ABSTRACT
Background: Leishmania major LmSTI1 is a conserved protein among different species of leishmania, and expressed in both amastigote and promastigote forms of L. major life cycle. It has previously been expressed in bacterial systems
Materials and Methods: To express LmSTI1 in the methylotrophic yeast Pichia pastoris [P. pastoris], the shuttle vector pPICZA containing gene lmsti1 was constructed under the control of the AOX1 promoter. The recombinant vector was electro-transformed into P. pastoris, and induced by 0.5% methanol in the buffered medium. The expression of the LmSTI1 protein was visualized in the total soluble protein of P. pastoris by 12% SDS-PAGE, and further confirmed by Western blotting with L.major-infected mouse sera and HRP-conjugated goat anti-mouse IgG as the first and secondary antibodies, respectively
Results: The expression level was 0.2% of total soluble proteins
Conclusion: It might be possible to use this formulation as a whole yeast candidate vaccine against cutaneous leishmanization
ABSTRACT
Background: Oncolytic herpes simplex virus [oHSV]-based vectors lacking gamma 34.5 gene, are considered as ideal templates to construct efficient vectors for [targeted] cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and gamma 34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors
Methods: Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein [GFP] and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines
Results: We generated three recombinant viruses, HSV-GFP, HSV-GR [Green-Red], and HSV-Red. The HSV-GFP showed two log higher titer [1010 PFU] than wild type [108 PFU]. In contrast, HSV-GR and HSV-Red showed one log lower titer [107 PFU] than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 [P<0.001]. Moreover, HSV-GFP showed higher infection potency [98%] in comparison with HSV-GR [82%]
Conclusion: Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2[nd] and 3[rd] generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy
Subject(s)
Oncolytic Viruses , Homologous Recombination , Flow Cytometry , Microscopy, FluorescenceABSTRACT
Understanding gene expression variations by using RNA transcript analysis methods during hepatic viral protein interactions with the IFN pathway in hepatic cell lines has recently gained importance. One of the most powerful techniques in gene expression quantification is quantitative real-time RT-PCR. Reference genes used as normalizer in this method may be affected across various experimental conditions or treatments. Hence, in the present study, the influence of IFN-treatment on the mRNA levels of common reference genes including ACTB, GAPDH, TBP, HPRT1 and HMBS was evaluated in Huh-7 or HepG[2] cell lines. Cells were treated with different concentrations of IFN- Then, using geNorm and NormFinder programs, we evaluated the expression stabilities of the above prominent reference genes in three sample groups that included each hepatic cell line and the total data sets. HPRT-1 and GAPDH were the most stable reference genes in the Huh-7 cell line, whereas ATCB, HMBS and GAPDH were the most stable in the HepG2 cell line. TBP was one of the least stable reference genes in the three studied groups. This investigation will provide appropriate reference genes for standardization of quantitative real-time PCR data in an IFN-?stimulated model of hepatocyte cell lines
ABSTRACT
To accomplish the worldwide demand for recombinant human erythropoietin [rHuEpo] as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris [P. pastoris] rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZ alpha A vector under control of AOX[1] promoter, downstream of the secretion-alpha-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin [1.0 mg/ml], followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDSPAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems