KR100542542B1 - A nucleotide sequence of HIV-1 subtype B genomic DNA from Korean, a molecular clone comprising the nucleotide sequence and a method for preparation thereof - Google Patents

Abstract

The present invention relates to a nucleotide sequence of genomic DNA of HIV-1 subtype B isolated from Koreans, a molecular clone comprising the same, and a method of manufacturing the same. Korean subtype B including a full length of HIV-1 genomic DNA using PCR recombination technology. It has an excellent effect on providing a molecular clone of.

In addition, the present invention can be used as a basis for domestic AIDS / HIV research by analyzing the nucleotide sequence of genomic DNA of Korean subtype B.

Therefore, the full-length clone of the domestic specific HIV-1 subtype B of the present invention can be used for the development of domestic HIV therapeutics and vaccines.

HIV, vaccine, cure, HIV-1 subtype B, molecular clone

Description

A nucleotide sequence of HIV-1 subtype B genomic DNA from Korean, a molecular clone comprising the nucleotide sequence and a method for preparation eg}

1 is a schematic diagram of the lineage analysis of the V3-V5 region of the env gene of HIV-1 for each subject for selecting a subject group belonging to the Korean subtype B group of the present invention.

2 is a schematic diagram of the phylogenetic tree of HIV-1 full-length genome isolated from Korean subject group.

Figure 3 is agarose gel photograph showing the results of long PCR amplification for 8.8 kb HIV-1 DNA fragment.

Figure 4 is a photograph of agarose gel showing the result of confirming that the 8.8 kb HIV-1 PCR product obtained after PCR amplification is HIV-1 DNA.

5 is a photograph of agarose gel showing the results of PCR amplification for 660bp of the 5 'LTR region and 1045bp HIV-1 DNA fragment of the 3' LTR region obtained after PCR amplification.

Figure 6 illustrates the recombination process of clones containing the HIV-1 genome full length using PCR recombination techniques.

Figure 7 shows the nucleotide sequence of the 3 'LTR portion of 5' LTR of HIV-1 of the target group KRB5041 selected as a Korean subtype B group.

FIG. 8 is a graph illustrating nucleotide sequence recombination of a target group KRB5041 selected from Korean subtype B group and another target group.

9 shows the nucleotide sequence of the LTR portion of the target group KRB5041 selected as the Korean subtype B group.

10 shows the amino acid sequence of env (V3) of subject group KRB5041 selected from the Korean subtype B group.

11 shows the amino acid sequence of nef of the target group KRB5041 selected from the Korean subtype B group.

The present invention relates to a nucleotide sequence of genomic DNA of HIV-1 subtype B isolated from Koreans, a molecular clone comprising the same, and a method of manufacturing the same. More specifically, the present invention is to select the target group belonging to the Korean subtype B group by analyzing the full-length sequence of HIV-1 and to isolate the genomic DNA from it and PCR using the template as a template for the HIV-1 genomic DNA The present invention relates to obtaining a full length and introducing the same into a vector to obtain a molecular clone including the full length of the HIV-1 genome, and analyzing the nucleotide sequence of the clone to investigate genetic properties.

HIV-1 has a large genetic and phenotypic diversity, leading to many differences in biological characteristics such as cell tropism, cytostatic effect, drug sensitivity, and pathogenicity in different strains. (Olivares et al. 1998). HIV differs in subtype patterns by continent and region (Louwagie et al. , 1995; Miura et al. , 1990), and these characteristics are likely to lead to differences in disease progression. Depending on the epidemic, natural history, such as the latency or survival of AIDS, may vary.

The reason for genetically high mutation rate in HIV is the high error rate of reverse transcriptase (preston et al ., 1988; Roberts et al ., 1988), and the recombination that occurs during virus replication (Clavel et al ., 1989; Coffin, 1979), and selective pressures produced by the host's immune system (Borrow et al ., 1997). Diversity of these sequences may include cell denaturation (O'Brien et al. , 1990; Shioda et al ., 1992; Westervelt et al ., 1991), replication kinetics (Fenyo et al ., 1988), and cell degeneration. To alter activity (Tersmette et al. , 1989), response to neutralizing antibodies (Albert et al ., 1990; Nara et al. , 1990), and response to cytotoxic T lympocytes (phillips et al. , 1991).

On the other hand, disease progression in people infected with HIV is characterized by an increase in viral load (Mellors et al ., 1996), an increase in viral transcription and metastasis (Connor et al ., 1994; Furtado et al ., 1995; Michael et al. , 1991; saksela et al ., 1994), the emergence of highly pathogenic viruses (Connor et al ., 1993; Mullins et al. , 1991), accelerated by changes in the host immune system (Klein et al . (1995), slow disease progression is caused by infection with inactivated viruses (Deacon et al. , 1995; Desrosiers 1992), or mutations of chemokine coreceptors (Smith et al ., 1997).

As such, the various antigenicities of HIV-1 pose a major problem in the development of AIDS vaccines (Esparza et al. , 1991; Osmanov et al. , 1996) . This study is aimed at elucidating the characteristics of HIV-1 of various subtypes and producing various molecular clones based on this, and trying to investigate the relationship between HIV gene expression and disease mechanism.

Molecular clones are an important method for studying the basic mechanisms of viral replication and etiology involved in the genetic diversity of HIV-1, providing systematic information on the biological and immunological properties of HIV-1 genes, and important data for the development of AIDS prevention vaccines. do.

It is not easy to obtain useful molecular clones due to the mutation of the viral genome in the host during HIV-1 infection, but if so, diagnosis and characterization of the HIV-1 virus, analysis of the efficacy of existing therapeutic agents, development of new therapeutic agents, and various It will be useful in the study of effective vaccine development according to subtype.

