|Year : 2003 | Volume
| Issue : 2 | Page : 77-81
Cloning of gag gene of HIV-1 subtype c (Indian strain) into a mammalian expression vector and in vitro expression studies
Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 110 029, India
Department of Microbiology, All India Institute of Medical Sciences, New Delhi - 110 029, India
PURPOSE: Acquired immunodeficiency syndrome caused by HIV-1 is one of the biggest health problems we are facing today. It is required to concentrate efforts towards designing a safe, effective and affordable vaccine candidate in the context of the growing epidemic worldwide. Recently the approach of DNA based immunogen has evoked a lot of enthusiasm in the preclinical models. METHODS: This study was designed towards a subtype C based gag DNA construct in the expression vector pJW4304. The gag and protease genes of HIV-1 subtype C were cloned into a mammalian expression vector pJW4304. The cloning strategy was designed so as to express a naturally processed form of the protein. Expression of gag protein by the construct pJWgagprotease49587 was evaluated by western blotting, p24 antigen capture ELISA and electron microscopy. RESULTS: Gag p24 was detected both in the supernatant and in the transfected cells. Extra cellular p24 protein was estimated by p24 antigen capture ELISA. Immunoblotting using HIV positive polyclonal sera further confirmed the expression and processing of gag gene. The 24kDa band has been observed in cell lysates, which indicates that the proper processing is taking place in the presence of protease. Virus like particles were seen budding from the cell membrane 24 and 48 hours post transfection by transmission electron microscopy. CONCLUSIONS: The recombinant construct pJWgagprotease49587 has shown good expression in vitro and therefore is a good candidate to study immunogenicity of the construct. Immunogenicity testing in mice is being carried out currently with this construct.
|How to cite this article:|
Chugh P. Cloning of gag gene of HIV-1 subtype c (Indian strain) into a mammalian expression vector and in vitro expression studies. Indian J Med Microbiol 2003;21:77-81
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Chugh P. Cloning of gag gene of HIV-1 subtype c (Indian strain) into a mammalian expression vector and in vitro expression studies. Indian J Med Microbiol [serial online] 2003 [cited 2020 Feb 18];21:77-81. Available from: http://www.ijmm.org/text.asp?2003/21/2/77/7979
Acquired immunodeficiency syndrome (AIDS) was first recognised as a new disease in 1981 and human immunodeficiency virus (HIV) has claimed about 20 million lives, and infected 56 million people worldwide. National AIDS control organization (NACO) has estimated that currently India has over 3.86 million HIV-1 infected persons including one million women and 50,000 infected infants. The current HIV pandemic in India threatens to exacerbate many of other health problems. With the advent of Highly Active Anti-Retroviral Therapy (HAART) it has become possible to control the disease to an extent, however these regimes are far from reach of most patients in developing countries like ours.
Designing an effective prophylactic and/or therapeutic vaccine is of high priority world wide, to control this epidemic. The biggest obstacle in realising this objective is that correlates of protective immunity for HIV have not been precisely identified. Besides, HIV continually evolves as a result of genetic mutation and recombination, thus an estimate of the significance of strain variation within individuals and among populations has to be taken when developing AIDS vaccines. Several strategies are being pursued to develop a suitable candidate vaccine against HIV including, recombinant subunit; combination (prime-boost strategies); synthetic peptide; virus-like particles; DNA; viral vectors; whole-killed and live-attenuated vaccines. In an effort to induce viable CTL and antibody responses, attention has turned to evaluating a combination vaccine approach referred to as the prime boost strategy. One of the most promising vaccine candidates is the use of DNA vaccine to “prime” the immune response, followed by a vaccinia-virus vector or administration of interleukins to “boost” that response. DNA based immunogens in prime boost strategy have shown good responses in the pre clinical models and have generated a great enthusiasm., The HIV-1 core protein gag has been targeted in various studies for elicitation of immune responses and had shown promising results.,, The gag gene is a relatively more conserved region of HIV-1 and various T cell epitopes have been mapped to it in various studies., The immense genetic variation in HIV-1 sequences from different parts of the world gives rise to the speculation that there might be antigenic variation conjunct with the genetic variation. In view of the above discussion we are aiming towards a gag based DNA vaccine construct from HIV-1 subtype C strains from local circulating types in India. In this paper we present the cloning and expression of gag gene of HIV-1 Indian subtype C.
