---

Background: Hyaluronidase A is an antigenic protein that is secreted by Streptococcus pyogenes. Nowadays, streptococcal infections are diagnosed by tracking down anti-hyaluronidase A antibodies. In this study, the attempt was made to generate recombinant hyaluronidase A in E. coli. Materials and Methods: In this experimental study, through designing specific primers and polymerase chain reaction (PCR), hyaluronidase A gene was amplified and after purification, it was sub-cloned in plasmid expression vector pET32a. Then pET32a-hylA was transferred to E. coli BL21-DE3-plySs. Protein generation induced by IPTG. The recombinant protein was purified by Ni-NTA kit and its concentration was assayed by Bradford method. Western-Blot analysis was run for verifying the recombinant hyaluronidase A. Results: The nucleotide sequencing of the gene amplified by PCR was the same as hyaluronidase A gene from Streptococcus pyogenes. Production of the recombinant hyaluronidase A via induction by pET32a-hylA plasmid was done through IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. The concentration of purified protein was 500µg/ml. analysis using a mouse anti-hyaluronidase A serum was reacted with the generated protein using Western-Blot analysis. Conclusion: Recombinant HylA protein can be generated in E.coli and the resulting protein maintains its antigenic properties desirably.

---

Background: Vibrio cholera is an important agent causing cholera in human. The expression of Flagellum and the movement of the bacterium are critical in the colonization and virulence of Vibrio cholera. FlaA gene is one the five genes encoding Flagellin which plays an important role in the activity and movement of the bacterium and its colonization which has a significant role in its immunogenicity. The aim of this study was to express and produce the recombinant FlaA protein in E.coli using Western blot method. Materials and Methods: In this experimental study, FlaA gene was proliferated by PCR method using the specific primers and cloned with BamHI and Xhol in pTz57R/T. Then it was proliferated and sequenced in DH5a vector of E.coli. The cloned FlaA gene was inserted into pGEX-4T-1 vector. The cloned vector was transformed to BL21-DE3 of E. coli and successfully expressed by induction of IPTG. The expressed protein was purified by GST affinity resin. For preparation of the primary antibody, the purified recombinant protein was injected to rats. Western blot assay method was used for determining the antigenicity of the recombinant FlaA. Results: Determination of gene sequencing showed that this gene has been proliferated properly and the antibody used in Western blot verified the production of the recombinant protein. Conclusion: The results of this study demonstrate that FlaA protein is immunogenic and can be evaluated in vaccine designing and as a diagnostic tool for detection of cholera infection.

---

Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa) could act as a cell penetrating peptide (CPP). In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method.

Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3) strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method.

Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it.

Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.