Globally, research on HIV was conducted by the WHO in 1992 to establish a Global Program on AIDS (GPA / WHO) as a laboratory network to isolate and characterize HIV, and to continuously observe HIV diversity worldwide. A research system is in place. In the United States, the National Institute of Allergy and Infectious Diseases (NIAID) established and operated the NIH AIDS Research and Reference Reagent Program in 1988, providing AIDS researchers with a variety of HIV isolates, infectious molecular clones, cell lines, and HIV. It provides all the basic samples related to HIV research, such as antigens and monoclonal antibodies, and serves as a central institution for HIV research. In addition, The Joint United Nations Program on HIV / AIDS (UNAIDS), the National Institute for Biological Agency and Control (NIBSC) in the UK, and the National Agency for AIDS Research in France provide a variety of research resources for AIDS research.

On the other hand, although there are various researches on HIV in Korea, most of them are in foreign isolates, and most of them are conducted through genes rather than genomes. There are many difficulties to follow up overall. Therefore, in order to study the characteristics of HIV-1 and to develop vaccines in Korea, it is required to introduce a systematic system to activate AIDS / HIV research by producing clones, supplying standardized samples suitable for domestic epidemic strains, and supplying them. .

Accordingly, it is an object of the present invention to provide a full length sequence of genomic DNA of HIV-1 subtype B that is prevalent in our country.

It is also an object of the present invention to provide a molecular clone comprising the full length of the genome of the HIV-1 subtype B and a method for producing the same for the development of domestic HIV therapeutics and vaccines.

The object of the present invention is to analyze the full-length sequence of HIV-1 to select a target group belonging to the Korean subtype B group to isolate genomic DNA and PCR using the template as a template 5'-LTR region of HIV-1, PCR products obtained near the 3'-LTR site and the full length were obtained and introduced into the vector to obtain a clone including the full length of the HIV-1 genome, and the nucleotide sequence of the clone was analyzed to investigate the genetic properties.

Hereinafter, the specific configuration of the present invention will be described.

The present invention comprises the steps of selecting a subject group belonging to the Korean subtype B group of HIV-1; Separating genomic DNA from the subject group; Obtaining a PCR product close to the 5'-LTR region, 3'-LTR region, and full length of HIV-1 using the genomic DNA as a template; Using the PCR product to obtain an HIV-1 genome full length and introducing it into a vector to clone; It consists of the sequencing step of the clone obtained above.

The method for preparing a full-length clone of HIV-1 subtype B isolated from Koreans of the present invention comprises the following steps:

By analyzing the full-length sequence of HIV-1, genomic DNA is isolated by selecting a target group belonging to Korean subtype B group,

10 cycles of 94 DEG C for 1 minute, 55 DEG C for 1 minute, and 72 DEG C for 8 minutes were performed using the genomic DNA as a template, and then 30 cycles of 94 DEG C for 15 seconds, 55 DEG C for 45 seconds, and 72 DEG C for 1 minute were repeated. In the PCR reaction condition which finally polymerizes at 72 ° C for 30 minutes, 5'-LTR of 660 bp was prepared using the primers shown in SEQ ID NOS: 1 and 2, and 1,045 bp 3 using the primers of SEQ ID NOs: 3 and 4 '-LTR, using the primers of SEQ ID NO: 5 and 6 to obtain three kinds of PCR products containing a length close to 8,800 bp,

From the PCR products, 9.8 kb of HIV-1 genome full length was obtained by secondary PCR, which was then cloned by introducing into a pCR 2.1 TOPO vector.

The present invention is characterized by providing a molecular clone Escherichia coli KRB 5041.7 KFCC-11332 comprising the full-length genome of HIV-1 subtype B isolated from Koreans.

According to an embodiment of the present invention, the clone of the present invention constructs a clone containing the full length of the HIV-1 coding gene using recombinant technology using PCR. This has the advantage that cloning with restriction enzymes can be cloned using fragments obtained by precisely cutting the desired site, but there are many limitations in finding available ones of restriction enzymes. Therefore, restriction enzyme positions are not considered to overcome such limitations. Do not use the advantages of recombinant technology using PCR.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

EXAMPLE

Example 1: Selection of subject group belonging to Korean HIV-1 subtype B group

To construct a molecular clone containing the full length of the genomic DNA of HIV-1 belonging to the HIV-1 subtype B group isolated from Koreans, 74 randomly selected domestic HIV infections from 1985 to 1999 were selected. The gene of the env region was analyzed.

As shown in FIG. 1, an analysis of the env (V3-V5) gene region from 74 randomly selected Korean AIDS infected individuals revealed that seven of the subjects forming a unique “Korean subtype B cluster” were identified. The entire HIV-1 gene was selected and analyzed. As a result, similar to the results shown in the env (V3-V5) gene region, it appeared to form mostly unique "Korean population". Based on the results, KRB5041, which constitutes a typical Korean population in the phylogenetic tree, was selected and used for the production of HIV-1 full-length clones. The phylogenetic tree is shown in FIG. 2.

Example 2: Isolation of Genomic DNA of Korean HIV-1 Subtype B Group

Isoquick, kit, and nucleic acid extraction kit No. MXT-020 were used to prepare cultured Peripheral Blood Mononuclear Cells (PBMCs) of the selected subjects to produce full-length clones of domestic epitopes selected according to the characteristics of the env gene. -100, ORCA Research Inc., WA, USA) was used to extract DNA and used as template for PCR.

To isolate genomic DNA, 100 μl of PBS (phosphate buffered saline) was added to the cultured PBMC, shaken sufficiently, and allowed to stand at room temperature for 5 to 15 minutes, followed by mixing the same amount of lysis solution. The nucleic acid was stabilized. After that, add 700 μl of extraction solution 2 to the lysate and mix well. Then, add 400 μl of extraction solution 3 and shake for 10 seconds at room temperature for 5 minutes at 15,000 rpm. Centrifuged. The supernatant containing nucleic acid was carefully transferred to a new tube and 3M sodium acetate corresponding to 0.1 times the supernatant was added. The same amount of isopropanol was added to the whole solution, and the mixture was gently mixed, followed by centrifugation at 15,000 rpm for 10 minutes. After centrifugation, all the solutions except the pellets were removed and 1 mL of 70% ethanol was added to the collected pellets, followed by centrifugation at room temperature for 5 minutes, and the supernatant was dried. The dried DNA was completely dissolved by adding 100 μl of sterile distilled water, and the amount of DNA was quantified using an absorbance spectrometer and stored frozen until PCR.