| ~ Materials and Methods|| |
Mammalian expression vector pJW4304 (gift from JI Mullins University of Washington, Seattle) was used which is pUC based vector comprising of CMV immediate early promoter enhancer, t-PA leader signal and bgh poly A site Col E1 origin of replication for propagation in E.coli and ampicillin resistance marker.
Cloning of gag gene into pJW4304
The integrated HIV1 proviral DNA from an infected individual was taken as template for PCR and a 4.3 kb (139 - 4495) Gag pol product was obtained by a set of nested PCRs using the following primers: MSF12 (5'AAA TCT CTA GCA GTG GCG CCC GAA CAG3'); Pol RP06 (5'AAA ACC ATC CAT TAG CTC TCC TTG AAA CAT3'); Gag FP01 (5'TTT GAC TAG CGG AGG CTA GCA GGA GAG AGA TGG GT3'); PolRP01 (5'CAT CCA TTA GCT CTC CTT GAA ACA TAC ATA3'). The amplification product was used to generate a clone using TA cloning pGEMT easy vector cloning system. This plasmid was double digested with Nhe1 and BamH1 enzymes resulting in release of a 2.3kb gag protease insert. This fragment was ligated to pJW4304 and transformation carried out in E.coli DH5 competent cells. Screening was carried out to obtain a clone of gag protease in pJW4304 [Figure - 1].
In vitro Expression studies
In vitro expression studies in COS-7 cells were carried out with clone pJWgagprotease49587. Lipofectin reagent (Gibco BRL) was incubated with 1.5 to 2 µg, plasmid DNA at a concentration of 10g/mL and was overlayed onto 40 to 50 % confluent Cos 7 cells . The cells were incubated with the transfection mix for 6,E.coli class="ref" name="ft8" href="#ref8">8,12 and 18 hours at 37oC and 5% CO2. After incubation with lipofectin DNA complexes, fresh medium was added to the plates (DMEM with 5% FCS, 2mM glutamine and antibiotics). The cells and supernates were harvested at different time points 24, 36, 48, 72 and 96 hours. Vector alone (pJW4304) and mock positive envelope plasmid pJWSK3 were used as controls in the study.
p24 Antigen capture ELISA
The supernatants were checked for presence of p24 by antigen capture ELISA (Innogenetics, Belgium). ELISA was performed as per the manufacturer's instructions. Standard curve was plotted for the absorbance recorded for standard provided in the kit and concentrations of supernate samples were determined from the curve. The negative controls included untransfected cells and cells transfected with vector alone (pJW4304) and mock positive (pJWSK3) control.
Western blot analysis
The cell lysates and supernates were run on a denaturing SDS PAGE and transferred onto nitrocellulose membrane by semidry transfer method. Immunoblotting was carried out using HIV positive sera as a source of polyclonal antibodies. The blot was blocked with 2.5% non-fat dry milk for two hours at room temperature. The blot was then washed thrice in TTBS (Tween-Tris buffered saline) and incubated in HIV-1 positive human polyclonal serum (at a dilution of 1:50). The blot was subsequently incubated with secondary antibody anti human IgG conjugated with alkaline phosphatase. BCIP NBT solution was used as substrate for AP activity.
