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WO2008095299A1 - Synthetic hiv-2 envelope gene that lead to optimized expression in bacteria for use in hiv-2 antibody immunoassays - Google Patents

Synthetic hiv-2 envelope gene that lead to optimized expression in bacteria for use in hiv-2 antibody immunoassays Download PDF

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Publication number
WO2008095299A1
WO2008095299A1 PCT/CA2008/000239 CA2008000239W WO2008095299A1 WO 2008095299 A1 WO2008095299 A1 WO 2008095299A1 CA 2008000239 W CA2008000239 W CA 2008000239W WO 2008095299 A1 WO2008095299 A1 WO 2008095299A1
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hiv
host
expression
gene
protein
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PCT/CA2008/000239
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French (fr)
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Steven Sui Hu
Catherine C. Q. Shi
Richard A. Galli
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Biolytical Laboratories Inc.
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Publication of WO2008095299A1 publication Critical patent/WO2008095299A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2

Definitions

  • the present invention relates to recombinant HIV-2 (Human Immunodeficiency Virus-2) antigens.
  • Recombinant antigens derived from the molecular cloning and expression in a heterologous expression system can be used as reagents for the detection of antibodies in body fluids from individuals exposed to HIV-2 isolates.
  • the nucleotide sequence of the pro viral genome has been determined for the HIV-2 ROD isolate was reported by Guyader et al. (Nature (1987) 326:662).
  • HIV-2 antigens have previously been obtained from the virus grown in tissue culture, or from a molecularly cloned genomic fragment expressed in heterologous hosts such as Escherichia coli.
  • tissue culture derived virus involves challenging, laborious, and bio-hazardous procedures of growing virus- infected cells and purifying proteins in highly sterile conditions under proper bio-containment facilities.
  • the expression of molecularly cloned HIV-2 genomic fragments overcomes the biohazard problem.
  • an HIV-2 genomic fragment from a single HIV-2 isolate with mammalian codons is expressed in a heterologous system, such as bacteria or yeast, and is limited to the use of available restriction sites present in the viral genome for cloning and expression.
  • codon triplet for each particular amino acid of the synthetic DNA sequence be used.
  • alternative codon(s) can be used to add or delete a particular restriction site.
  • the sequence should include unique restriction sites which can be used to delete a specific fragment or sequence, or substitute a particular sequence in case of an error in the original synthesis.
  • Poor expression can be the result of many factors, including but not limited to the specific nucleic acid sequence of the gene to be expressed, the fact that the mammalian codons of the gene sequence to be expressed may not be efficiently transcribed and translated in a particular heterologous system, and the secondary structure of the transcribed messenger RNA.
  • the use of synthetic DNA fragments can increase expression in heterologous systems.
  • Recombinant antigens which are derived from the molecular cloning and expression of synthetic DNA sequences in heterologous hosts are provided. Synthetic DNA sequences coding for the recombinant antigens of the invention are further provided. The synthetic DNA sequences selected for expression of various HIV antigens are based on the amino acid sequence of either a single isolate or several isolates, optimized for expression in Escherichia coli by specific codon selection. The synthetic DNA sequence gives higher expression of the particular antigen encoded. These antigens can be substituted for viral antigens derived from tissue culture or other recombinant technology procedures for use as diagnostic reagents.
  • the present invention can be utilized to synthesize HIV-2 transmembrane envelope genes using bacterial favorite codons.
  • FIG. 1 illustrates the alignment of the BLHIV- 18 gene amino acid sequence with the sequence of the HIV isolates HIV-2 ROD;
  • FIG. 2 is a list of HIV-2 synthetic primers and oligonucleotides
  • FIG. 3 illustrates the assembly of 4 oligonucleotides to form the synthetic HIV-2 envelope gene, and its cloning into pET41a, designated pBLHIV-3;
  • FIG. 4 is a schematic diagram of the cloning of HIV-2 envelope gene from pBLHIV-3 into expression vectors pMAL c2E to generate pBLHIV-18;
  • FIG. 5 illustrates the DNA and amino acid sequence of fusion protein of MBP and synthetic HIV-2 gene in BLHIV-18, indicating restriction enzymes used to assemble the gene;
  • FIG. 6 depicts a recombinant BLHIV-18 protein Enzyme Immunoassay (EIA) test result
  • FIG. 7 depicts a recombinant BLHIV- 18 protein flow-through immunoassay test result.
  • Synthetic DNA fragments of the HIV-2 genome can be synthesized based on their corresponding amino acid sequences. By comparing the particular region of interest between different isolates, a sequence can be selected which is different from any sequence that exists in nature, because the sequence is a compilation of the sequences from various isolates (reference to FIG. 1). Other properties can be built into the sequence. For example, codons can be switched for optimal expression in bacteria, specific restriction sites can be introduced, and other restriction sites can be removed. In addition, the sequence should have specific restriction sites at both 5' and 3' ends of the fragment to facilitate cloning in a particular vector. Synthetic DNA fragments can be synthesized as follows: (1) select a unique protein sequence, (2) reverse translate to determine complementary DNA sequence, (3) optimize codons for bacterial expression, and (4) introduce and/or remove specific restriction sites.
  • Vector systems which can be used include plant, bacterial, yeast, insect, and mammalian expression systems. It is preferred that the codons are optimized for expression in the system used.
  • the proteins and polypeptides provided by the invention, which are cloned and expressed in heterologous systems can be used for diagnostic purposes.