Example 3 Preparation of Molecular Clones Including Full Length of HIV-1 Genomic DNA Using PCR Recombination Technique

In order to produce a molecular clone containing the HIV-1 full length of the present invention, a clone was prepared by modifying a recombinant method using PCR. To this end, three steps of PCR were performed. First, the genomic DNA obtained above was used as a template, and PCR was performed according to the method of Fang (1996) using primers of FGF61 (forward: 5'-TAGTCAGTGTGGAAAATCTCTAGCAGTG-3 ') and FGR94 (reverse; 5'-CTCGATGTCAGCAGTTCTTGAAGTACTC-3'). Was carried out. PCR reactions were performed with 10 mM Tris-HCl, 50 mM KCl, pH8.3, 8 μl of dNTP (2.5 mM each), 0.125 mM MgCl ₂ , 0.4 pmole of each primer, and 5 U / μl of extack polymerase ( 1 μl of DNA (0.1-1 μg / μl) was added to a mixed solution of 0.5 μl of Ex Taq polymerase (manufactured by Takara Corporation), and 50 μl of distilled water was titrated to perform PCR. PCR reaction conditions were performed 10 cycles of 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 8 minutes, then repeated 30 cycles of 94 ℃ 15 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minutes, and then 30 at 72 ℃ Finally polymerized for minutes.

In order to identify the DNA fragments amplified by PCR, electrophoresis was performed for 50 minutes at 80 V with TAE buffer solution in 1% agarose gel, followed by staining with ethidium bromide solution to confirm the PCR product (Fig. 3). Lane 1 of FIG. 3 shows a size marker, lane 2 shows KRB5041, and lane 3 shows negative control.

As shown in Figure 3, the PCR product of the above-selected subject group showed a single band of 8.8 kb.

PCR was performed on the env and pol sites, which are structural gene regions of HIV-1, from the 8.8 kb PCR products of the near-full length, and confirmed whether the products were DNA of HIV-1 on agarose gel.

As shown in FIG. 4, each of the subjects showed 1.2 kb of env and 1.2 kb of pol as expected, thereby confirming that the PCR product was HIV-1.

Although a clone close to the full length was obtained from the representative domestic epidemic, the clones of the 5'LTR and 3'LTR sites were also intended to clarify the characteristics of the entire genome. Amplification of the 5 'LTR site (660bp) was used by modifying the method of Salminen et al . (2000), PCR reaction solution and reaction conditions are the same as above. At this time, primers were used by modifying MSF13 (forward: 5'-GGCTAGTCTAGATGGAAGGGCTAATTTGGTCCCAAA-3 ') and MSR7 (reverse: 5'GCATGCGGATCCGTTCGGGCGCCACTGCTAGAGATT-3').

For amplification of 3'-LTR and Nef gene (1045 bp), primers were added to Nef6 (forward: 5'-TCGCCACACCTAGAAGAATAAG-3 ') and MSR8 (reverse: 5'-GCATGCGAATTCCTGCTAGAG ATTTTCCACACTGA-3'). Was used.

As shown in the PCR results of FIG. 5, PCR products of 3'LTR (1,045bp) region partially overlapped with 5'LTR (660bp) and nef gene were obtained, respectively.

In order to produce a clone including the HIV-1 full length of the present invention, a 5 'LTR (660 bp) portion, a portion near the full length (8,800 bp), and a 3' LTR (1,045 bp) portion of the HIV-1 obtained above Three different PCR products of different sizes were purified using Qiagen gel Extraction Kit. Purified products were PCR products of 5'-LTR (660 bp) and 3'LTR (1045 bp) sites to 2 μg, 8.8 kb PCR product 0.1 μg, 2.5 U rTth polymerase, 0.004 A second PCR was performed under conditions of U pfu polymerase, 10 μm dNTP, 1.2 mM Mg (OAc) ₂ , and 1 × XL PCR buffer to obtain an HIV-1 full length genome.

DNA confirmed to be HIV-1 gene was cloned using pCR 2.1-TOPO TA cloning kit (Invitrogen).

First, 3 'A overhanging process was performed at 72 ° C. for 15 minutes with addition of dATP and Taq polymerase to increase the insertion efficiency of the purified PCR product into the pCR 2.1 TOPO vector for pCR 2.1-TOPO cloning. 2 μl of PCR product, 2 μl of distilled water, and 1 μl of vector containing topoisomerase were mixed and allowed to stand at room temperature for 5 minutes, followed by 30 seconds at 42 ° C. in TOP 10 cells, which are competent cells. Heat treated and left on ice for 2 minutes. Then 250 μl / mL of SOC medium was added and incubated for 1 hour at 150 rpm in a shaker. The cells transformed in LB plate medium containing ampicillin (ampicillin, 5 μg / μl) smeared with X-gal (50 μg / μl) were plated and incubated at 37 ° C. for 14-16 hours. After incubation, the transformed white colonies were inoculated in LB liquid medium containing ampicillin and incubated for 14-16 hours in a 37 ° C shake incubator.

The production process of the clone including the full length of HIV-1 is shown in FIG.

In order to determine whether the clones obtained from the transformation resulted in the full length of the HIV-1 genomic DNA, the plasmid of each clone was isolated. Isolation of the plasmid was extracted using alkaline lysis method (alkaline lysis, Birnboin and Doly, 1979; Ish-Horowicz and Burke, 1981). First, the suspension was centrifuged at 8,000 rpm to collect the cells, and the pellet collected with 100 μl of the I solution (50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0)) was collected again. Suspended. Then 200 μl of the II solution (0.2 N NaOH, 1% SDS) was added to lyse the cells and the III solution (60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid, 28.5 mL of distilled water) 150 DNA was stabilized by treating with μl. Cell debris was removed by centrifugation at 15,000 rpm and DNA was recovered by treating the same volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) solution.

The recovered DNA was washed with 70% ethanol, dried and then dissolved in distilled water containing RNase (20 μg / μl). DNA was digested with EcoRI to confirm the insertion of each gene.

