JP4461211B2 - Cells for measuring HIV infectivity titer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an animal cell capable of expressing a secreted reporter protein by HIV-1 infection, more specifically, an HIV-1 infected secreted reporter protein that can be used for measurement of HIV-1 infectivity titer. It relates to animal cells that can be expressed.
[0002]
[Prior art]
In 1996, by E. Berger et al., In addition to CD4 molecule, which is the main viral receptor on the cell side, T cell-directed HIV-1 expressed CXCR4, macrophage-directed HIV-1 Has been shown to use CCR5 as a co-receptor, and since then, HIV-1 orientation and viral entry processes have been elucidated (Feng, Y. et al., HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G-protein-coupled receptor. Science 272: 872-877, 1996; Alkhatib, G. et al., CC-CCR5, a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272: 1955-1958, 1996; Choe, H. et al., The beta-chemokine receptor CCR3 and CCR5 facillitate infection by primary HIV-1 isolates.Cell 85: 1135-1148, 1996; Debg, H. et al., Identification of a major co-receptor for primary isolates of HIV-1.Nature 381: 661-666, 1996; and DRagic, T. et al., HIV-1 entry into CD4 + cells is mediated by the chemokine receptor CC-CCR5. Nature 381: 667-673, 1996). As the measurement system for HIV-1 infection, the measurement system based on ELISA for the gag protein p24, which is the structural protein of HIV-1, and the measurement system based on the measurement of reverse transcriptase activity have been used. It is not a quantitative system that reflects the infectious value itself.
[0003]
As an infectious titer measurement system currently used, a measurement system using CD4-expressing HeLa cells (HeLa CD4 / LTR-bGal; commonly known as MAGI cells) developed by Emerman et al. Is known (Kimpton, J. et al. , Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. J. Virol. 66: 2232-2239, 1992). This cell line incorporates an expression unit containing β-galactosidase with an SV40 nuclear translocation signal downstream of the HIV-1 LTR and is more potent than the Tat protein, a transcriptional activator expressed by infected HIV-1. This measurement system utilizes the expression of β-galactosidase enzyme in infected cell nuclei by driven HIV-1 Promoter LTR. This cell is called Multiple nuclear Activation of Galactosidase Indicator cell Assay (MAGI Assay) because it can count HIV-1-infected multinucleated cells in situ, and has been applied to many experimental systems. However, MAGI parental HeLa cells constitutively express CXCR4, so the infectious titer of T cell-directed HIV-1 can be measured, but CCR5 is not expressed, so macrophage-directed HIV- There was a problem that it could not be applied to many clinical isolates including 1.
[0004]
Therefore, for the purpose of conferring CCR5 expression ability on MAGI cells, CCR5 was incorporated downstream of the promoter of Elongation Factor 1α, and an expression vector pEFBOSbsrCCR5 that can be selected by Blasticidine was constructed, and this was transfected into MAGI cells. , CCR5-expressing MAGI cells (MAGIC-5: MAGI-CCR5) were established by limiting dilution (Masashi Tsuji, Asato Kojima: CCR5-expressing HeLa / CD4-LTR-beta- suitable for HIV-1 infectivity titer measurement) Establishment and application of Gal cells The 10th Annual Meeting of the AIDS Society of Japan, December 1997, Kumamoto, Tatsumi, M. and Kojima, A .: Establishment of HeLa / CD4-LTR-bGal expressing CCR5 suitable for infectivity assay of T- and M-tropic HIV-1 and its application to clinical isolation and cloning of virus. The XIIth International Congress of AIDS, Geneve, Switzerland, June, 1998). This cell line was found to be able to count not only T-cell-directed HIV-1 but also macrophage-directed HIV-1 infectivity titers, and unlike MAGI cells, clones with greatly reduced false positives could be selected.
[0005]
About HIV-1 infectivity titration, virus cloning, virus isolation from clinical specimens and establishment of infectious molecular clones using this MAGIC-5 cell line,
Masashi Tsuji, Asato Kojima: Establishment and application of CCR5-expressing HeLa / CD4-LTR-beta Gal cells suitable for HIV-1 infectivity titer 10th Annual Meeting of the Japan Society for AIDS Research, December 1997, Kumamoto;
Tatsumi, M. And Kojima, A .: Establishment of HeLa / CD4-LTR-bGal expressing CCR5 suitable for infectivity assay of T- and M-tropic HIV-1 and its application to clinical isolation and cloning of virus.The XIIth International Congress of AIDS, Geneve, Switzerland, June, 1998;
Masashi Tsuji Establishment of infectious HIV-1 molecular clones using MAGIC-5A cells The 13th Japan Society for AIDS Research December 1998 Yokohama;
Tatsumi, M. Efficient molecular cloning of infectious HIV-1 subtype C by MAGIC-5 virus cloning and long PCR. The XIthe International Congress of Virology, Sydney, Australia, August, 1999;
Reiko Hachiya, Saori Aizawa, Mari Tanaka, Yukiko Takahashi, Yoshihiro Hirabayashi, Setsuko Ida, Masashi Tsuji, Shinichi Oka Regarding anti-HIV drug resistance testing using CCR5-expressing Hela / CD4-LTR-beta Gal cells (MAGIC5 clone 1-10) Examination The 13th Japan AIDS Society December 1999 Tokyo;
Has been reported. Since this cell line forms the basic foundation for measuring the infectious titer of HIV-1, it was assumed that it could be applied to many HIV-1 research areas. Currently, it has already been applied to drug-resistant Phenotype Assay and anti-HIV drug screening.