  • proteins from any source including bacterial, yeast, insect, plant or mammalian, can be used with the synthetic DNA fragments of the invention to produce fusion proteins. Those which are expressed efficiently in their respective expression systems are especially preferred because they can enhance the expression of the synthetic fragment of the fusion protein.
  • HIV-2 envelope gene is inserted into a pMAL vector downstream from the mal E gene, which encodes maltose-binding protein (MBP).
  • MBP maltose-binding protein
  • the vector uses the strong Ptac promoter to express large amount of the fusion protein.
  • the fusion protein is then purified by one-step affinity purification specific for MBP.
  • the synthetic DNA sequences of the present invention derived from HIV-2 isolate and optimized for expression in E. coli provides continuous availability and uniformity of HIV-2 antigen preparations which will recognize test sera from individuals exposed to HIV-2 isolate.
  • the recombinant antigen expression is very stable since E. coli codons have been used for its synthesis.
  • the insertion of specific restriction sites allows addition, deletion, or substitution in important antigenic epitopes in the coding sequences, a property difficult to achieve when naturally occurring HIV sequences are utilized for expression. Construction of synthetic genes also allows the addition of protein sequences at either amino- or carboxyl-terminus of the gene thereby allowing the development of novel reagents.
  • a fusion gene can be produced comprising a fusion between HIV epitopes and other protein.
  • the HIV-2 envelope synthetic gene comprises the HIV-2 gp36 sequence and MBP sequence, which can be expressed at high levels in heterologous host systems such as E. coli.
  • Media such as LB broth, IPTG (isopropyl-.beta.-D-thiogalactoside), glycerol,
  • Dithiothreitol SigmaMarker wide molecular weight range, Lysozyme, Anti-human IgG peroxidase conjugate and protein molecular weight standards were purchased from Sigma; Some restriction enzymes, T4 DNA ligase were purchased from Invitrogen. Pefect DNA Markers, 0.05-10 kbp were purchased from Novagen; Molecular biology agerose, 30% Acrylamide/Bis Solution, N,N,N',N',-
  • TEMED Tetramethylethylenediamine
  • SDS sodium dodecylsulfate
  • Bio-Safe Coomassie Stain Tetramethylethylenediamine
  • PfuUltra hign-fedelity DNA polymerase was purchased from Stratagene.
  • dNTP set, Sequenase Version 2.0 T7 DNA Polymerase and Klenow, Exonuclease-Free were purchased from GE Healthcare.
  • SureBlue TMB microwell peroxidase substrate, Wash solution concentrate (20X) were purchased from ICPL.
  • Some restriction enzymes, Amylose resin were purchased from New Enhland Bio Labs.
  • DH5 ⁇ competent cells was purchased from Invitrogen.
  • pET expression system 41 was purchased from Novagen, E. coli Kl 2 TBl cells, pMAL- c2E vectors were purchased from New Enhland BioLabs. All of bioLytical HIV-2 synthetic primers and oligonucleotides (reference to FIG. 2) were synthesized by Sigma-Genosys.
  • the subfragment-1 designated BLSP-9 through BLSP-10, were synthesized along with additional sequences containing a BamH I restriction site at the 5' end and a
  • the subfragment encoding the N-terminal gp36 amino acid sequence comprised two overlapping oligonucleotides with 16 bp complementary ends. When annealed, the end served as primers for the extension of the complementary strands. 1 ug of each of the two oligonucleotides was annealed. 10 U Sequenase was added and incubated 30 min at 30 degrees C, then heated the reaction at 70 degrees C. for 10 min to inactive the DNA polymerase. The reaction was subsequently digested with the BamH I and Nco I and checked by electrophoresis on a 2% agerose gel. The subframent 1 was further extracted and purified by UltraClean 15 DNA purification kit (Mo
  • the subfragment-2 designated BLSP-11 through BLSP-12, were synthesized along with additional sequences containing a Nco I restriction site at the 5' end and a EcoR I restriction site at the 3' end to facilitate molecular cloning of the individual sub fragments.
  • the sub fragment encoding the gp36 amino acid sequence comprised two overlapping oligonucleotides with 15 bp complementary ends. When annealed, the end served as primers for the extension of the complementary strands. 1 ug of each of the two oligonucleotides was annealed. 10 U Sequenase was added and incubated 30 min at 30 degrees C, then heated the reaction at 70 degrees C. for 10 min to inactive the DNA polymerase.
  • the reaction was subsequently digested with the Nco I and EcoR I and checked by electrophoresis on a 2% agerose gel.
  • the subframent-2 was further extracted and purified by UltraClean 15 DNA purification kit (Mo Bio) for cloning use.
  • the subfragment-1 with a BamH I restriction site at the 5' end and a Nco I restriction site at the 3' end and the subfragment-2 with Nco I restriction site at the 5' end and a EcoR I restriction site at the 3' end were ligated into the vector pET41a (Novagen) that had been digested with BamH I and EcoR I and gel-isolated.
  • the ligation product was used to transform DH5 ⁇ competent cells (clone pBLHIV-3). The desired clone was identified by digestion with BamH I and EcoR I. and checked on a 1% agerose gel.
  • the pBHIV-18 encodes the recombinant HIV-2 envelope protein that was consisted of fusion protein of MBP and 106 amino acids of HIV-2 gp36 (reference to FIG. 5).
  • the construction of pBLHIV-18 was accomplished as follows.
  • a PCR reaction (50 ul) was set up with Stratagene PfuUltra hign-fedelity DNA polymerase (2.5 U) and IX buffer along with 10 mM each dNTP, 100 ng primer
  • BLSP-24 100 ng primer BLSP-28, and 1 ng pBLHIV-3 miniprep DNA.