The transformant (full length clone) containing the full length of the HIV-1 subtype B isolated from Koreans obtained from the above was named Escherichia coli KRB 5041.7, and the Korean Microbiological Conservation Center on June 17, 2004. Deposited with accession number KFCC-11332.

Example 4 Genome Analysis of Clones Containing the Present HIV-1 Full Length

In order to investigate the characteristics of the HIV-1 full-length genome that is popular in Korea, sequencing was performed on the clones obtained according to the above-mentioned 3. For genome analysis of the clone including the present invention HIV-1 full length obtained in Example 3, the cloned plasmid DNA as a template was sequenced using an ABI PRISM Dye terminator cycle sequencing ready reaction kit (Perkin Elmer).

Sequencing PCR was performed by adding 1.7 μl of distilled water to 1 μl of PEG-purified DNA sample, 2 μl of premixture, and 0.3 μl of 10 pmol / μl sequencing primer. 25 cycles were performed in minutes. 15 µl of distilled water, 0.1-fold sodium acetate and 2-fold ethanol were added to the PCR product, and the precipitate was centrifuged at 15,000 rpm for 15 minutes. The resultant was washed with 70% ethanol, dried, denatured by adding 2 μl of sequencing dye at 90 min for 2 minutes, and loaded onto a sequencing gel to determine nucleotide sequence.

The determined sequences were sorted by the clustal V method and the neighbor-joining method using the MegAlign program in the DNA star package (DNA Star package) to calculate the genetic distance between HIV-1 isolates. By analyzing the phylogenetic tree of the isolates, their flexibility relationship was analyzed. Variations in the nucleotide sequence and amino acid sequence were sorted using the MegAlign program and analyzed for the relationship between the study group and the molecular genetic variation. In addition, the analysis was performed using several HIV-1 sequences reported in the Los Alamos Database.

A comparison of the genes of KRB5041.7 with reference subtypes reported in foreign countries showed that domestic isolates form a unique Korean population. In order to determine the cause of the unique Korean population, the characteristics of the entire gene of clone KRB5041.7 were examined and shown in FIG. 7 (see SEQ ID NO: 7).

In addition, to investigate whether the recombinant phenomenon consisting of the recently reported mosaic genome appears in domestic isolates was analyzed using the Recombinant Identification Program (RIP) provided by the Los Alamos Database (Fig. 8).

As shown in FIG. 8, compared to all subtypes of HIV-1 M type, KRB5041, the domestic epidemic strain, showed the highest homology with HXB2, the subtype B reference strain in the whole genome, and recombination with other subtypes was observed in any gene region. It wasn't.

In addition, KRB5041, which is typical of Korean subtype B, was compared with a gene reported from a representative foreign subtype B reference strain reported previously.

gag pol env vif vpu vpr tat rev nef Average HXB2 94.1 95.5 86.2 91.7 81.7 92.1 87.6 81.6 84.5 88.3 pNL43 94.6 95.5 86.4 91.9 84.1 88.2 88.1 81.6 84.5 88.3 JRFL 93.7 95.6 84.9 93.4 89.0 93.5 88.5 85.5 86.6 90.1 MN 91.9 94.7 94.5 92.7 91.1 91.4 89.9 89.5 89.3 90.6 NY5 94.1 95.3 87.8 91.7 - 93.8 89.0 88.2 - 91.4 OYI 93.9 95.3 83.5 91.7 88.2 93.8 89.4 90.8 87.4 90.4 RF 91.3 95.0 84.6 91.2 - 92.4 87.2 78.9 83.3 88.0 Weau160 91.9 94.6 84.1 92.7 89.8 91.8 88.5 82.9 85.6 89.1

As shown in Table 1, there was a difference between each gene, but showed about 80-95% homology. None of the eight reference strains compared showed significant homology with any of the reference strains, and each gene showed even homology among all reference strains.

The genes were analyzed by region as follows:

(1) LTR: The most unique feature of the subject group belonging to the Korean group B in the LTR region is a phenomenon in which TATAA is transformed into TAAAA in the TATA box at the -23 position (FIG. 9). Compared with the various reference strains, these results are limited to subtype E, indicating that subtype E is present in the LTR region of domestic isolates. In addition, as TATAA is transformed into TAAAA, there is a tendency to overlap in the TCF-1α region, which has been argued that this is a result of mutations to enhance the modified function of the TATA box. In addition, the NF-kB site, which is not absolutely necessary for viral replication at the LTR site, which stimulates LTR activity and responds to cellular activation signals, is relatively well conserved in KRB5041 and is a glycine-rich site that is a gene of the virus. SPI ^I , SPI ^II and SPI ^III sites involved in regulation and replication were also well conserved. In addition, the TAR site, which is known to affect the secondary structure by inducing the binding of Tat trans activator protein and cellular protein of the virus, has a +23 position C, which is different from U in the foreign subtype B reference strain. Characteristic nucleotide sequence shown in. As a result of the analysis of the nucleotide sequence of the LTR region, the overall tendency was similar to that of subtype B, but the characteristic nucleotide sequence of subtype E appeared in some sites, and the effect of the change of base on the virus function was more advanced. It should be identified through

(2) gag : As a result of analyzing the gene of gag region of about 1.5 kb, in the nuclear localization signal of amino acid composition from 25 to 33, the 30th amino acid is represented by K, unlike R, which appears in domestic isolate, similar to foreign isolate B. The 203rd amino acid was identified as D appearing in other types except B type. The amino acids 285 to 304, known as homologous regions of the gag gene, were well conserved, and the 398th and 427th amino acids of the zinc finger, the basic region, were replaced with I and N, respectively.

(3) pol : In the pol gene region encoding the expression of reverse transcriptase, protease and integrase, KRB5041.7 shows that the YMDD motif, which plays an important role in the catalytic activity of reverse transcriptase, was completely conserved and the activity of the protease coding site There was no mutation at the site and substrate binding sites. In the zinc finger region of the integrase coding region, the 20th amino acid is composed of I, which is different from V in foreign isolates, and the 62nd amino acid of the central catalytic domain is represented by N. Was consistent with the The second amino acid of the non-specific DNA binding site was composed of KRB5041.7 million M, although all other strains were composed of I.

