Difference between revisions of "Part:BBa K1621004"

Latest revision as of 11:44, 15 September 2015

gag/tat/pol/env - polyepitopic antigen derived from HIV-1

Figure 1: Schematic view of HIV

Figure 3: Western Blot of HIV multi-epitopic antigen. HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. For blotting, anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa. HIV=Human Immunodeficiency Virus

This part contains the coding sequence of a polyepitopic antigen used for the specific detection of antibodies against Human Immunodeficiency Virus-1 (HIV-1; figure 1). A number of immunogenic epitopes derived from the polyprotein gag/tat/pol/env have been combined in a single nucleotide sequence. Thus, the antibody binding properties remain the same as for the individual proteins but the catalytical functions cannot be fulfilled (Jafarpour et al., 2014).

Originally, this sequence was designed for the development of a polyepitopic DNA vaccine strategy. This strategy relies on the concept of combining immunogenic epitopes for vaccination to focus the immune response on conserved and crucial epitopes (Memarnejadian et al., 2009).
Accordingly, those epitopes have been chosen by means of immunogenicity and conservancy as well as the maintenance of their antibody binding affinities without the rest of the protein. Selection of the epitopes was based on bioinformatic analyses (Jafarpour et al., 2014).

Figure 2: Expression vector for the polytopic peptide derived from HIV. The part was expressed in E. coli using the vector backbone of pET22b+. Fusion of a C-terminal His-tag enabled gravity flow affinity purification. HIV=Human Immunodeficiency Virus.

The proteins the epitopes derive from contribute to various processes in the viral life cycle. The gag gene encodes for the precursor forms of several cellular and nuclear proteins that are markers for very early stages of an infection. env encoded proteins mediate, for example, the attachment to CD4 T cells and can be found from early stages until the end of the disease. Many enzymes that are crucial for the pathogenesis of the virus are encoded by pol. These include, among others, the reverse transcriptase and integrase, important for integration of viral genes into the host genome. The tat gene encodes for the TAT protein which increases viral transcription (Goepfert, 2013).
All in all, the combination of epitopes derived from those proteins enables targeting several different stages of the viral life cycle and thus bears great potential for next generation vaccines (Goepfert, 2013). Additionally, it is a useful tool for the detection of antibodies developed in response to HIV-1 infections at multiple stages.

Cloning the part's sequence into an expression vector (figure 2) enables efficient overexpression of this polyepitopic peptide in Escherichia coli. E.coli BL21 pLys cells were grown in LB medium (Luria Bertani) containing ampicillin and chloramphenicol over night at 28°C. Then the expression was induced with 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside).
The protein was purified by His-tag based affinity purification and analyzed by SDS PAGE afterwards. Figure 3 shows the result of Western Blot analysis. Thus, the polypeptide was efficiently overexpressed and purified without losing its antibody binding affinities.

References

Goepfert PA (2003). Making sense of the HIV immune response. Top HIV Med 11(1):4–8.

Jafarpour N, Memarnejadian A, Aghasadeghi MR, Kohram F, Aghababa H, Khoramabadi N, Mahdavi M (2014). Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice. Mol Biol Rep 41:5207–5214.