  • the reaction was incubated at 95 degrees C. for 120 seconds then amplified with 30 cycles of 94 degrees C. for 30 seconds; 54 degrees C. for 45 seconds; 72 degrees C. for 60 seconds. Then the BLSP-24/BLSP-28 PCR product was gel isolated.
  • the BLSP-24/BLSP-28 PCR product was digested with Kpn I and BamH I and ligated into pMAL c2E (BioLabs), which had been digested with Kpn I and BamH I.
  • the ligation product was used to transform TBl Kl 2 competent cells (clone pB LHIV- 18).
  • the desired clone was identified by digestion with Kpn I and BamH I and checked on a 1% agerose gel.
  • Miniprep DNA was prepared from an overnight culture of clone BLHIV-18 and sequenced with the oligonucleotide primers Ml 3 forward by Cortec DNA Service Laboratories, Inc.
  • Amylose column buffer was prepared by dissolving 1.2 g of Trizma base, 11.7 g of NaCl, and 372 mg of EDTA in 900 ml of Milli-Q water. Once the chemicals were completely dissolved, 700 ul of 1 mM DTT was added and pH was adjusted to 7.4 (within 0.1) with 10 N HCl. This solution should not be used if stored for longer than 30 days.
  • Amylose elute buffer was prepared by dissolving 1.2 g of Trizma base, 11.7 g of NaCl, 372 mg of EDTA, and 3.42 g of Maltose in 900 ml of Milli-Q water. Once the chemicals were completely dissolved, 700 ul of 1 mM DTT was added and the pH of the solution was adjusted to 7.4 (within 0.1) with IO N HCl. This solution should not be used if stored for longer than 30 days.
  • LB Broth with glucose was prepared by dissolving 20 g of LB broth and 2 g of glucose in 1 L of Milli-Q water, adjusting the pH to 7.2, and sterilizing by autoclaving. The solution was allowed to cool and Ampicillin was added to a final concentration of 0.05 mg/ml.
  • the cultures were induced by adding 1 ml of 200 mM IPTG to each flask.
  • the flasks were covered and incubated for 4 hours at 37 degrees C. and 180 RPM.
  • the cells were harvested by centrifugation in the Sorvall RC-5B at 5000 RPM for 20 minutes. If cells were not required for immediate purification, they were stored at -20 degrees C.
  • the cell pellet was thawed and resuspended in a total of 20 ml of Novagen BugBuster protein extraction reagent (100 ml/1 L culture ratio) with 20 ul of 100 mM PMSF Stock, 40 ul of 1 M DTT and 10 ul of Benzonase (Novagen).
  • the cell suspension was incubated on a shaking platform for 1 hour at room temperature.
  • the extract should not be viscous at the end of the incubation.
  • the solution was centrifuged in a Sorvall RC-5B at 9000 RPM in a GS-3 rotor for 50 min. Then the pellet was resuspended in 30 ml of Inclusion Body Wash Solution with 40 ul of 1 M DTT.
  • the solution was sonicated for 6 X 15 seconds (60 Sonic Dismembrator Fisher Scientific). The solution was then centrifliged in a Sorvall RC-5B at 9000 RPM in a GS-3 rotor for 50 min. The crude recombinant BLHIV-18 protein (supernatant) was stored at 4 degrees C. until further process.
  • the crude recombinant BLHIV-18 protein was diluted 1 :5 with Amylose column buffer. 15 ml of diluted crude extract was loaded at a flow rate of 0.2 ml/min to 3 ml of Amylose resin column, which was equilibrated with 8 column volumes of Amylose Column Buffer. The column was washed with 8 -12 column volumes of Amylose Column Buffer. 10 column volumes of the Amylose Elute Buffer were run through the column. The fractions were collected and stored at 4 degrees C.
  • the purity of the peak fractions was determined by running 10 ul aliquots of each fraction on a 12% SDS-PAGE gel under reducing conditions. The peak fractions were pooled and further concentrated. The protein concentration of this solution was determined, and then 5 ug/lane of sample were run on a 12% SDS-PAGE to check the purity. Applying the recombinant BLHIV- 18 protein in the Enzyme Immunoassay (EIA) assay and flow-through immunoassay would be discussed in Example 3.
  • EIA Enzyme Immunoassay
  • EIA Enzyme Immunoassay
  • the present example demonstrates the utility of the invention for providing an EIA for the detection of HIV-2 antibodies employing the recombinant BLHIV-18 protein.
  • the recombinant BLHIV- 18 protein was prepared as described in Example 2.
  • the EIA assay may follow any variety of testing formats known to those of skill in the art, given the information of the present disclosure.
  • the recombinant BLHIV- 18 protein was used to coat NUNC MaxiSorp microtiter wells at 500 ng/well. Following 1 hour of coating in 37 degrees C, the wells were "blocked" with 1 % BSA, and rinsed 5 times with PBS buffer. 5 ul of serum or plasma was added to individual wells containing 100 ul of reaction buffer (PBS containing 1% BSA). The following steps are typical of an EIA format involving incubation for 20 min at 37 degrees C, 5 rinses with PBS buffer, and incubation with goat anti- human IgG-HRP conjugate (SIGMA) for 20 min at 37 degrees C. The wells then were rinsed 5 times and the SureBlue TMB substrate (KPL) was added.
  • SIGMA goat anti- human IgG-HRP conjugate
  • the plate was read on an ELISA plate reader at the optical density at 450 nm. Controls included wells with HIV-2 positive and negative specimen. The cut-off value for a positive result was set at 0.200 Absorbance Units above the average absorbance obtained from the negative control.