(4) env: env is a gene expressed from a 160 kD spliced mRNA that is divided into gp41 and gp120 by intracellular proteases and interacts between HIV and the CD4 receptor on the cell surface by specific domains. It is an important structural gene of HIV. gp120 has intrachain disulfide bonds by nine highly conserved 18 cysteine residues, five hypervariables (V1-V5) and five conserved regions (C1-C5). Analysis of the nucleotide sequence of the KRB5041.7 clone showed a high mutation at the V1 region and a single amino acid deletion when compared to the subtype B reference HXB2. Asp, amino acid 207, known to be important for replication, was well conserved, and four motifs at the end of the V3 site, known as the glycoprotein neutralizing domain, consisted of GPGS (Fig. 10). N-glycosylation site was found to be present more than other sites.

(5) vif : This region is composed of 192 amino acids and is expressed at high levels in the cytoplasm of HIV-infected cells and is a gene involved in enhancing the infectivity of HIV-1 virions. The cysteines at positions 114 and 133, which serve as the central region of the site, are well conserved and the amino acid motif SLQYLA, which is not mutated, is well conserved.

(6) vpr : vpr is a gene product consisting of 96 amino acids and is known to be involved in nuclear influx and cell control. KRB5041.7 did not show mutations at +29 to +36, the major sites involved in virion combinations, and +85 to +95, the sites involved in cell cycle arrest.

Vpu : This gene region regulates a series of processes that enhance the release of virions from infected cells and induce selective disruption of intracellular vesicles. In particular, phosphorylation of serine residues 53 and 57 is known to be an essential part of CD4 degradation. For KRB5041.7, amino acids at both positions were well conserved and at position 35 in the α-helix structure involved in the cytoplasmic tail attachment of CD4 Unlike R, which appears in other foreign subtypes B, the amino acids consisted of K.

(8) nef : Analysis of the nef gene of KRB5041.7 showed a flexible relationship with other isolates in the phylogenetic tree, and myristo acid binding mystic acid to N-terminal glycine in amino acid composition. The EEEE motifs of the myristoylation site and the acid cluster region were well preserved unchanged, and the GPGI (V), a β-turn motif, was GPGV and showed the same pattern as other domestic isolates (Fig. 11).

(9) tat : The tat gene is a protein that interacts with the RNA loop structure called the tat-responsive element in the 3'LTR and is known to boost HIV replication. Particularly, when the 58th amino acid is substituted for alanine to threonine, it has been reported to affect replication. However, no amino acid substitution was observed in KRB5041.7, and amino acid 21, which is a cysteine-rich site, was composed of P. And amino acid sequences appearing in subtypes such as G. The 62nd N is the amino acid found in subtype A, and the above results are similar to those of other domestic isolates. The 9th, 46th, and 78th amino acids, on the other hand, were composed of D, Y, and P, respectively, unlike other domestic isolates. However, in spite of some of these nucleotide variations, they were also located at the edge of subtype B, just like the results of other gene sites.

(10) rev : The analysis of the rev gene of KRB5041.7 revealed that position 16 is I in subtype A, which is characteristic in domestic isolates, unlike V in other isolates. rev is a gene that activates the RNA loop structure called the rev response element located in the viral envelope mRNA to enter the cytoplasm from the nucleus into the cytoplasm of the full-length viral protein required for virus progeny formation. There was no variation.

As described through the above examples, the present invention relates to a nucleotide sequence of genomic DNA of HIV-1 subtype B isolated from Koreans, a molecular clone comprising the same, and a method for preparing the same, and HIV-1 using PCR recombination technology. There is an excellent effect of providing molecular clones of Korean subtype B, including the full length of the genome.

In addition, the present invention can be used as a basis for domestic AIDS / HIV research by analyzing the nucleotide sequence of genomic DNA of Korean subtype B.

Therefore, the full-length clone of the domestic specific HIV-1 subtype B of the present invention can be used in the development of domestic HIV therapeutics and vaccines is a very useful invention in the pharmaceutical industry.

