Expression of Leishmania major LmSTI1 in Yeast Pichia Pastoris

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

Construction and production of Foxp3- Fc [IgG] DNA vaccine/fusion protein

Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell [DC] based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc[IgG] can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I [cross priming]

Methods: DNA construct containing fragment C [Fc] portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli [E. coli] strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining

Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells. Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification

Conclusion: Due to successful expression of Foxp3-Fc [IgG], it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means

Increased plasma levels of soluble CD27 among HIV/HCV co-infected and HIV/HCV/GBV-C triply infected subjects

CD27 is a biomarker associated with both T-cells and B-cells activation .Plasma soluble CD27 [sCD27] was identified as a marker of disease outcome in Human Immunodeficiency Virus [HIV] infection .Testing of plasma sCD27 represents a good tool to monitor the change of immune activation during HIV infection.We sought to analyses role of Hepatitis C Virus [HCV] and also GB Virus type C [GBV-C] co-infections on HIV-related immune activation, through measuring sCD27 plasma levels.Blood samples from a total of 86 patients with HIV infection were taken. Plasmas were analyzed for HCV using serologic test and GBV-C by reverse transcriptase polymerase chain reaction [RT-PCR]. CD4+ and CD8+T-cell counts were evaluated by CD3/CD4+ and CD3/CD8+ double staining of whole blood followed by flow cytometric analysis .Then Cross-sectional comparison of sCD27 plasma levels was carried out among patients : HIV [n=20], HIV/ GBV-C [n=14], HIV/ [HCV] [n=26] and HIV/HCV/GBV-C [n=26].Plasma level of sCD27 was higher in HIV/HCV/GBV-C patients as compared to HIV mono-infected patients [p= 0.006] and based on results there was significant differences in the plasma levels of sCD27 between HIV-infected individuals with and without HCV coinfection [P=0.017] and also correlation between sCD27 and percent of CD4+T-cells was in highest level among HIV/HCV co-infected patients group [r= -0.59 [p=0.001]]. High levels of sCD27 among HIV/HCV patients argues in favor of sCD27 plasma level determination for monitoring of clinical features among HIV/HCV coinfected patients

Overexpression and purification of c-terminal fragment of the passenger domain of hap protein from nontypeable haemophilus influenzae in a highly optimized escherichia coli expression system

Nontypeable Haemophilus influenzae [NTHi] is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae [H. influenzae] Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called "HapS" and a 45 kDa Cterminal translocator domain called "Hapbeta". Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain [CBD] which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24alpha-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography [IMAC] using Ni-NTA columns. The highest expression level of target protein was achieved when CBD expressing E. coli BL21 [DE3] cells were grown at 37°C in 2xTY medium with 1.0 mM IPTG at mid-log phase [OD[600 nm] equal to 0.6] for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than%97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process