  • the HIV-2 antibody test consists of a synthetic filtration membrane positioned a top, an absorbent material within a plastic cartridge.
  • the membrane was blotted 1 ul containing 2 ug of the recombinant BLHIV-18 protein of the invention prepared as described in Example 2, which reacted with HIV-2 antibodies in the specimen to produce a distinct visual signal on the membrane.
  • the membrane also included a human IgG-capture control which consisted of a protein- A treated spot capable of binding IgG antibodies normally presented in blood and blood components. If the control spot did not appear, the test was considered invalid.
  • the test was performed by adding 50 ul of the blood, serum, or plasma specimen to the vial of Sample Diluent which lysed the red blood cells. This specimen diluent solution was then poured onto the well of the Membrane Unit. HIV-2 antibodies, if present in the specimen, were captured by the recombinant BLHIV-18 protein on the filtration membrane. Color Developer was then added to the Membrane Unit. The Color Developer reacted with the captured antibodies to generate a distinct blue dot at the location of the control spot and, in the case that HIV-2 antibodies were present in the specimen, a blue dot also appeared at the location of the test spot on the membrane. In the final step, the Clarifying Solution was then added to the membrane to decrease background color in order to make the control and test spots more distinct.

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Abstract

A unique HIV-2 envelope gene using E. CoIi favorite codon, resulting in high-level expression in E. coli. The HIV-2 envelope protein thus expressed in E. coli can be used to detect HIV-2 antibody in in vitro diagnostic devices.

Description

SYNTHETIC HIV-2 ENVELOPE GENE THAT LEAD TO OPTIMIZED EXPRESSION IN BACTERIA FOR USE IN HIV-2 ANTIBODY
IMMUNOASSAYS
BACKGROUND OF THE INVENTION
The present invention relates to recombinant HIV-2 (Human Immunodeficiency Virus-2) antigens. Recombinant antigens derived from the molecular cloning and expression in a heterologous expression system can be used as reagents for the detection of antibodies in body fluids from individuals exposed to HIV-2 isolates.
The nucleotide sequence of the pro viral genome has been determined for the HIV-2 ROD isolate was reported by Guyader et al. (Nature (1987) 326:662).
HIV-2 antigens have previously been obtained from the virus grown in tissue culture, or from a molecularly cloned genomic fragment expressed in heterologous hosts such as Escherichia coli. The tissue culture derived virus involves challenging, laborious, and bio-hazardous procedures of growing virus- infected cells and purifying proteins in highly sterile conditions under proper bio-containment facilities. The expression of molecularly cloned HIV-2 genomic fragments overcomes the biohazard problem. Generally, an HIV-2 genomic fragment from a single HIV-2 isolate with mammalian codons is expressed in a heterologous system, such as bacteria or yeast, and is limited to the use of available restriction sites present in the viral genome for cloning and expression.
Sixty-one distinct nucleotide codons code for 20 amino acids giving rise to the degeneracy in the genetic code. Researchers have reported the frequencies of codons used in nucleic acids for both eukaryotic and prokaryotic organisms. (Grantham et al., Nucleic Acids Res. [1980] 9:r43; Gouy et al., Nucleic Acids Res. [1982] 10:7055; Sharp et al., Nucleic Acids Res. [1986] 14:7737.) Sequences from the entire E. coli genome, with examples of sequences from the chromosome, transposons, and plasmids, have been analyzed. These sequences code for structural proteins, enzymes and regulatory proteins. Correlation has been shown between the degree of codon bias within a particular gene and the level of gene expression.
It is preferred that the same codon triplet for each particular amino acid of the synthetic DNA sequence be used. However, alternative codon(s) can be used to add or delete a particular restriction site. The sequence should include unique restriction sites which can be used to delete a specific fragment or sequence, or substitute a particular sequence in case of an error in the original synthesis.
Poor expression can be the result of many factors, including but not limited to the specific nucleic acid sequence of the gene to be expressed, the fact that the mammalian codons of the gene sequence to be expressed may not be efficiently transcribed and translated in a particular heterologous system, and the secondary structure of the transcribed messenger RNA. The use of synthetic DNA fragments can increase expression in heterologous systems.
SUMMARY OF THE INVENTION
Recombinant antigens which are derived from the molecular cloning and expression of synthetic DNA sequences in heterologous hosts are provided. Synthetic DNA sequences coding for the recombinant antigens of the invention are further provided. The synthetic DNA sequences selected for expression of various HIV antigens are based on the amino acid sequence of either a single isolate or several isolates, optimized for expression in Escherichia coli by specific codon selection. The synthetic DNA sequence gives higher expression of the particular antigen encoded. These antigens can be substituted for viral antigens derived from tissue culture or other recombinant technology procedures for use as diagnostic reagents.
The present invention can be utilized to synthesize HIV-2 transmembrane envelope genes using bacterial favorite codons.
The foregoing was intended as a broad summary only and of only some of the aspects of the invention. It was not intended to define the limits or requirements of the invention. Other aspects of the invention will be appreciated by reference to the detailed description of the preferred embodiment and to the claims. BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be better understood with reference to the drawings in which:
FIG. 1 illustrates the alignment of the BLHIV- 18 gene amino acid sequence with the sequence of the HIV isolates HIV-2 ROD;
FIG. 2 is a list of HIV-2 synthetic primers and oligonucleotides;
FIG. 3 illustrates the assembly of 4 oligonucleotides to form the synthetic HIV-2 envelope gene, and its cloning into pET41a, designated pBLHIV-3;
FIG. 4 is a schematic diagram of the cloning of HIV-2 envelope gene from pBLHIV-3 into expression vectors pMAL c2E to generate pBLHIV-18;
FIG. 5 illustrates the DNA and amino acid sequence of fusion protein of MBP and synthetic HIV-2 gene in BLHIV-18, indicating restriction enzymes used to assemble the gene;
FIG. 6 depicts a recombinant BLHIV-18 protein Enzyme Immunoassay (EIA) test result; and
FIG. 7 depicts a recombinant BLHIV- 18 protein flow-through immunoassay test result.