<110> National Institute of Health <120> A nucleotide sequence of HIV-1 subtype B genomic DNA from Korean,          a molecular clone comprising the nucleotide sequence and a method          for preparation according <150> KR2003-0044521 <151> 2003-07-02 <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> sense sequence for PCR amplification of 5'LTR <400> 1 ggctagtcta gatggaaggg ctaatttggt cccaaa 36 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> antisense sequence for PCR amplification of 5 'LTR <400> 2 gcatgcggat ccgttcgggc gccactgcta gagatt 36 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense sequence for PCR amplification of sequence with 3'LTR <400> 3 tcgccacacc tagaagaata ag 22 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> antisense sequence for PCR amplification of sequence with 3'LTR <400> 4 gcatgcgaat tcctgctaga gattttccac actga 35 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> sense sequence for PCR amplification of seqence containing near          full length <400> 5 tagtcagtgt ggaaaatctc tagcagtg 28 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> antisense sequence for PCR amplification of sequence containing          near full length <400> 6 ctcgatgtca gcagttcttg aagtactc 28 <210> 7 <211> 9737 <212> DNA <213> Homo sapiens (Korean) <400> 7 tggaagggct aatttggtcc caaaagagac aagatatact tgatttatgg gtatatcaca 60 cacaaggcta cttccctgat tggcagaact acacaccagg gccaggggtc agatatccac 120 tgacctttgg gtggtgcttc aagctagtac cagttgatcc agaaaagtta gaagaggcca 180 ctgtaggaga gaacaactgc ttgttacatc ccataaacac gcatggaatg gatgacccgg 240 agaaagaagt gttacagtgg aagtttgaca gccgcctggc atttcatcac ttggcccgag 300 agaaacatcc ggagttctac aaggactgct gacactgaac tttctacaag ggactttccg 360 ctggggactt tccaggggag gcgtggcctg ggcgggaccg gggagtggcg agccctcaga 420 tgctgcataa aagcagctgc tttctgcctg tactgggtct ctctggttag accagatccg 480 agcctgggag ctctctggct gactagggaa cccactgctt aagcctcaat aaagcttgcc 540 ttgagtgctt taagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct 600 cagactcttt tagttcgtgt ggaaaatctc tagcagtggc gccccgaacg gggacgaaag 660 cgaaagtaga accagagaag ctctctcgac gcaggactcg gcttgctgaa gccgcgcacg 720 gcaagaggcg aggggcggcg accggtgagt acgccgaaat ttttgactag cagaggctag 780 aaggagagag atgggtgcga gagcgtcaat attaagcggg ggaaaattag atagatggga 840 aaaaattcgg ttaaggccag ggggaaagaa aaagtataag ttaaaacatc tagtatgggc 900 aagcagggag ctagaacgat tctcaattaa ccctggcctg ttagaaacat cagaaggctg 960 tagacaaata ttggaacagc tacaatcagc ccttaagaca ggatcagaag aacttaaatc 1020 attatttaat acagtagcaa ccctctattg tgtacatcaa gggatagata taaaagacac 1080 caaggaagct ttagataaga tagaggaaga gcaaaacaaa agtaagaaaa aggcacagca 1140 agcaacagct gacacaggaa acagcagtca ggtcagccaa aattacccta tagtgcagaa 1200 cctccagggg caaatggtac atcaggcaat atcacctaga actttaaatg catgggtaaa 1260 agtagtagaa gagaaggctt tcagcccaga agtgataccc atgttttcag cattagcaga 1320 aggagccacc ccacaagatt taaacactat gctaaacaca gtggggggac atcaagcagc 1380 tatgcaaatg ttaaaagata ccatcaatga ggaagctgca gaatgggata gattgcatcc 1440 aatacatgca gggcctattg caccaggcca gatgagagaa ccaaggggaa gtgacatagc 1500 aggaactact agtacccttc aggaacaagt aggatggatg acaagtaatc cacctatccc 1560 agtaggagaa atctataaaa gatggataat cctgggatta aataaaatag tgagaatgta 1620 tagtcctacc agcattctgg acataagaca aggaccaaag gaacccttta gagactatgt 1680 agaccggttc tataaaactc taagagctga gcaagcgtca caggatgtaa aaaattggat 1740 gacagaaacc ttgttggtcc aaaatgcaaa cccagactgt aagactattt taaaagcatt 1800 gggaccagca gctacactag aagaaatgat gacagcatgt cagggagtgg gaggacccag 1860 ccataaagca agagttttgg ctgaagcaat gagccaagca acaggttcag ctactataat 1920 gatgcaggga ggcaatttca agaaccaaag aaagcctgtt aagtgtttca attgtggcaa 1980 aatagggcac atagccaaaa attgcagggc ccctaggaaa aagggctgtt ggaaatgtgg 2040 aaaggaagga caccaaatga aagactgtaa tgagagacag gctaattttt tagggaagat 2100 ctggccttcc aacaaaggaa ggccagggaa ttttctccag agcagaccag agccaacagc 2160 cccaccagaa gagagcttca ggtttgggga agagacagca actccctctc agaagcagga 2220 gccgatagac aaggaccacc tgtatccttt agcttccctc aaatcactct ttggcaacga 2280 cccctcgtca caataaagat aggggggcaa acaaaggaag ctctattaga tacaggagca 2340 gatgatacag tattagaaga aatgagttta ccagggagat ggaaaccaaa aatgataggg 2400 ggaattggag gttttatcaa agtaagacag tatgatcaga taactataga aatctgtgga 2460 cataaagcta taggtacagt attagtagga cctacacctg tcaacataat tggaagaaat 2520 ctgttgactc agattggctg tactttaaat ttccccatta gtcctattga aactgtgcca 2580 gtaaaattaa agccaggaat ggatggccca aaagttaaac aatggccatt aacagaagaa 2640 aaaataaagg cattagtaga aatttgtaca gaattggaaa aggaaggaaa aatttcaaaa 2700 attgggcctg aaaatccata taatactcca gtatttgcca taaagaaaaa agacagtact 2760 aaatggagaa aattagtaga tttcagagaa cttaataaga gaactcaaga cctctgggaa 2820 gttcaattag gaataccaca tccagcaggg ttaaaaaaga aaaaatcggt aacagtactg 2880 gatgtgggtg atgcatactt ttcagttccc ttagatgaaa acttcaggaa gtatactgca 2940 ttcaccatac ctagtataaa caatgagaca ccaggaatta gatatcagta caatgtgctt 3000 ccacaggggt ggaaaggatc accagcgata ttccaaagta gcatgacaaa aatcttagag 3060 ccttttagaa aacaaaatcc agacatagtt atctatcaat acatggatga tttatatgta 3120 ggatctgact tagaaatagg gcagcataga acaaaaatag aggaactgag acaacatctg 3180 tggaagtggg ggttttacac accagacaaa aaacatcaga aagaaccccc attcctttgg 3240 atgggttatg aactccatcc tgataagtgg acagtacagc ctatagtgct accagaaaaa 3300 gacagctgga ctgtcaatga catacagaag ttagtgggaa aactgaattg ggcaagtcag 3360 atttatgcag ggattaaagt aaagcagtta tgtaaactcc ttaggggaac caaggcacta 3420 acagaagtag taccactaac agaagaagca gagctagaac tggcagaaac cagggagatt 3480 ctaaaagaac cagtacatgg agtttattat gacccatcaa aagacttaat agcagaaata 3540 cagaagcagg ggcaaggcca atggacatat caaatttatc aagagccatt taaaaatctg 3600 aaaacaggaa aatatgcaaa aatgagaagt gcccacacta atgatgtaaa acaattaaca 3660 gaggtagtgc aaaaaatagc cacagaaagc atagtaatat ggggaaagac tcctaaattt 3720 agactaccca tacaaaaaga aacatgggaa gcatggtgga cagagtattg gcaggccact 3780 tggattcctg agtgggagtt tgtcaatacc cctcctttag tgaaattatg gtaccagtta 3840 gaaaaagaac ccatagtagg agcagaaact ttctatgtag atggagcagc taatagggaa 3900 actagattag gaaaagcagg atatgtcact gacagaggaa gacaaaaggt tgtctcccta 3960 ccggacacaa ccaatcagaa gactgagtta caagcaattc atctagcttt gcaggattcg 4020 ggattagaag taaacatagt aacagactca caatatgcat taggaatcat tcaagcacaa 4080 ccagataaaa gtgaatcaga gttagtcagc caaataatag agcagttaat aaaaaaggaa 4140 aaggtctacc tggcatgggt accagcacac aaaggaattg gaggaaatga acaaatagat 4200 aaattagtca gtactggaat caggaaagtg ctatttttag atggaataga taaggcccaa 4260 gaagagcatg aaaaatatca cagtaattgg agagcaatgg ctagtgattt taacctgcca 4320 