Molecular cloning and biologically active production of IpaD N-terminal region

Hesaraki, Mahdi; Saadati, Mojtaba; Hamed, Hossein A.; Olad, Gholamreza; Heiat, Mohammad; Malaei, Fatemeh; Ranjbar, Reza

doi:10.1016/j.biologicals.2013.03.002

Cited by 8 publications

(3 citation statements)

References 21 publications

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“…The recombinant vector was transformed into competent Agrobacterium tumefaciens GV-3101 strain by heat shock [25]. Kanamycin and rifampin were used to select colonies harboring recombinant plasmid and Agrobacterium (and not other possible bacteria contamination like transformed E. coli), respectively.…”

Section: Agroinfilterationmentioning

confidence: 99%

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato

et al. 2018

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Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis. PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals.

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Section: Agroinfilterationmentioning

confidence: 99%

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato

et al. 2018

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“…16 Expression and solubility of rDKK-1 was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% gel, as described previously. 17,18 Purification and Refolding of rDKK-1…”

Section: Experimental Analysis Cloning and Expression Of The Chimericmentioning

confidence: 99%

In Silico Structural Prediction and Production of a Chimeric Recombinant Dickkopf-1 (DKK-1) Antigen

Malaei

Rasaee

Latifi

et al. 2019

IJAAI

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Dickkopf (DKK) family of proteins are known as antagonists for the Wnt-β-catenin signaling pathway. It is suggested that the Dickkopf-1 (DKK-1) has a role in several diseases such as hepatocellular carcinomas, hepatoblastomas, Wilms’ tumors, lung cancer and Myeloma bone disease. The aim of the present study was to produce a chimeric-recombinant DKK-1 protein in order to induce immune response against the antigen. The recombinant Dickkopf-1 (rDKK-1) protein was designed using bioinformatics analysis. The standard methods were used for cloning, expression and purification. The structure of recombinant protein was analyzed by spectroscopy methods. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were performed to confirm the recombinant protein using a commercial anti-DKK-1 (whole protein) polyclonal antibody. The immunogenicity of the recombinant DKK-1 was assessed by immunizing, intraperitoneally, BALB/C mice four times with the 31-kDa and 45-kDa purified rDKK-1 cloned in pET28a and pET32a vectors respectively. The antibody titer was measured in due course of time. Stronger immunogenic parts of the protein were selected based on in-silico predictions and recombinant protein was successfully designed. The chimeric gene was sub-cloned, expressed, purified and refolded. The purified protein was confirmed by Western blotting and ELISA. The three dimensional structural was confirmed by CD spectrum and predicted structures by bioinformatics tools, revealed the stability of helix structures. rDKK-1 protein was capable of inducing immune response with high titer antibody and excessive humoral immune response. No significant difference was observed between immunization by 31-kDa and 45-kDa antigen.

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“…However, it was also broadly used to test vaccine candidates, particularly against Shigella spp. [114,115]. Furthermore, similar to mice and rats, guinea pigs are used to evaluate the toxicity of vaccine candidates.…”

Section: Further Rodent Animal Models Used For Human Vaccine Developmentmentioning

confidence: 99%

Rodents as pre-clinical models for predicting vaccine performance in humans

Riese

Trittel

Schulze

et al. 2015

Expert Review of Vaccines

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Vaccines represent a key building block for establishing a successful and sustainable control strategy against infectious diseases. Vaccine development often depends on the availability of correlates for protection and reliable animal models for the screening, selection and prioritization of potential vaccine candidates. This is performed according to their immunogenicity, efficacy and safety profiles in pre-clinical studies, which are also critical for identification of candidate antigens, selection of an optimal delivery system and design of appropriate vaccine formulations. Thus, pre-clinical studies in animal models are a prerequisite for addressing crucial issues and generating a solid pre-clinical package for the approval of clinical trials. This review addresses the strengths, limitations and perspectives of rodents as a vaccine development and pre-clinical validation tool.

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Molecular cloning and biologically active production of IpaD N-terminal region

Cited by 8 publications

References 21 publications

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato

In Silico Structural Prediction and Production of a Chimeric Recombinant Dickkopf-1 (DKK-1) Antigen

Rodents as pre-clinical models for predicting vaccine performance in humans

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