DETAILED DESCRIPTION OF THE INVENTION
Synthetic DNA fragments of the HIV-2 genome can be synthesized based on their corresponding amino acid sequences. By comparing the particular region of interest between different isolates, a sequence can be selected which is different from any sequence that exists in nature, because the sequence is a compilation of the sequences from various isolates (reference to FIG. 1). Other properties can be built into the sequence. For example, codons can be switched for optimal expression in bacteria, specific restriction sites can be introduced, and other restriction sites can be removed. In addition, the sequence should have specific restriction sites at both 5' and 3' ends of the fragment to facilitate cloning in a particular vector. Synthetic DNA fragments can be synthesized as follows: (1) select a unique protein sequence, (2) reverse translate to determine complementary DNA sequence, (3) optimize codons for bacterial expression, and (4) introduce and/or remove specific restriction sites.
Vector systems which can be used include plant, bacterial, yeast, insect, and mammalian expression systems. It is preferred that the codons are optimized for expression in the system used. The proteins and polypeptides provided by the invention, which are cloned and expressed in heterologous systems can be used for diagnostic purposes.
Other proteins from any source, including bacterial, yeast, insect, plant or mammalian, can be used with the synthetic DNA fragments of the invention to produce fusion proteins. Those which are expressed efficiently in their respective expression systems are especially preferred because they can enhance the expression of the synthetic fragment of the fusion protein.
In the present invention, HIV-2 envelope gene is inserted into a pMAL vector downstream from the mal E gene, which encodes maltose-binding protein (MBP). This results in the expression of an MBP-fusion protein. The vector uses the strong Ptac promoter to express large amount of the fusion protein. The fusion protein is then purified by one-step affinity purification specific for MBP.
The synthetic DNA sequences of the present invention, derived from HIV-2 isolate and optimized for expression in E. coli provides continuous availability and uniformity of HIV-2 antigen preparations which will recognize test sera from individuals exposed to HIV-2 isolate. The recombinant antigen expression is very stable since E. coli codons have been used for its synthesis. Moreover, the insertion of specific restriction sites allows addition, deletion, or substitution in important antigenic epitopes in the coding sequences, a property difficult to achieve when naturally occurring HIV sequences are utilized for expression. Construction of synthetic genes also allows the addition of protein sequences at either amino- or carboxyl-terminus of the gene thereby allowing the development of novel reagents. For example, a fusion gene can be produced comprising a fusion between HIV epitopes and other protein. More specifically the HIV-2 envelope synthetic gene comprises the HIV-2 gp36 sequence and MBP sequence, which can be expressed at high levels in heterologous host systems such as E. coli.
The following examples further describe the invention. The examples are not intended to limit the invention in any manner.
Reagents and Enzymes
Media such as LB broth, IPTG (isopropyl-.beta.-D-thiogalactoside), glycerol,
Dithiothreitol, SigmaMarker wide molecular weight range, Lysozyme, Anti-human IgG peroxidase conjugate and protein molecular weight standards were purchased from Sigma; Some restriction enzymes, T4 DNA ligase were purchased from Invitrogen. Pefect DNA Markers, 0.05-10 kbp were purchased from Novagen; Molecular biology agerose, 30% Acrylamide/Bis Solution, N,N,N',N',-
Tetramethylethylenediamine (TEMED) and sodium dodecylsulfate (SDS), Bio-Safe Coomassie Stain were purchased from BioRad Laboratories. PfuUltra hign-fedelity DNA polymerase was purchased from Stratagene. dNTP set, Sequenase Version 2.0 T7 DNA Polymerase and Klenow, Exonuclease-Free were purchased from GE Healthcare. SureBlue TMB microwell peroxidase substrate, Wash solution concentrate (20X) were purchased from ICPL. Some restriction enzymes, Amylose resin were purchased from New Enhland Bio Labs.
Host Cell, Vectors and bioLytical HIV-I Synthetic Primers and Oligonucleotides
Subcloning efficiency DH5α competent cells was purchased from Invitrogen. pET expression system 41 was purchased from Novagen, E. coli Kl 2 TBl cells, pMAL- c2E vectors were purchased from New Enhland BioLabs. All of bioLytical HIV-2 synthetic primers and oligonucleotides (reference to FIG. 2) were synthesized by Sigma-Genosys.
General Methods
All restriction enzyme digestions were performed according to suppliers' instructions. At least 5 units of enzyme were used per microgram of DNA, and sufficient incubation was allowed to complete digestions of DNA. Plasmid isolations from E. coli strains used QIAprep Spin Miniprep Kit (Qiagen). Gel extraction of DNA fragments used MinElute Gel Extraction Kit (Qiagen) and UltraClean 15 DNA purification kit (Mo Bio). Standard buffers were used for T4 DNA ligase and
Sequenase Version 2.0 T7 DNA Polymerase. Standard procedures were used for in vitro amplification of DNA by the Polymerase Chain Reaction, Gel electrophoresis of DNA, SDS-PAGE, and Preparation and transformation of competent E. Coli using calcium chloride (Maniatis et al., Molecular Cloning. A Laboratory Manual third edition [New York: Cold Spring Harbor, 2001]). Standard procedure was used for gene synthesis (Maniatis et al., Short Protocols in molecular biology Molecular third edition [John Wiley & Sons, Inc., 1995]).