cctatagtag caaaagaaat agtagccagc tgtgataaat gtcagctaaa aggagaagcc 4380 atgcatggac aagtagactg tagtccagga atatggcagc tagattgtac acatctagaa 4440 ggaaaaatta tcctggtagc agttcatgta gccagtggat atatagaagc agaagttatc 4500 ccagcagaga cagggcagga aacagcatac tttctcttaa aattagcagg aagatggcca 4560 gtaaaaacaa tacatacaga caatggcagc aatttcacca gtaatacggt taaggccgcc 4620 tgttggtggg cagggatcaa ccaggaattt ggcattccct acaatcccca aagtcaaggg 4680 gtagtagaat ctatgaataa agaattaaag aaaattatag ggcaggtaag agatcaggct 4740 gaacatctta agacagcagt gcaaatggca gtattcatcc acaattttaa aagaaaaggg 4800 gggattgggg gttacagtgc aggggaaaga atagtagaca tgatagcaac agacatacaa 4860 accaaagaac tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac 4920 agcagagatc cactttggaa aggaccagca aagcttctct ggaaaggtga aggggcagta 4980 gtaatacaag ataatagtga cataaaagta gtgccaagaa gaaaagtaaa gatcattagg 5040 gattatggaa aacagatggc aggtgatgat tgtgtggcaa gtagacagga tgaggattaa 5100 aacatggaaa agtttagtaa aacaccacat gtatatttca aagaaagcta agggatgggt 5160 ctataaacat cactatgaaa gcaatcatcc aagaataagt tcagaagtac acatcccact 5220 aggggatgct aaattagtaa taacaacata ttggggtctg catacaggag aaagagaatg 5280 gcatttgggt cagggagtct ccatagaatg gaggaaaaag agatataaca cacaagtaga 5340 ccctgaccta gcagacaaac taattcacct gcattatttt gattgttttt cagactctgc 5400 cataagacaa gccatattag gatattcagt taggcatagc tgtgaatatc aagcaggaca 5460 taacaaggta gggtctctac agtacttggc actaacagca ttaatagcac caaaaaagat 5520 aaggccacct ttgcctagtg ttaagaagct aacagaggat agatggaaca agccccagaa 5580 gaccaagggc cacagaggga gccataccat gcatggacac tagagctctt agaggaactt 5640 aagaatgaag ctgttagaca ttttcctagg ccatggctcc atagcttagg acaatatatc 5700 tatgaaactt atggggatac ttgggaagga gtaggagcca caataagaat tctgcaacaa 5760 ctgctgttta ctcatttcag aattggatgc caccatagca gaataggcat tactcgacag 5820 aggagagcaa ggaatggagc cagtagatcc tagactagat ccctggaagc atccaggaag 5880 tcagcctaag actccttgta ccaaatgcta ttgtaaaaaa tgttgctttc attgtcaagt 5940 ttgcttcata acaaaaggct taggcatata ctatggcagg aagaagcgga gacagcgacg 6000 aagagctcct tgggacaata agaatcatca agttcctcta ccaaagcagt aagtaatata 6060 tgtgatgcaa cctttagtga tattagcaat agtagcatta gtagtagcaa taataatagc 6120 aatagttgtg tggtccatag tattaataga atataggaaa atattaaagc aaagaaaaat 6180 agacaggtta attgagagaa taagcgaaag agcagaagac agtggcaatg aaagtgaagg 6240 agatcaggaa gaatcatcag cactggtgga gatggggcac gatgctcctt gggatgtgga 6300 tgatctgtag tgctatagga agatcgtggg tcacagtcta ttatggggta cctgtgtgga 6360 gagaagcaac caccacccta ttttgtgcat cagatgccaa agcatatgac acagaggtgc 6420 ataatgtttg ggccacacat gcctgtgtac ccatagaccc caacccacaa gaagtagtat 6480 tggaaaatgt gacagaaaaa tttaacatgt ggaaaaataa catggtagaa cagatgcatg 6540 aagatataat caatttatgg gatcaaagcc taaagccatg tgtaaaatta actccactct 6600 gtgttacttt aaattgtagt gatgtgaaga atgttactaa aaccaatagt agtagggagg 6660 gaatgatgga ggaaggagga atgaaaaact gctctttcaa tgttaccaca gatataatag 6720 ataaggtgaa ggaagaatat gctctttttt ataaacttga tgtagtatca atagataata 6780 caagctatac attgataaac tgtaactcct caaccattac acaggcctgt ccaaaggtgt 6840 cctttgaacc aattcccata cattattgta ccccggctgg ttttgcgatt ctacagtgta 6900 atgataagaa gttcaatgga tcaggaccat gcagaaatgt cagcacagta caatgtacac 6960 atggaattag gccagtagta tcaactcaac tgctgttaaa tggcagtcta gcagaagaag 7020 agatagtaat tagatctgaa aatttcacgg acaatgctaa aaccataata gtacagctga 7080 atgcatctgt acaaattaat tgtacaagac ccaacaacaa tacaagaaaa agtataagaa 7140 taggaccagg gagtacattt tatgcaacag gagacataat aggagatata agacaagcac 7200 actgtaacat tagtggagca gaatggaaca acactttaag acagctagtt ataaaattag 7260 gaaaacaatt tgagaataaa acaatagcct ttaaacaatc ctcaggaggg gacccagaga 7320 ttgtaatgca cagttttaat tgtggagggg aatttttcta ctgtaataca acacaactgt 7380 ttaatagtac ttggaaaatg tttaatagta cttggaatag tactgaaggg ttaaatataa 7440 ctgaaggaaa tatcacactc ccatgtagaa taaaacaaat tataaacaga tggcaggaag 7500 taggaaaagc aatgtatgcc cctcccatca gcggacaaat tagatgttca tcaaatatta 7560 cagggctgct attaacaaga gatgggggaa ctgggaatga gaccagaggg actgagatct 7620 tcagacctgg ggggaggaga tatgagggac aattggagaa gtgaattata taaatataaa 7680 gtagtaagaa ttgagccatt agggatagca cccaccgagg caaggagaag agtggtgcag 7740 agagaaaaaa gagcagtggg aacaatagga gctatgttcc ttgggttctt gggagcagca 7800 ggaagcacta tgggcgcagc gtcactgacg ctgacggtac aggccagact attattgtct 7860 ggtatagtgc aacagcaaaa taatttgctg agagttattg aggcgcaaca gcatatgttg 7920 caactcacag tctggggcat caagcagctc caggcaagag tcctagctgt ggaaagatac 7980 ctaaaggatc aacagctcct ggggatttgg ggatgctctg gaaagctcat ctgcaccact 8040 aatgtgcctt ggaatactag ttggagtaat aaatctttgg atactatctg acctggatgg 8100 acctggatgg agtgggacaa agaaattaac aattacacaa gcttaatata caacttactt 8160 gaagaatcgc agaaccaaca agaaaagaat gaacaagatt tattgaaatt ggatgaatgg 8220 gcaagtttgt ggaattggtt tgacatatca cgatggctat ggtatataaa aatattcata 8280 ataatagtag gaggcttggt aggtttaaga ataatttttg ctgtgctttc tatagtgaat 8340 agagttaggc agggatactc accattatca ttccagaccc acttcccaag cccgagggga 8400 cccggcaggc caggagaaat cgaaggagaa ggtggagaag aagacagaga tggatccgat 8460 cgcttagtgc atggattctt aacagtcgtc tgggtcgacc tgcgaagcct gtgcctcttc 8520 agctaccgcc acttgagaga cttactctta attctagcga ggggtgtgga acttctggga 8580 ctcagggggt ggaaaatcct caaatattgg tggaatctcc tgcagtactg gagccaggaa 8640 ctaaagaaga gagctgttag tttgcttaat gccacagcta tagcagtagc tgaggggaca 8700 gataggatta tagaattagt acaaagagct tttagagcta ttcgccacat acctagaaga 8760 ataagacagg gcttggaaag ggctttgcta taagatgggt ggcaagtggt caaaacgtgg 8820 tgtccctgga tgggaacgtg taagggaaag aatgagacga actgagcctg agccagcagc 8880 agaaggggtg ggagcagtat ctcgagacct ggaacggcat ggagcaatca caagtagtaa 8940 tacagcaagt aacaatgctg attgtgcctg gctggaagca caagaggagg aggaggtggg 9000 ttttccagtc agacctcagg tacccttaag accaatgact tacaagggag ctttagatct 9060 tagccacttt ttaaaagaaa aggggggact ggaagggcta atttggtccc aaaagagaca 9120 agatatactt gatttatggg tatatcacac acaaggctac ttccctgatt ggcagaacta 9180 cacaccaggg ccaggggtca gatatccact gacctttggg tggtgcttca agctagtacc 9240 agttgatcca gaaaagttag aagaagccac tgtaggagag aacaactgct tgttacaccc 9300 cataaacacg catggaatgg atgacccgga gagagaagtg ttacagtgga agtttgacag 9360 ccgcctagca tttcatcaca tggcccgaga gaaacatccg gagttctaca aggactgctg 9420 acattgagct ttctacaagg gactttccgc tggggacttt ccaggggagg cgtggcctgg 9480 gcgggaccgg ggagtggcga gccctcagat gctgcataaa agcagctgct ttctgcctgt 9540 actgggtctc tctggttaga ccagatcaga gcctgggagc tctctggctg actggggaac 9600 ccactgctta agcctcaata aagcttgcct tgagtgcttt aagtagtgtg tgcccgtctg 9660 ttgtgtgact ctggtaacta gagatccctc agactctttt agttcgtgtg gaaaatctct 9720 agcagtggcg cccgaac 9737

