WO2008095298A1

WO2008095298A1 - Synthetic hiv-i envelope gene that lead to optimized expression in bacteria for use in hiv-i antibody immunoassays

Info

Publication number: WO2008095298A1
Application number: PCT/CA2008/000238
Authority: WO
Inventors: Steven Sui Hu; Catherine C. Q. Shi; Richard A. Galli
Original assignee: Biolytical Laboratories Inc.
Priority date: 2007-02-09
Filing date: 2008-02-08
Publication date: 2008-08-14
Also published as: CA2575000A1; WO2008095299A1

Abstract

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

Description

SYNTHETIC HIV-I ENVELOPE GENE THAT LEAD TO OPTIMIZED EXPRESSION IN BACTERIA FOR USE IN HIV-I ANTIBODY

IMMUNOASSAYS

BACKGROUND OF THE INVENTION

The present invention relates to recombinant HIV (Human Immunodeficiency Virus) 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 various HIV isolates.

The nucleotide sequence of the pro viral genome has been determined for several HIV isolates, including HIV-I strains HTLV-III (HIV BHlO) (Ratner et al., Nature (1985) 313:277); ARV-2 (Sanchez-Pescador et al., Science (1985) 227:484); LAV (Wain- Hobson et al., Cell (1985) 40:9); and CDC-451 (Desai et al., Proc. Natl. Acad. Sci. USA (1986) 83:8380).

HIV 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 genomic fragments overcomes the biohazard problem. Generally, an HIV genomic fragment from a single HIV 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 has been difficult to obtain expression in heterologous systems of some of the HIV proteins, such as the HIV-I envelope antigen gp41. Several researchers have tried deleting the hydrophobic regions of the HIV-I gp41 to increase expression levels. UK Patent Application GB 2188639 discloses an HTLV-III gag/env gene protein wherein the env fragment of the DNA sequence deleted codons corresponds to the first hydrophobic region of the gp41 protein. U.S. Pat. No. 4,753,873 discloses a peptide fragment that is encoded by a nucleotide sequence wherein the nucleotides coding for a first and second hydrophobic region of HTLV-III gp41 are deleted.

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 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-I transmembrane envelope genes using bacterial favorite codons. Another aspect of the invention involves the linkage of sequences comprising different HIV epitopes to express with high efficiency.

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 QF THE DRAWINGS

The present invention will be better understood with reference to the drawings in which:

FIG. 1 illustrates the alignment of the BLHIV-21 gene amino acid sequence with the sequences of the four HIV different isolates of HTLV-III (HIV BHlO), LAV-I , ARV- 2/SF2 and CDC-451 ;

FIG. 2 is a list of bioLytical HIV-I synthetic primers and oligonucleotides;

FIG. 3 illustrates the assembly of 4 oligonucleotides to form the synthetic HIV-I gp41 part 1 , and its cloning into pBLHIV-8, designated pB LHIV-12;

FIG. 4 illustrates the assembly of 2 oligonucleotides to form the synthetic HIV-I gp41 part 2, and its cloning into pBLHIV-1 , designated pBLHIV-13;

FIG. 5 is a schematic diagram of the cloning of HIV-I envelope gene from pB LHIV- 12 and pBLHIV-13 into vectors pBLHIV-11 to generate pBLHIV-16;

FIG. 6 is a schematic diagram of the cloning of HIV-I envelope gene from pBLHIV- 16 into vectors pBL-1 to generate pBLHIV-21 ;

FIG. 7 illustrates the DNA and amino acid sequence of the full length synthetic BLHIV-21 gene, indicating restriction enzymes used to assemble the gene; FIG. 8 depicts a recombinant BLHI V-21 protein Enzyme Immunoassay (EIA) test result; and

FIG. 9 depicts a recombinant BLHIV-21 protein flow-through immunoassay test result.

DETAILED DESCRIPTION OF THE INVENTION

Synthetic DNA fragments of the HIV 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. For example, the synthetic HIV-I envelope protein described in Example 1, is based on the amino acid sequence of four different HfV 1 isolates, namely, HTLV-III (HIV BHlO), LAV-I, ARV-2 and CDC-451 (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.

A preferred expression system pBL-1 utilizes the lambda pRpL vector system for HIV-I envelope protein. This expression system is controlled by the temperature- sensitive lambda repressor cl857 and expressed at high levels.

The synthetic DNA sequences of the present invention, derived from several HIV isolates and optimized for expression in E. coli provides continuous availability and uniformity of HIV antigen preparations which will recognize test sera from individuals exposed to genetically distinguishable variant HIV isolates. 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.