EXAMPLES
Example 1
Construction of HIV -2 Clones
Synthesis and Cloning of HIV-2 gp36 (reference to FIG. 3)
A. Synthesis of Subfragment- 1
The subfragment-1 , designated BLSP-9 through BLSP-10, were synthesized along with additional sequences containing a BamH I restriction site at the 5' end and a
Nco I restriction site at the 3' end to facilitate molecular cloning. The subfragment encoding the N-terminal gp36 amino acid sequence comprised two overlapping oligonucleotides with 16 bp complementary ends. When annealed, the end served as primers for the extension of the complementary strands. 1 ug of each of the two oligonucleotides was annealed. 10 U Sequenase was added and incubated 30 min at 30 degrees C, then heated the reaction at 70 degrees C. for 10 min to inactive the DNA polymerase. The reaction was subsequently digested with the BamH I and Nco I and checked by electrophoresis on a 2% agerose gel. The subframent 1 was further extracted and purified by UltraClean 15 DNA purification kit (Mo
Bio) for cloning use.
B. Synthesis of Subfragment-2
The subfragment-2, designated BLSP-11 through BLSP-12, were synthesized along with additional sequences containing a Nco I restriction site at the 5' end and a EcoR I restriction site at the 3' end to facilitate molecular cloning of the individual sub fragments. The sub fragment encoding the gp36 amino acid sequence comprised two overlapping oligonucleotides with 15 bp complementary ends. When annealed, the end served as primers for the extension of the complementary strands. 1 ug of each of the two oligonucleotides was annealed. 10 U Sequenase was added and incubated 30 min at 30 degrees C, then heated the reaction at 70 degrees C. for 10 min to inactive the DNA polymerase. The reaction was subsequently digested with the Nco I and EcoR I and checked by electrophoresis on a 2% agerose gel. The subframent-2 was further extracted and purified by UltraClean 15 DNA purification kit (Mo Bio) for cloning use.
C. Cloning of Sub fragment- 1 and Subfragment-2
The subfragment-1 with a BamH I restriction site at the 5' end and a Nco I restriction site at the 3' end and the subfragment-2 with Nco I restriction site at the 5' end and a EcoR I restriction site at the 3' end were ligated into the vector pET41a (Novagen) that had been digested with BamH I and EcoR I and gel-isolated. The ligation product was used to transform DH5α competent cells (clone pBLHIV-3). The desired clone was identified by digestion with BamH I and EcoR I. and checked on a 1% agerose gel. Miniprep DNA was prepared from an overnight culture of clone pBLHIV-3 and sequenced with the oligonucleotide primers T7 Terminator reverse by Cortec DNA Service Laboratories, Inc. Construction of pBLHIV-18 (reference to FIG. 4)
The pBHIV-18 encodes the recombinant HIV-2 envelope protein that was consisted of fusion protein of MBP and 106 amino acids of HIV-2 gp36 (reference to FIG. 5). The construction of pBLHIV-18 was accomplished as follows.
A. PCR
A PCR reaction (50 ul) was set up with Stratagene PfuUltra hign-fedelity DNA polymerase (2.5 U) and IX buffer along with 10 mM each dNTP, 100 ng primer
BLSP-24, 100 ng primer BLSP-28, and 1 ng pBLHIV-3 miniprep DNA. The reaction was incubated at 95 degrees C. for 120 seconds then amplified with 30 cycles of 94 degrees C. for 30 seconds; 54 degrees C. for 45 seconds; 72 degrees C. for 60 seconds. Then the BLSP-24/BLSP-28 PCR product was gel isolated.
B. Cloning
The BLSP-24/BLSP-28 PCR product was digested with Kpn I and BamH I and ligated into pMAL c2E (BioLabs), which had been digested with Kpn I and BamH I. The ligation product was used to transform TBl Kl 2 competent cells (clone pB LHIV- 18). The desired clone was identified by digestion with Kpn I and BamH I and checked on a 1% agerose gel. Miniprep DNA was prepared from an overnight culture of clone BLHIV-18 and sequenced with the oligonucleotide primers Ml 3 forward by Cortec DNA Service Laboratories, Inc.
Example 2
Procedure for Producing and Purifying Recombinant BLHIV- 18 Protein
Preparation of Required Solutions:
Amylose column buffer was prepared by dissolving 1.2 g of Trizma base, 11.7 g of NaCl, and 372 mg of EDTA in 900 ml of Milli-Q water. Once the chemicals were completely dissolved, 700 ul of 1 mM DTT was added and pH was adjusted to 7.4 (within 0.1) with 10 N HCl. This solution should not be used if stored for longer than 30 days.
Amylose elute buffer was prepared by dissolving 1.2 g of Trizma base, 11.7 g of NaCl, 372 mg of EDTA, and 3.42 g of Maltose in 900 ml of Milli-Q water. Once the chemicals were completely dissolved, 700 ul of 1 mM DTT was added and the pH of the solution was adjusted to 7.4 (within 0.1) with IO N HCl. This solution should not be used if stored for longer than 30 days.
LB Broth with glucose was prepared by dissolving 20 g of LB broth and 2 g of glucose in 1 L of Milli-Q water, adjusting the pH to 7.2, and sterilizing by autoclaving. The solution was allowed to cool and Ampicillin was added to a final concentration of 0.05 mg/ml.
