Construction of prokaryotic expression plasmid of fusion protein including porin A and porin B ofNeisseria gonorrhoeae and its expression inE. coli

2004 ◽  
Vol 24 (5) ◽  
pp. 417-420
Author(s):  
Liao Fang ◽  
Song Qifa ◽  
Wan Mufen
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Expression Plasmid ◽  
Ofneisseria Gonorrhoeae ◽  
Porin B

scholarly journals Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein

2005 ◽  
Vol 151 (3) ◽  
pp. 449-464 ◽  
Author(s):  
J. Franke ◽  
W. N. Batts ◽  
W. Ahne ◽  
G. Kurath ◽  
J. R. Winton
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Sequence Motifs

Expression of Recombinant Human Cytomegalovirus Fusion Proteins pp150-pp65 Fragments and its Application

2013 ◽  
Vol 634-638 ◽  
pp. 1313-1318
Author(s):  
Yu Fen Jin ◽  
Yan Lei Li ◽  
Yan Hua ◽  
Xiao Gang Zhang ◽  
Ting Yu
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Human Cytomegalovirus ◽  
Colloidal Gold ◽  
Fusion Proteins ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Serum Samples ◽  
Igg Antibody ◽  
Prokaryotic Expression System

Objective To evaluate the effectiveness of prokaryotic expression of fusion proteins pp150-pp65 of human cytomegalovirus (hCMV) for its application as antigen, the fusion protein of pp150-pp65 were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column for preparing the colloidal gold kit. Methods Using DNA from HCMV strain as template, the genes encoding pp150 and pp65 protein fragment were amplified by PCR technique, respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pET28a-pp150-pp65 was transformed into E.Coil.BL21(DE3) and induced to express with IPTG. The expressed fusion protein was characterized by SDS-PAGE and western blot after purified, then we used the purified fusion protein to develop combined detection kit of IgM/IgG antibody against HCMV (colloidal gold method) with Beijing Innovita Bio-tech Co., Ltd for detecting the samples, compared with the imported kits (ELISA). Results The gene of fusion fragment pp150-pp65 was correctly amplified and the recombinant vector was successfully constructed. The purified protein with the molecular weight of 45KD had good antigenicity by western blot. The protein was subjected to assay with an ELI SA capture kit in its specific and sensitive assay based on colloidal gold nanoparticles, testing of 600 serum samples indicated that this kit had a sensitivity of 92.7%;, specificity of 83.1%, crude consistency o f 90.2%, compared to the imported HCMV-IgG kit; the sensitivity of the kit was 88.1%, specificity was 89.2%, coarse consistency was 88.5%, compared to the imported HCMV-IgM kit; Conclusion In this experiment, the HCMV antigen with high purity and specificity (pp150-pp65 recombinant protein) was prepared effectively through genetic engineering technology. Compared to imported reagents, the colloidal gold kit consisting of fusion protein in a capture assay had high sensitivity and specificity. Preliminary clinical use warrants further development and use of this kit. Furthermore, it provides a technological basis for detection of HCMV in different stages of clinical infection.

MicroRNA-181a Targets TEL/AML1 Expression and Impairs Cell Proliferation in t(12;21) Acute Lymphocytic Leukemia (ALL) Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 766-766 ◽  
Author(s):  
Christopher Hickey ◽  
Sebastian Schwind ◽  
Heiko Becker ◽  
Houda Alachkar ◽  
Ramiro Garzon ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Fusion Gene ◽  
Luciferase Reporter ◽  
Control Vector ◽  
Expression Plasmid ◽  
Antileukemic Effect ◽  
Reh Cells ◽  
Dose Dependent

Abstract Abstract 766 MicroRNAs are short non-coding RNA sequences that hybridize by imperfect base-pairing more commonly to the 3′ untranslated region (UTR) of coding target mRNAs, thereby inducing target degradation or translation inhibition into the encoded proteins. Recently, we reported a microRNA signature that is associated with clinical outcome in acute myeloid leukemia (AML) patients [Marcucci G, et al. New Engl J Med 2008;358:1919-28], whose backbone was constituted by miR-181 family members. The higher the miR-181a and miR-181b levels, the lower the risk for relapse and/or death, thereby suggesting miR-181 mediated tumor suppressor activity. By utilizing the TargetScan 5.1 algorithm, we identified RUNX1 (AML1), a transcription cofactor involved in normal hematopoieis and in several genetic aberrations in both AML and ALL, as one of the predicted targets of miR-181a. In t(12;21)(p13;q22), one of the most frequent chromosomal abnormalities (20-25%) in pediatric B-cell ALL, almost the entire coding sequence of RUNX1 (AML1) including the putative miR-181a binding site within the 3′-UTR fuses with the 3′ end of ETV6 (TEL) generating a fusion gene most commonly reported as TEL/AML1. The gene encodes a fusion protein that has been shown to contribute to lymphoid leukemogeneis. Since RUNX1 and TEL/AML1 share the same 3′-UTR we hypothesized that increased levels of miR-181a might downregulate leukemogeneic levels of the fusion protein thereby resulting in a microRNA-mediated antileukemic effect. To demonstrate that RUNX1 and TEL/AML1 are targets of miR-181a, we generated a pGL3 luciferase reporter construct containing the miR-181a binding seed sequence of the RUNX1 and TEL/AML1 3′-UTR. This construct was cotransfected into HEK293T cells along with a miR-181a expressing plasmid. An expression vector lacking miR-181a coding region served as the “empty” control vector. Nearly a 30% reduction in luciferase activity was achieved in a dose-dependent fashion by increasing the amount of miR-181a expression plasmid from 1 μg to 10 μg, thereby confirming RUNX1 and TEL/AML1 as targets of miR-181a. Then, to demonstrate further miR-181a-mediated downregulation of endogenous RUNX1, we transiently transfected the AML cell line THP-1, that harbors RUNX1 wild type with the miR-181a expression plasmid. Analyzing cell lysates by Western blotting, we showed a miR-181a-dependent RUNX1 protein downregulation; expression levels were reduced to 56% and 72% upon treatment with 1 μg or 5 μg of the miR-181a expressing plasmid, respectively, compared to control vector treated cells. Next, miR-181a expressing plasmid or the control vector were transiently transfected into the TEL/AML1-positive ALL cell line REH. Western blot analysis revealed 55% reduction in fusion protein expression in cells transfected with the miR-181a expressing plasmid compared to controls. Quantitative RT-PCR confirmed the increased ectopic miR-181a expression in the cells and the concurrent reduction in TEL/AML1 RNA levels. Finally, MTS assays were utilized to assess the cellular proliferation of REH cells transfected with various concentrations (5-15 μg) of miR-181a or control vector. Cellular proliferation was decreased in a dose-dependent manner up to nearly 50% in miR-181a transfected REH cells, suggesting an antiproliferative activity of miR-181a in TEL/AML1-positive cells. In conclusion, we demonstrated that RUNX1 and TEL/AML1 are targets of miR-181a. Ectopic miR-181a can effectively target the ALL-associated TEL/AML1 fusion gene, decrease the level of the encoded fusion protein and induce a significant antileukemic effect. The use of synthetic miR-181a and/or agents capable of increasing endogenous miR-181a levels have the potential to be novel therapeutic approaches in the treatment of t(12;21) ALL, and in vivo clinical studies are underway. Disclosures: No relevant conflicts of interest to declare.

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