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, 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 KPL. Some restriction enzymes 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 Blue -2 plasmid, pET expression system 29 were purchased from Novagen. pBluescript II KS+ purchased from Stratagene. pBL-1 vector was obtained from

Chinese Center for Disease Control and Prevention. All of bioLytical HIV-I 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-I Clones

Synthesis and Cloning of HIV-I gp41 Part 1 (reference to FIG. 3)

A. Synthesis of Subfragment-1

The subfragment-1 located downstream from pBLHIV-21 , designated pBLSP- 15 through pBLSP-16, were synthesized along with sequences containing a BamH I restriction site at the 5' end and a AIwN I restriction site at the 3' end to facilitate molecular cloning. The subfragment encoding the N-terminal gp41 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 at 70 degrees C. for 10 min to inactive the DNA polymerase. The reaction was subsequently digested with the BamH I and AIwN I and checked by electrophoresis on a 2% agerose gel. The subframent 1 was extracted and purified by UltraClean 15 DNA purification kit (Mo Bio) for further cloning use.

B. Synthesis of Subfragment-2

The subfragment-2 located downstream from pBLHIV-21, designated BLSP- 17 through BLSP-18, were synthesized along with sequences containing a AIwN I restriction site at the 5' end and a Hind III restriction site at the 3' end to facilitate molecular cloning of the individual subfragments. The subfragment encoding the gp41 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 at 70 degree C. for 10 min to inactive the DNA polymerase. The reaction was subsequently digested with the Hind III and AIwN I and checked by electrophoresis on a 2% agerose gel. The subframent-2 was extracted and purified by UltraClean 15 DNA purification kit (Mo Bio) for further 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 AIwNl restriction site at the 3' end and the subfragment-2 with AIwNl restriction site at the 5' end and a Hind III restriction site at the 3' end were ligated into the vector pBLHIV-8 that had been digested with BamH I and Hind III and gel- isolated. The ligation product was used to transform DH5α competent cells (clone pBLHIV-12). The desired clone was identified by digestion with BamH

I and Hind III and checked on a 1.5% agerose gel. Miniprep DNA was prepared from an overnight culture of clone pBLHIV-12 and sequenced with the oligonucleotide primers T7 Promoter forward and T3 Promoter reverse by Cortec DNA Service Laboratories, Inc.

Synthesis and Cloning of HIV gp41 Part 2 (reference to FIG. 4)

A. Synthesis of Sub fragment

The subfragment located downstream from pBLHIV-21 , designated BLSP-22 through BLSP-23, were synthesized along with sequences containing a EcoR I restriction site at the 5' end and a Xho I restriction site at the 3' end to facilitate molecular cloning of the individual subfragments. The subfragment encoding the gp41 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 at 70 degrees C. for 10 min to inactive the DNA polymerase. The reaction was subsequently digested with the EcoR I and Xho I and checked by electrophoresis on a 2% agerose gel. The subframent 1 was extracted and purified by UltraClean 15 DNA purification kit (Mo Bio) for further cloning use.

B. Cloning of Subfragment

The subfragment with a EcoR I restriction site at the 5' end and a Xho I restriction site at the 3' was ligated into the vector pBLHIV- 1 that had been digested with EcoR I and Xho I and gel-isolated. The ligation product was used to transform DH5α competent cells (clone pBLHIV- 13). The desired clone was identified by digestion with EcoR I and Xho I and checked on a 2% agerose gel. Miniprep DNA was prepared from an overnight culture of clone pBLHIV- 13 and sequenced with the oligonucleotide primers T7 Promoter forward and T3 Promoter reverse by Cortec DNA Service Laboratories, Inc.

Construction of pBLHIV-16 (reference to FIG. 5)

pBLHIV- 12 that had been digested with BamH I and EcoR I and pBLHIV- 13 that had been digested with EcoR I and Xho I were ligated into the vector pB LHIV-11 that had been digested with BamH I and Xho I and gel-isolated. The ligation product was used to transform DH5α competent cells (clone pBLHIV- 16). The desired clone was identified by digestion with BamH I and Xho I and checked on a 2% agerose gel. Miniprep DNA was prepared from an overnight culture of clone BH IV- 16 and sequenced with the oligonucleotide primers T7 Promoter forward and T7 Terminator reverse by Cortec DNA Service Laboratories, Inc. pBLHIV- 16 encoded the recombinant HIV-I envelope protein that was consisted of S tag, 38 amino acids of env gpl20 (HIV-I group M), and 192 amino acids of env gp41 (HIV-I group M), which HIV-I env gp41 amino acids 6-30 were substituted with a single Serine, amino acids 169-193 were deleted.