Growth of and Induction of pBLHIV-18 TBl Kl 2
A 250 ml Flask containing 50 ml LB Broth (0.05mg/ml Ampicillin) was innoculated with 0.02 ml of pBLHIV-18 TBl K12 glycerol stock and grown overnight in the incubator shaker at 37 degrees C. and 180 RPM. Each of 500 ml flasks containing 200 ml of LB Broth with glucose was innoculated with 10 ml of culture grown in the previous step. Cultures were grown in the incubator shaker at 37 degrees C. and 180 RPM until the O.D. at 600 nm was between 0.7 and 1.0 (3 hours).
The cultures were induced by adding 1 ml of 200 mM IPTG to each flask. The flasks were covered and incubated for 4 hours at 37 degrees C. and 180 RPM. The cells were harvested by centrifugation in the Sorvall RC-5B at 5000 RPM for 20 minutes. If cells were not required for immediate purification, they were stored at -20 degrees C.
Induction was analyzed on a 12% SDS-PAGE gel under reducing conditions.
Extraction and Isolating of Recombinant BLHIV-18 Protein:
The cell pellet was thawed and resuspended in a total of 20 ml of Novagen BugBuster protein extraction reagent (100 ml/1 L culture ratio) with 20 ul of 100 mM PMSF Stock, 40 ul of 1 M DTT and 10 ul of Benzonase (Novagen). The cell suspension was incubated on a shaking platform for 1 hour at room temperature. The extract should not be viscous at the end of the incubation. The solution was centrifuged in a Sorvall RC-5B at 9000 RPM in a GS-3 rotor for 50 min. Then the pellet was resuspended in 30 ml of Inclusion Body Wash Solution with 40 ul of 1 M DTT.
In order to enable efficient resuspension, the solution was sonicated for 6 X 15 seconds (60 Sonic Dismembrator Fisher Scientific). The solution was then centrifliged in a Sorvall RC-5B at 9000 RPM in a GS-3 rotor for 50 min. The crude recombinant BLHIV-18 protein (supernatant) was stored at 4 degrees C. until further process.
Purification of Crude Recombinant BLHIV- 18 Protein on Amylose Column:
The crude recombinant BLHIV-18 protein was diluted 1 :5 with Amylose column buffer. 15 ml of diluted crude extract was loaded at a flow rate of 0.2 ml/min to 3 ml of Amylose resin column, which was equilibrated with 8 column volumes of Amylose Column Buffer. The column was washed with 8 -12 column volumes of Amylose Column Buffer. 10 column volumes of the Amylose Elute Buffer were run through the column. The fractions were collected and stored at 4 degrees C.
The purity of the peak fractions was determined by running 10 ul aliquots of each fraction on a 12% SDS-PAGE gel under reducing conditions. The peak fractions were pooled and further concentrated. The protein concentration of this solution was determined, and then 5 ug/lane of sample were run on a 12% SDS-PAGE to check the purity. Applying the recombinant BLHIV- 18 protein in the Enzyme Immunoassay (EIA) assay and flow-through immunoassay would be discussed in Example 3.
Example 3
Diagnostic Utility of Recombinant BLHIV-18 Protein
A. Enzyme Immunoassay (EIA) with of Recombinant BLHIV- 18 Protein
The present example demonstrates the utility of the invention for providing an EIA for the detection of HIV-2 antibodies employing the recombinant BLHIV-18 protein. The recombinant BLHIV- 18 protein was prepared as described in Example 2. The EIA assay may follow any variety of testing formats known to those of skill in the art, given the information of the present disclosure.
The recombinant BLHIV- 18 protein was used to coat NUNC MaxiSorp microtiter wells at 500 ng/well. Following 1 hour of coating in 37 degrees C, the wells were "blocked" with 1 % BSA, and rinsed 5 times with PBS buffer. 5 ul of serum or plasma was added to individual wells containing 100 ul of reaction buffer (PBS containing 1% BSA). The following steps are typical of an EIA format involving incubation for 20 min at 37 degrees C, 5 rinses with PBS buffer, and incubation with goat anti- human IgG-HRP conjugate (SIGMA) for 20 min at 37 degrees C. The wells then were rinsed 5 times and the SureBlue TMB substrate (KPL) was added. Following addition of stop buffer, the plate was read on an ELISA plate reader at the optical density at 450 nm. Controls included wells with HIV-2 positive and negative specimen. The cut-off value for a positive result was set at 0.200 Absorbance Units above the average absorbance obtained from the negative control.
The results in FIG. 6 showed the reactivity of the recombinant BLHlV- 18 protein of the invention with HIV-2 positive and negative specimen in the EIA assay.
B. A Flow-through Immunoassay for the Rapid Detection of HIV-2 with Recombinant BLHIV- 18 Protein
A flow-through immunoassay for the presence of antibodies to HIV-2 in test serum, plasma and whole blood was performed as follows: The HIV-2 antibody test consists of a synthetic filtration membrane positioned a top, an absorbent material within a plastic cartridge. The membrane was blotted 1 ul containing 2 ug of the recombinant BLHIV-18 protein of the invention prepared as described in Example 2, which reacted with HIV-2 antibodies in the specimen to produce a distinct visual signal on the membrane. The membrane also included a human IgG-capture control which consisted of a protein- A treated spot capable of binding IgG antibodies normally presented in blood and blood components. If the control spot did not appear, the test was considered invalid.