Claims (3)

By analyzing the full-length sequence of HIV-1, genomic DNA is isolated by selecting a target group belonging to Korean subtype B group, 10 cycles of 94 DEG C for 1 minute, 55 DEG C for 1 minute, and 72 DEG C for 8 minutes were performed using the genomic DNA as a template, and then 30 cycles of 94 DEG C for 15 seconds, 55 DEG C for 45 seconds, and 72 DEG C for 1 minute were repeated. In the PCR reaction condition which finally polymerizes at 72 ° C for 30 minutes, 5'-LTR of 660 bp was prepared using the primers shown in SEQ ID NOS: 1 and 2, and 1,045 bp 3 using the primers of SEQ ID NOs: 3 and 4 '-LTR, using the primers of SEQ ID NO: 5 and 6 to obtain three kinds of PCR products containing a length close to 8,800 bp, Preparation of a full-length clone of HIV-1 subtype B isolated from Koreans, characterized by obtaining 9.8 kb of HIV-1 genome full length from the PCR products and cloning it by introducing it into a pCR 2.1 TOPO vector Way. Nucleotide sequence of HIV-1 genomic DNA of HIV-1 subtype B isolated from Korean population shown in SEQ ID NO: 7. Escherichia coli KRB 5041.7 KFCC-11332, which comprises the genome full length of HIV-1 subtype B produced by the method of claim 1 and isolated from Koreans listed in SEQ ID NO: 7.

Images