Memarnejadian A, Roohvand F, Arashkia A, Rafati S, Shokrgozar MA (2009). Polytope DNA vaccine development against hepatitis C virus: a streamlined approach from in silico design to in vitro and primary in vivo analyses in BALB/c mice. Protein Pept Lett 16(7):842–850.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 [[File:freiburg_2015_schematicHIV.png|200px|thumb|right|'''Figure 1: Schematic view of HIV''']]
-[[File: 2015_Freiburg_pET_1703.jpg|300px|thumb|right|'''Figure 2: Expression vector for the polytopic peptide derived from HIV.''' The part was expressed in pET22b+ with a C-terminal His-tag for affinity purification. HIV=Human Immunodeficiency Virus.]]
+[[File: 2015_Freiburg_WB_17.png|200px|thumb|right|'''Figure 3: Western Blot of HIV multi-epitopic antigen.''' HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. For blotting, anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa. HIV=Human Immunodeficiency Virus]]
-This part contains the coding sequence of a polytopic antigen used for the specific detection of antibodies against Human Immunodeficiency Virus-1 (HIV-1; figure 1). A number of immunogenic epitopes derived from the polyprotein ''gag/tat/pol/env'' have been combined in a single nucleotide sequence. Thus, the antibody binding properties remain the same as for the individual proteins but the catalytical functions cannot be fulfilled (Jafarpour ''et al.'', 2014).
+This part contains the coding sequence of a polyepitopic antigen used for the specific detection of antibodies against Human Immunodeficiency Virus-1 (HIV-1; figure 1). A number of immunogenic epitopes derived from the polyprotein ''gag/tat/pol/env'' have been combined in a single nucleotide sequence. Thus, the antibody binding properties remain the same as for the individual proteins but the catalytical functions cannot be fulfilled (Jafarpour ''et al.'', 2014).
 <br>
 <br>
-Originally, this sequence was designed for the development of a polytopic DNA vaccine strategy. This strategy relies on the concept of combining immunogenic epitopes for vaccination to focus the immune response on conserved and crucial epitopes (Memarnejadian ''et al.'', 2009).
+Originally, this sequence was designed for the development of a polyepitopic DNA vaccine strategy. This strategy relies on the concept of combining immunogenic epitopes for vaccination to focus the immune response on conserved and crucial epitopes (Memarnejadian ''et al.'', 2009).
 <br>
 Accordingly, those epitopes have been chosen by means of immunogenicity and conservancy as well as the maintenance of their antibody binding affinities without the rest of the protein. Selection of the epitopes was based on bioinformatic analyses (Jafarpour ''et al.'', 2014).
+[[File: 2015_Freiburg_pET_1703.jpg|300px|thumb|left|'''Figure 2: Expression vector for the polytopic peptide derived from HIV.''' The part was expressed in ''E. coli'' using the vector backbone of pET22b+. Fusion of a C-terminal His-tag enabled gravity flow affinity purification. HIV=Human Immunodeficiency Virus.]]
-[[File: 2015_Freiburg_WB_17.png|200px|thumb|left|'''Figure 3: Western Blot of HIV multi-epitopic antigen.''' HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. For blotting, anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa. HIV=Human Immunodeficiency Virus]]
-The proteins the epitopes derive from contribute to various processes in the viral life cycle. The ''gag'' gene encodes for the precursor forms of several cellular and nuclear proteins that are markers for very early stages of an infection. ''env'' encoded proteins mediate, for example, the attachment to CD4 T cells and can be found from early stages until the end of the disease. Many enzymes that are crucial for the pathogenesis of the virus are encoded by ''pol''. These include among others the reverse transcriptase and integrase, important for integration of viral genes into the host genome. The ''tat'' gene encodes for the TAT protein which increases viral transcription (Goepfert, 2013).
+The proteins the epitopes derive from contribute to various processes in the viral life cycle. The ''gag'' gene encodes for the precursor forms of several cellular and nuclear proteins that are markers for very early stages of an infection. ''env'' encoded proteins mediate, for example, the attachment to CD4 T cells and can be found from early stages until the end of the disease. Many enzymes that are crucial for the pathogenesis of the virus are encoded by ''pol''. These include, among others, the reverse transcriptase and integrase, important for integration of viral genes into the host genome. The ''tat'' gene encodes for the TAT protein which increases viral transcription (Goepfert, 2013).
 <br>
-All in all, the combination of epitopes derived from those proteins enables to target several different stages of the viral life cycle and thus bears great potential for next generation vaccines (Goepfert, 2013). Additionally, it is a useful tool for the detection of antibodies developed in response to HIV-1 infections at multiple stages.
+All in all, the combination of epitopes derived from those proteins enables targeting several different stages of the viral life cycle and thus bears great potential for next generation vaccines (Goepfert, 2013). Additionally, it is a useful tool for the detection of antibodies developed in response to HIV-1 infections at multiple stages.
 <br>
 <br>
-Cloning the part's sequence into an expression vector (figure 2) enables efficient overexpression of this polyepitopic peptide in ''Escherichia coli''. ''E.coli'' BL21 pLys cells were grown in LB medium (Luria Bertani) containing ampicillin and chloramphenicol over night at 28°C. Then, the expression was induced with 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside).
+Cloning the part's sequence into an expression vector (figure 2) enables efficient overexpression of this polyepitopic peptide in ''Escherichia coli''. ''E.coli'' BL21 pLys cells were grown in LB medium (Luria Bertani) containing ampicillin and chloramphenicol over night at 28°C. Then the expression was induced with 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside).
 <br>
 The protein was purified by His-tag based affinity purification and analyzed by SDS PAGE afterwards. Figure 3 shows the result of Western Blot analysis. Thus, the polypeptide was efficiently overexpressed and purified without losing its antibody binding affinities.