Construction of pBLHIV-21 (reference to FIG. 6)

pBLHIV-21 encoded the recombinant HIV-I envelope protein that was consisted of 38 amino acids of gpl20 (HIV-I group M), and 192 amino acids of gp41 (HIV-I group M), which HIV-I gp41 amino acids 6-30 were substituted with a single Serine, amino acids 169-193 were deleted (reference to FIG. 7). The construction of pBLHIV-21 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-26, 100 ng primer BLSP-27, and 1 ng pBLHIV-16 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; 50 degrees C. for 45 seconds; 72 degrees C. for 120 seconds. Then the BLSP-26/BLSP-27 PCR product was gel isolated.

B. Blunt Ends A blunt ends reaction (20 ul) was set up with GE Healthcare Klenow, Exonuclease- Free (5 U) and IX klenow buffer along with 25 mM each dNTP and 4 ug of each of the vector pBL-1 , which was digested with EcoR I and gel-isolated and the BLSP- 26/BLSP-27 PCR product, which was gel isolated. The reaction was incubated at room temperature for 15 minutes. And the reaction was stopped by heating to 75 degrees C. for 10 minutes.

C. Cloning

The blunt ends BLSP-26/BLSP-27 PCR product was digested with Pst I and ligated into the blunt ends pBL-1 (EcoR I cut) that had been digested with Pst I. The ligation product was used to transform DH5α competent cells (clone pBLHIV-21). The desired clone was identified by digestion with EcoR I and BamH I and checked on a 1% agerose gel. Miniprep DNA was prepared from an overnight culture of clone pBLHIV-21 and sequenced with the oligonucleotide primers BLSP-35 forward and BLSP-36 reverse by Cortec DNA Service Laboratories, Inc. Example 2

Procedure for Producing and Purifying Recombinant BLHIV-21 Protein

In this and the following examples, all chemicals were obtained from Sigma Chemical Corporation Canada unless otherwise noted.

Preparation of Required Solutions:

An LB solution was prepared by dissolving 20 g LB Broth in 1 liter of Milli-Q water, adjusting the pH to 7.2, and sterilizing by autoclaving. Milli-Q water was obtained by filtration of water through a Milli-Q water system (Millipore Corp.). The solution was allowed to cool and Ampicillin was added to bring the final concentration to 0.05 mg/ml. Inclusion Body Wash Solution:

PH 8.0 50 mM Tris-HCl buffer including 50 mM NaCl, 5 mM EDTA, 0.5% Triton

XlOO, 0.02% NaN₃

Solubilization Buffer:

PH 9.6 Carbonate buffer including 50 mM NaCl, 5 mM EDTA, 8 M Urea, ImM DTT and 0.02% NaN₃

Growth of and Induction of pBLHIV-21 DH5α:

LB Broth (50 ml) was inoculated with 0.02 ml of a pBLHIV-21 DH5α glycerol stock and then grown overnight in an incubator shaker at 37 degrees C. and 180 RPM. The 10 ml overnight culture was added to 200 ml LB solution containing 0.05 mg/ml Ampicillin in a 500 ml Flask and the mixture was grown until the optical density was 0.7 to 1.0 at 600 nra (3 hours). The temperature of the culture aliquot was quickly raised to 42 degrees C. The cultures were incubated at 42 degrees C. and 180 RPM for 4 hours. The cells were harvested by centrifugation in a Sorvall RC-5B centrifuge at 5000 RPM for 20 minutes. If the cells were not required for immediate purification, they were stored at -20 degrees C. Extraction and Purification of Inclusion Bodies:

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 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 20 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 centrifuged in a Sorvall RC-5B at 9000 RPM in a GS-3 rotor for 50 min. The supernatant was collected and stored at 4 degrees C. until an aliquot (10 ul) had been analyzed by 12% Polyacrylimide Gel Electrophoresis (PAGE). The pellet was resuspend in 10 ml of room temperature Solubilization Buffer. The solubilized recombinant antigens were clarified by centrifugation in a Sorvall RC-5B at 9000 RPM in a GS-3 rotor for 50 min. The crude recombinant BLHIV-21 protein was stored at 4 degrees C. until further process.

Purification of Recombinant BLHIV-21 Protein by Electroelution:

Preparation of Required Solution:

Sample Buffer (SDS- reducing buffer): 0.06 M Tris-HCL, PH 6.8, 10% Glyderol, 2% SDS and 0.025% Bromophenol Blue

Preparative SDS Polyacrylamide Gels (37 mm ID) were prepared as described in the BIO-RAD Model 491 Prep Cell Instruction Manual. 100 ml (10-11 cm) of a 12% Separating Gel solution and 20 ml (2.5 cm) of Stacking Gel solution were used to pour the gels. The Model 491 Prep Cell (BIO-RAD) was assembled and operated according to the Instruction Manual. Prior to loading, 4 ml of Sample Buffer and 200ul of 1 M DTT were added to 4 ml of crude recombinant BLHIV-21 protein obtained in the previous step. The loading sample was heated to 95 degrees C. for 4 minutes (this was done by transferring small aliquots of the sample into Eppendorf tubes and placing them into a heat block). A total of 8.2 ml of sample was loaded onto each preparative gel. After loading, the Model 491 Prep Cells was operated under a constant power of 12 W, for 20 h, with an elution buffer flow rate of 60 ml/h (1 ml/min), and fractions were each collected for 8 min.

