scholarly journals HIV-1 P24 EXPRESSION IN TRANSPLASTOMIC TOBACCO PLANTS

2020 ◽  
Vol 15 (2) ◽  
pp. 52-68
Author(s):  
Loc Tuong Phan ◽  
Ho Huu Nguyen ◽  
Thanh Thi Nguyen
Keyword(s):  
Western Blot ◽  
Southern Blot ◽  
Gel Filtration ◽  
Target Site ◽  
Gel Filtration Chromatography ◽  
Tobacco Plants ◽  
Transplastomic Tobacco ◽  
P24 Protein ◽  
High Concentrations ◽  
Hiv 1

Expression of HIV-1 p24 gene in chloroplasts was achieved in a tobacco variety V2 (Virginia TBE2). Through PCR and Southern blot analyses, it was demonstrated that the transgene integrated into the target site in the chloroplasts, between trnfM and trnG. Western blot results showed that HIV-1 p24 gene expressed in transplastomic tobacco plants. p24 protein accumulations were detected by ELISA in the range from 1.7% to 6.3% TSP and the high concentrations in the leaves near the top. p24 protein was purified by gel filtration chromatography demonstrated that the purification is 9.694 folds and the performance is 31.94%, however, protein p24 largely was inactive after purification.

scholarly journals Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay

2015 ◽  
Vol 24 (1) ◽  
pp. 14-8
Author(s):  
Ade P.R. Simaremare ◽  
Budiman Bela ◽  
Andi Yasmon ◽  
Fera Ibrahim
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Coli Strain ◽  
Expression And Purification ◽  
Purification System ◽  
P24 Protein ◽  
Native Condition ◽  
Different Strains ◽  
Hiv 1 ◽  
Induction Temperature

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition. Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.

scholarly journals Production of HIV-1 p24 Protein in Transgenic Tobacco Plants

2002 ◽  
Vol 20 (2) ◽  
pp. 131-136 ◽  
Author(s):  
G Gary Zhang ◽  
Lauren Rodrigues ◽  
Benjamin Rovinski ◽  
K Andrew White
Keyword(s):  
Transgenic Tobacco ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants ◽  
P24 Protein ◽  
Hiv 1

scholarly journals Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

2013 ◽  
Vol 57 (10) ◽  
pp. 4743-4750 ◽  
Author(s):  
Mark Contarino ◽  
Arangassery R. Bastian ◽  
Ramalingam Venkat Kalyana Sundaram ◽  
Karyn McFadden ◽  
Caitlin Duffy ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Gel Filtration ◽  
Etiologic Agent ◽  
Host Cells ◽  
Dependent Manner ◽  
Cell Infection ◽  
C Terminus ◽  
P24 Protein ◽  
Env Protein ◽  
Hiv 1

ABSTRACTHuman immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)xlinker (wherexis 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.

scholarly journals Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit

2004 ◽  
Vol 2 (5) ◽  
pp. 389-399 ◽  
Author(s):  
Dominique Rumeau ◽  
Noëlle Bécuwe-Linka ◽  
Audrey Beyly ◽  
Patrick Carrier ◽  
Stéphan Cuiné ◽  
...  
Keyword(s):  
Large Subunit ◽  
Zinc Content ◽  
Tobacco Plants ◽  
Transplastomic Tobacco

scholarly journals Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 180
Author(s):  
Lucia Sarcina ◽  
Giuseppe Felice Mangiatordi ◽  
Fabrizio Torricelli ◽  
Paolo Bollella ◽  
Zahra Gounani ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Covalent Binding ◽  
Hiv Infections ◽  
Selectivity Ratio ◽  
Selective Detection ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Label Free Detection ◽  
P24 Protein ◽  
Hiv 1

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

scholarly journals Analysis of serum lipoproteins by high performance gel filtration chromatography.

1996 ◽  
Vol 45 (1) ◽  
pp. 103-106 ◽  
Author(s):  
Takashi KITAMURA ◽  
Seiji ITO ◽  
Yoshio KATO ◽  
Keiko SASAMOTO ◽  
Mitsuyo OKAZAKI
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Serum Lipoproteins ◽  
Gel Filtration Chromatography
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