes. Camel milk demonstrated the ability to improve general fatigue, health and liver function (ALT and AST levels); ALT was reduced in ~88% of patients and AST was reduced in all patients subsequent to drinking camel milk for four months. The majority of patients responded positively to camel milk treatment; RNA viral load decreased in 13 out of the 17 patients (76.47%) and one patient exhibited undetected viremia following camel milk treatment. The anti-HCV antibodies profile and isotyping were significantly decreased (P<0.05) in immunoglobulin (Ig)G1 following treatment in 70–76% of patients. However, the treatment was ineffective in 23.53% of patients who experienced no reduction in RNA viral load following treatment with camel milk. In conclusion, whole camel milk treatment demonstrated efficacy in vivo; the viral load in the majority of patient sera was reduced and the IgG isotype profile was converted to Th1 immunity.

BackgroundIsopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or variable protein yields. Auto-induction allows for production of much higher target protein yield and cell mass than conventional procedures using induction with IPTG without monitoring cell growth then adding IPTG at the appropriate cell density. This method involves special media recipes that promote growth to high density and automatically induce expression of target protein from T7 promoter. Consensus interferon is a synthetic artificially engineered interferon having an amino acid sequence that is a rough average of the sequences of all natural human alpha interferon subtypes and has greater potency than other interferons even the pegylated versions. The purpose of this study was high-level expression of human consensus interferon-alpha (cIFN-α) in E. coli using an auto-induction protocol. The cIFN-α gene was cloned into pET101/D-TOPO expression vector under the T7 promoter transcriptional regulation. Expression was optimized with respect to temperature and length of incubation in shake flask cultures. The antiviral potency and anticancer activity of cIFN-α were evaluated in comparison to IFN-α2a.ResultsThe expressed cIFN-α protein in auto-induction T7 system was found mostly in soluble fraction of the cell lysate (about 70% of yield in total cell lysate) after lowering incubation temperature to 25°C or 30°C. Protein expression was maximal after 24 h incubation at 25°C or 30°C. After purification via single-step chromatography using DEAE-Sepharose, the yield was 270 mg/L in shake flask E. coli cultures which is much higher than IPTG-inducible T7 expression system and other systems according to available data. The synthesized cIFN-α was biologically active as confirmed by its anticancer and antiviral effects and was significantly more potent than IFN-α2a.ConclusionsThe auto-induction process was reliable and convenient for production of cIFN-α protein in E. coli, and can be adapted for large-scale therapeutic protein production.

Open Life Sci. 2017; 12: 143-155 was at 450 rpm. Overall, the results suggest that caprylic acid purification of camel serum IgG is more effective and safe than ammonium sulfate method in simplicity, purity, and lower non-IgG proteins in the final preparation with lower protein aggregates.Keywords: Camelus dromedarius; camel IgG purification; caprylic acid; temperature; stirring IntroductionCamels produce several immunoglobulin classes in their serum, colostrum and milk, including IgM, IgA and IgG (IgG1, IgG2 and IgG3) [1][2][3]. Camels are special animals in antibody production as they possess a unique type of antibody in their serum which lack light chains as well as the heavy chain constant domain "CH1", so-called heavychain antibodies [4]. IgG1 occurs in the classical structure form of antibodies, while IgG2 and IgG3 represent heavy-chain antibodies [4], and they form about 75% of the IgG in camel serum [5]. Heavy-chain antibodies in camels are significantly effective in antigen recognition; in spite of losing their light chains [6][7][8], also, they have lower molecular weight than conventional antibodies. In addition, these antibodies are less immunogenic than other mammalian antibodies [9], that means when they are injected into experimental models, it will be less likely to induce adverse reactions during the course of treatment [10]. The above pharmaceutical features recommend the camel antibodies as a substitute for other animals' therapeutic antibodies [11,12], or alternatively, it can be converted to a complete humanized antibody [13].Antibody purification from different biological fluids such as serum, ascites and milk is an important step in the production of antibodies used for diagnostic, therapeutic, and research applications [14]. There are different methods for purification and the first reported method for purifying camel IgG from serum, by , used protein A-Sepharose and protein G-Sepharose. More Abstract: The present study aimed to describe and standardize a simple and efficient protocol for purification of camel IgG from serum, which can be applied for Camilidae antibody production in research laboratories, the preindustrial stage. Camel serum IgG was separated with caprylic acid and ammonium sulfate, then the effect of four variables studied: caprylic acid concentration, pH, stirring time, and stirring intensity. Camel IgG prepared by standardized caprylic acid fractionation method for camel serum was compared with commercial anti-sera products. Camel IgG purification from undiluted sera using caprylic acid at concentration of 8% v/v gave the best results. Purification at different pH values using caprylic acid at 8% v/v revealed that pH 5.5 was optimal. Investigating purification at different stirring time intervals using 8% v/v caprylic acid at pH 5.5 demonstrated that stirring for 90 min gave the optimum results. Finally, studying purification at different stirring intensities using 8% v/v caprylic acid at pH 5.5 for 90 min, the best stirring intensity

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