Aliquots of sample were collected after the elution of the Bromophenol Blue. These aliquots were run on a 12% SDS polyacrylamide gel in order to determine which of them contained the 27.3 kd recombinant BLHIV-21 protein. The fractions containing recombinant BLHIV-21 protein were then 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 recombinant BLHIV-21 protein in the Enzyme Immunoassay (EIA) assay and flow-through immunoassay would be discussed in Example 3.

Example 3

Diagnostic Utility of Recombinant BLHIV-21 Protein

A. Enzyme Immunoassay (EIA) with of Recombinant BLHIV-21 Protein

The present example demonstrates the utility of the invention for providing an EIA for the detection of HIV-I antibodies employing the recombinant BLHIV-21 protein. The recombinant BLHIV-21 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-21 protein was used to coat NUNC MaxiSorp microtiter wells at 500 ng/well. Following 1 hour of coating in 37 degrees C, Ihe 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-I 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. 8 showed the reactivity of the recombinant BLHIV-21 protein of the invention with HIV-I positive and negative specimen in the EIA assay.

B. A Flow-through Immunoassay for the Rapid Detection of HIV-I with Recombinant BLHIV-21 Protein

A flow-through immunoassay for the presence of antibodies to HIV-I in test serum, plasma and whole blood was performed as follows: The HIV-I 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 each of the recombinant BLHIV-21 protein of the invention prepared as described in Example 2, which reacted with HIV-I 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-I antibodies, if present in the specimen, are captured by the recombinant BLHIV-21 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-I 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. 9 showed the reactivity of the recombinant BLHtV-21 protein of the invention with HIV-I 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

CLAIMSWhat is claimed is:

1. A HIV-I synthetic gene comprising the following DNA sequence:

1 ATGGATATGC GTGATAACTG GCGTAGCGAA CTCTACAAAT ATAAAGTAGT

51 AAAAATTGAA CCATTAGGAG TAGCACCGAC CAAGGCAAAG CGCCGCGTGG

101 TGCAGCGCGA AAAACGCGCA GTGGGAATTG GATCCCGCCA GTTATTGTCT 151 GGCATCGTTC AACAACAGAA CAATTTGCTG CGCGCTATTG AGGCGCAACA 201 ACACCTGTTG CAGCTGACAG TCTGGGGCAT CAAGCAACTG CAAGCACGTA 251 TCCTGGCTGT GGAACGCTAC ATCTTGGATC AACAGCTGCT GGGGATTCAA 301 GGTTGTTCGG GTAAACTGAT TTGCACCACT GCTGTGCCGT GGAACGCTAG 351 TTGGAGTAAT AAATCTCTGG AACAGATTTG GAATAACATG ACCTGGATGG 401 AGTGGGACAG AGAAATTAAC AATTACACAA GCTTGATTCA CTCCTTAATT 451 GAAGAATCGC AAAACCAGCA AGAAAAGAAT GAACAAGAAT TATTAGAGCT 501 GGATAAATGG GCAAGTTTGT GGAATTGGTT TAACATTACA AATTGGCTGG 551 AATTCGTGAA TCGCGTTCGT CAGGGATATT CACCATTATC GTTTCAGACC 601 CACCTCCCAA TCCCGCGTGG ACCGGACCGT CCGGAAGGAA TTGAAGAAGA 651 AGGTGGAGAG CGTGACCGCG ACCGTTCCAT TCGCTTAGTG AACGGCTCGT 701 AA

2. The synthetic gene of Claim 1 encoding for a polypeptide having the following amino acid sequence:

NH2-MDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQREKRAVGIGSRQLLS GIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYILDQQLLGIQ GCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLI EESQNQQEKNEQELLELDKWASLWNWFNITNWLEFVNRVRQGYSPLSFQT HLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGS*-COOH

3. An expression vector containing the synthetic gene of Claim 1 , where said gene encodes for a polypeptide having the following sequence:

4. The expression vector of Claim 3 wherein heterologous gene expression is regulated by the lambda pRpL 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 DH5α.

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-I 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-I 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-I 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.