The test was performed by adding 50 ul of the blood, serum, or plasma specimen to the vial of Sample Diluent which lysed the red blood cells. This specimen diluent solution was then poured onto the well of the Membrane Unit. HIV-2 antibodies, if present in the specimen, were captured by the recombinant BLHIV-18 protein on the filtration membrane. Color Developer was then added to the Membrane Unit. The Color Developer reacted with the captured antibodies to generate a distinct blue dot at the location of the control spot and, in the case that HIV-2 antibodies were present in the specimen, a blue dot also appeared at the location of the test spot on the membrane. In the final step, the Clarifying Solution was then added to the membrane to decrease background color in order to make the control and test spots more distinct.
The results in FIG. 7 showed the reactivity of the recombinant BLHlV-18 protein of the invention with HIV-2 positive and negative specimen in the flow-through immunoassay.
It will be appreciated by those skilled in the art that the preferred and alternative embodiments have been described in some detail but that certain modifications may be practiced without departing from the principles of the invention.

Claims

What is claimed is:
L A HIV-2 synthetic gene comprising the following DNA sequence:
1 5'-CTGCTCGATG TGGTGAAACG CCAACAAGAG CTGCTGCGTC TGACCGTTTG 51 GGGCACCAAA AATCTCCAAG CGCGTGTGAC CGCGATTGAA AAATATCTGG 101 AAGATCAGGC GCAACTGAAT GCGTGGGGTT GTGCGTTTCG CCAGGTGTGC 151 CATACCACCG TGCCATGGGT GAACGATAGC CTGGCCCCGG ATTGGGATAA 201 TATGACCTGG CAAGAGTGGG AGAAGCAAGT GCGCTACCTC GAAGCGAACA 251 TTAGCAAAAG TCTCGAACAG GCGCAGATTC AGCAGGAAAA AAACATGTAT 301 GAACTGCAGA AACTGAATTAA-3'
2. The synthetic gene of Claim 1 encoding for a polypeptide having the following amino acid sequence:
NH2-LLDWKRQQELLRLTVWGTKNLQARVTAIEKYLEDQAQLNAWGCAFRQVC HTTVPWVNDSLAPDWDNMTWQEWEKQVRYLEANISKSLEQAQIQQEKNMY ELQKLN*-COOH
3. An expression vector containing the synthetic gene of Claim 1 , where said gene encodes for a polypeptide having the following sequence:
NH2-LLDWKRQQELLRLTVWGTKNLQARVTAIEKYLEDQAQLNAWGCAFRQVC HTTVPWVNDSLAPDWDNMTWQEWEKQVRYLEANISKSLEQAQIQQEKNMY ELQKLN*-COOH
4. The expression vector of Claim 3 wherein heterologous gene expression is regulated by the Ptac Promoter.
5. A host transformed with the expression vector of Claim 3.
6. The transformed host of Claim 5 wherein said host is E. coli.
7. The host of Claim 6 wherein said host is E. coli Stain Kl 2 TBl.
8. A method for performing an immunoassay comprising the steps of: contacting a biological test sample with a polypeptide or protein composition according to Claim 2; and detecting an immunological complex formed between antibodies to HIV-2 in said biological test sample and said peptide or protein composition, characterized in that the presence of said complex being indicative of the presence of antibodies to HIV-2 in said biological sample.
9. The method according to Claim 8, characterized in that said polypeptide or protein composition is immobilized to a solid support.
10. The method according to Claim 9, characterized in that said solid support is a nitrocellulose membrane.
11. The method according any one of Claims 8 to 10, characterized in that the presence of said complex is determined by the addition of an indicator reagent.
12. The method according to Claim 11 , characterized in that said indicator reagent is a signal-generating component attached to a specific binding molecule capable of binding to a human HIV-2 antibody.
13. The method according to Claim 12, characterized in that said signal- generating component is Indigo Blue.
14. The method according to Claim 9, characterized in that said solid support is an assay plate comprising a multiplicity of microtiter wells.
15. The method according any one of Claims 8, 9, and 14, wherein a step comprises binding of labeled antibodies to human Ig and the specific binding of said labeled antibodies are measured.
16. The method according to Claim 15, wherein said labeled antibodies are identified by an enzyme label.
17. The method according to Claim 16, wherein said enzyme label is Anti-Human
IgG HRP conjugate.
PCT/CA2008/000239 2007-02-09 2008-02-08 Synthetic hiv-2 envelope gene that lead to optimized expression in bacteria for use in hiv-2 antibody immunoassays WO2008095299A1 (en)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDRE S. ET AL.: "Increased Immune Response Elicited by DNA Vaccination with a Synthetic gp120 Sequence with Optimized Codon Usage", JOURNAL OF VIROLOGY, vol. 72, no. 2, 1998, pages 1497 - 1503, XP002073767 *
HAAS J. ET AL.: "Codon usage limitation in the expression of HIV-1 envelope glycoprotein", CURRENT BIOLOGY, vol. 6, no. 3, 1996, pages 315 - 324, XP000619599 *
HODINKA R.L. ET AL.: "Detection of Human Immunodeficiency Virus Antibodies in Oral Fluid", CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, vol. 5, no. 4, 1998, pages 419 - 426 *
HOLLER T.P. ET AL.: "HIV1 integrase expressed Escherichia coli from a synthetic gene", GENE, vol. 136, 1993, pages 323 - 328, XP023797227, DOI: doi:10.1016/0378-1119(93)90488-O *
MAKRIDES S.C.: "Strategies for Achieving High-Level Expression of Genes in Escherichia coli", MICROBIOLOGICAL REVIEWS, vol. 60, no. 3, 1996, pages 512 - 538, XP002922140 *

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