Electron microscopy of transfected COS-7 cells
Transmission electron microscopy (TEM) was done as previously described with minor modifications. Briefly, transfected cells were scraped off from the plates and fixed in 1% glutaraldehyde solution for two hours on ice. Cells were then washed thrice with PBS and treated with 1% osmium tetroxide in PBS for two hours. Washing was done thrice and then the sample was dehydrated by treatment with acetone followed by clearing with toluene. After that infiltration was done with toluene araldite mixture at room temperature then infiltration was repeated at 50oC. The sample embedded in the epoxy resin was sectioned and viewed by TEM.
| ~ Results|| |
Construction of pJWgagprotease49587
Gag and protease genes of HIV-1 subtype C were cloned together in pJW4304. For this first complete gag and pol genes were cloned into the pGEMTeasy-cloning vector by PCR based cloning. Gag and protease fragment was extracted from the clone by double digestion by Nhe1 and BamH1, and ligated into the expression vector pJW4304. The recombinant clone pJWgagprotease49587 was confirmed for the presence of required gene fragment by various digestions and PCR amplification products for gag and protease genes.
p24 Antigen Capture ELISA
The amount of protein secreted in the medium by transfected cells was assessed by p24 antigen capture ELISA. The p24 antigen was detectable at 24 hours posttransfection and showed a gradual increase in expression upto 48 hours and thereafter a decline in the amount of secreted protein. The negative controls included untransfected cells and cells transfected with vector pJW4304. None of the control supernates (negatives and mock positive pJWSK3) showed reactivity in the assay. Up to 110-pg/mL protein was detected in the supernates [Figure - 2].
| ~ p24 Estimation in Transfection Supernates|| |
The transfection cell lysates were run on a SDS PAGE and transferred to nitrocellulose membrane for immunoblotting using HIV positive sera as a source of polyclonal antibodies to HIV proteins. The 24 kDa band was detected in the 24 and 48 transfection cell lysates indicating that the 55 kDa gag precursor was being cleaved into respective products. The negative control and mock positive cell lysates did not show any such band [Figure - 3].
Electron microscopy of transfected cells
Numerous virus like particles were seen budding out of the cell membrane and outside the membrane. The morphology of these budding particles corresponded to that expected of virus like particles. These virus like particles were observed in COS-7 cells transfected with pJWgagprotease49587 at 24 and 48 hours posttransfection. The average size of the particles was determined to be 140-nm to160 nm [Figure:4]. The negative and mock positive controls in the study did not show such particles.
| ~ Discussion|| |
In this study, our objective was to produce a HIV-1 subtype C gag based plasmid immunogen from local circulating strains of India. DNA vaccines are known to elicit both cell mediated and humoral immune responses in different disease models. The biggest advantage being native production of the target gene thus allowing post-translational modifications and presentation through both MHC I and II pathways. The gene under consideration in this study was selected owing to the following reasons. It is well established that the core protein is one of the most conserved regions in the HIV-1 genome. In course of HIV-1 infection it is understood that the cytotoxic T lymphocyte (CTL) responses are responsible to bring down the initial burst of viremia. In many studies with long term survivors CTL epitopes have been mapped to the gag region. These studies have shown that maintenance of memory to gag epitopes and various other epitopes could be responsible for virus control. The gag gene is expressed as a precursor protein p55, which is processed into its components by the viral protease; hence we also cloned the protease gene of HIV-1. Protease is encoded by N terminal region of the pol gene of HIV-1 starting within the gag in a -1 frameshift from the gag transcript. The gagprotease construct was cloned under the control of CMV immediate early promoter enhancer of the expression vector pJW4304. Upon evaluation of expression, cells and supernates both showed that gag was being produced and processed into the respective components. Since gag gene was cloned in continuation with the t-PA leader signal sequence in the expression vector it was expected that the protein will be secreted from the cells. However in the event of virus like particle formation also, the virus like particles (VLPs) will be released into the cell culture. In this study numerous VLPs were found in cells at 24 and 48 hours posttransfection indicating good expression. With these observations it could be expected that the construct pJWgagprotease49587 will express fairly good amount of antigen in vivo as well leading to efficient priming of the immune system, thus evoking immune responses, which is our ultimate objective. In the above study, we have successfully cloned gag-protease gene of HIV-1 subtype C from Indian strain into expression vector pJW4304 and confirmed the expression of cloned genes by different methods, thus validating it for further use as a DNA vaccine construct for immunogenicity testing in animal studies.
| ~ Acknowledgements|| |
The author thanks the Jai Vigyan mission, Department of Biotechnology, Government of India for funding this research and University Grants Commission for providing the fellowship.
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