Molecualr Cloning of the capsular antigen F1 of Yersinia pestis in pBAD/gIII plasmid.

Yersinia pestis which is the causative agent of pneumonic plague and distributed in all continents has led to many deaths during the history. Because of its high mortality rate, it must be diagnosed and treated at the earliest time post infection and therefore, rapid diagnostic tests are required. In the present study, we cloned the coding sequence of F1 capsular antigen of the bacteria in the pBAD/gIII plasmid for later expression and purification of the protein to produce poly and monoclonal antibodies against this antigen, and subsequently to develop rapid and efficient diagnostics tools for Y. pestis infections.

Expression of the recombinant plasminogen activator (reteplase) by a non-lytic insect cell expression system.

Reteplase is a potent thrombolytic agent which is widely used in the management of acute myocardial infarction and stroke. It belongs to the third generation of the thrombolytic drugs and has been derived from native human tissue plasminogen activator by removing three domains of it and keeping the Kringle 2 and Serine protease domains. However, the high cost of this drug, has limited the application of this drug especially in the developing and third world countries. The most laborious steps in the bacterial production of this drug is its purification and refolding steps which keep the process yield low and the cost high. Therefore, in the present study we evaluated the expression of reteplase by a non-lytic insect cell expression system. Following cloning and transfection procedures, recombinant Sf9 insect cell clones expressing the reteplase protein were selected. Primarily, the expression was verified by dot-blot analysis and subsequently it was confirmed by Western Blotting showing a band of about 45 kD on nitrocellulose membrane. The biological activity of the expressed protein was also evaluated and showed to be about 29 IU/ml. This confirmed the possibility of expression and the correct folding of the expressed protein. Hence, optimization of the expression followed by purification of the protein could be the next steps of the study.

Assessment of selective toxicity of insect cell expressed recombinant A1-GMCSF protein toward GMCSF receptor bearing tumor cells.

One of the emerging therapeutic strategies for targeted treatment of most cancers is the use of immunotoxins which are fusion proteins consisted of a targeting and a toxic moieties. We previously showed that the recombinant A254-GMCSF fusion protein selectively kills acute myeloblastic leukemia cells which harbor a large number of granulocyte-macrophage colony stimulating factor (GMCSF) receptors. Since further in vitro and preclinical studies require large amounts of this fusion protein free from any troublesome material like lipopolysacharide, we selected the insect cell expression system. Thus, the coding sequences of the A254-GMCSF and its truncated form, A247-GMCSF, were cloned and expressed by Sf9 cells. Subsequently, specific cytotoxicity of the purified proteins was evaluated on GMCSF receptor positive cell lines. SDS-PAGE and Western blot analysis of the expressed A254GMCSF and A247GMCSF fragments revealed bands of about 60 kD which were larger than the theoretically predicted size of about 47 kD. Deglycosylation analysis showed that these proteins are N-glycosylated by the insect cells. However, any other post-translation modification of the proteins by insect cells could be the reason for higher molecular weight of the fragments. Cytotoxicity assays showed specific killing activity of these proteins on HL60 and U937 cell lines with IC(50)s ranging 2-2.5 µg/ml. These IC(50) values are much higher than those obtained from bacterially expressed A254-GMCSF (80 ng/ml) which could be due to any modification performed by insect cells on the fusion proteins.

Effects of polygonum aviculare herbal extract on proliferation and apoptotic gene expression of MCF-7.

BACKGROUND AND THE PURPOSE OF THE STUDY: One of the most common malignancies in women is breast cancer. Although several treatments for breast cancer are available, application of herbal medicine as a supplementary treatment is a new option to help curing the disease. In this study anticancer effects of Polygonum avicular herbal extract was investigated. METHODS: Polygonum avicular extract was obtained by methanol. MCF-7 cell line was treated with different concentrations of Polygonum avicular (50, 100, 150, 200, 250, 300,350 400 ng/µl) for different time lengths ( 6, 12, 24, and 48 hrs). MTT assay and Flow Cytometry were used to evaluate cell proliferation and apoptosis, respectively. RT-PCR was also carried out to evaluate the expression of apoptotic genes. RESULTS AND DISCUSSION: Results showed that Polygonum avicular induced cytotoxicity in MCF-7 cell line at concentrations higher than 300 ng/µl and this was confirmed by the highest rate of cell death as measured by Trypan Blue and MTT assays. RT-PCR results showed up-regulation of P53 and down-regulation of Bcl-2 proteins which implied the ability of Polygonum avicular to induce apoptosis in MCF-7 cells and confirmed its anticancer property. Further studies are required to evaluate effects of the extract on other apoptotic genes.