[0006]
Although it is quick, simple and economical compared to the commonly used measurement of HIV-1 gag protein p24 by ELISA or the measurement of reverse transcriptase activity, the HIV infectious titer measurement using this cell line can be performed under a microscope. Since it was a measurement by counting cell nuclei stained with X-gal, it was not suitable for rapid processing of multiple specimens.
[0007]
[Problems to be solved by the invention]
The present invention has been made to solve the above-described problems of the prior art. That is, the present invention is a cell suitable for measuring the infectivity titer of HIV-1 virus itself, and is applicable to many clinical isolates including not only T-cell-directed HIV-1 but also macrophage-directed HIV-1. An object to be solved is to provide a cell that can be applied, is quick, simple, and economical, and enables rapid processing of multiple specimens.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have selected secreted alkaline phosphatase as a reporter protein that allows easy acquisition of various enzyme active substrates, high measurement sensitivity, and simplification of measurement techniques. Then, we tried to establish indicator cells into which alkaline phosphatase (SEAP) secreted into the culture supernatant by HIV infection was incorporated as a reporter gene. Then, the HIV sensitivity of this established cell line was measured with a colorimetric reaction using p-Nitrophenol phosphate as a substrate, fluorescence emission using MUP as a substrate (Clonetech), and chemiluminescence measurement system using CSPD as a substrate (Toyobo Co. Ltd). As a result of comparison with Blue Cell Count of MAGIC-5 cells, this cell line showed high correlation with MAGIC-5 in HIV-1 sensitivity. In addition, this cell line was sensitive to a wide range of HIV-1 isolated by peripheral blood lymphocyte culture, and could be detected regardless of the Tropism of the virus, and the detection sensitivity was sufficiently high. The present invention has been completed based on these findings.
[0009]
That is, according to the present invention, an animal cell capable of expressing a secreted reporter protein by HIV-1 infection is provided.
Preferably, the animal cell of the present invention has HIV-1 virus receptor CD4 and HIV-1 virus co-receptor.
Preferably, the animal cell of the present invention has CXCR4 and / or CCR5 as HIV-1 virus co-receptors, and more preferably has both CXCR4 and CCR5 as HIV-1 virus co-receptors.
[0010]
Preferably, the secretory reporter protein is a protein that can be detected by any one or more measurement systems of colorimetric reaction, fluorescence, or chemiluminescence, and more preferably alkaline phosphatase, luciferase, or peroxidase.
[0011]
Preferably, the animal cell of the present invention is obtained by transforming an animal cell with an expression vector having a gene encoding a secreted reporter protein downstream of the HIV-1 LTR sequence.
Preferably, the animal cell of the present invention is a cell derived from a mammalian animal cell, more preferably a cell derived from a human Hela cell or a human HOS cell.
Particularly preferred animal cells of the invention are those having the accession number FERM P-18078.
[0012]
The animal cells of the present invention can be used for the determination of HIV-1 infectivity.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
The animal cell of the present invention is characterized in that it can express a secreted reporter protein by HIV-1 infection.
The animal cell of the present invention is not particularly limited as long as it can be infected by HIV-1.
Usually, since HIV-1 virus receptor CD4 is required for HIV-1 infection, the animal cell of the present invention preferably has HIV-1 virus receptor CD4. In addition, as described above in the present specification, in addition to CD4 molecule, which is a major viral receptor on the cell side, HIV-1 is directed to CXCR4, macrophage-directed in the process of HIV-1 entry into target cells. Since HIV-1 uses CCR5 as a co-receptor, the animal cells of the present invention preferably also have these co-receptors. When it is intended to analyze a wide range of HIV-1 infectivity, it is preferable to use animal cells expressing both CXCR4 and CCR5.
[0014]
These HIV-1 virus receptor CD4 and HIV-1 virus co-receptor may be used as they are if they are originally retained and expressed by the starting animal cell. If the starting cell does not express them, a recombinant expression vector in which a gene encoding the above receptor or co-receptor is incorporated into a suitable expression vector is introduced into the starting animal cell, so that the receptor or The co-receptor can be expressed in the animal cell.
[0015]
The kind of the starting cell that can be used for establishing the animal cell of the present invention is not particularly limited, but is preferably a mammal (for example, a primate such as a human or a monkey, or a rodent such as a mouse, a rat or a hamster). And more specifically, human-derived Hela cells or human-derived HOS cells.
[0016]
As a technique for selecting cells expressing the receptor or co-receptor of interest, an antibiotic resistance gene can be expressed together with the receptor or co-receptor in a recombinant expression vector containing the receptor or co-receptor. And cells that express the target receptor or co-receptor can be selected by culturing in a medium containing the antibiotic. A limiting dilution method can be used as a method for cloning a target cell as a single cell derived from one cell.
[0017]
For example, when a HeLa cell that constitutively expresses CXCR4 is used as a starting cell, a mouse retroviral vector carrying an antibiotic resistance gene (for example, a neomycin (Neo) resistance gene) is transformed into the HeLa cell. Transform to express CD4, optionally transform LTR-βGal expression unit with hygromycin resistance gene and select growing cells. Further, the above cells were transformed with an expression vector for imparting CD4 expression and Puromycin resistance to the transformant for enhancing CD4 expression, and an expression vector for imparting CCR5 expression and Blastocitidine resistance to the transformant. Clone the cells of interest. The target cells cloned by this manipulation are resistant to a total of four selective drugs, G418, Hygromycin, Puromycin and Blastcitidin.
[0018]
The animal cell of the present invention is characterized in that it can express a secreted reporter protein by HIV-1 infection. The animal cell of the present invention expresses a secreted reporter protein by HIV-1 infection, and the expressed secreted reporter protein is secreted into the cell and released into the culture medium. By adding a sample containing HIV-1 to the culture system containing the animal cells of the present invention, collecting the culture solution, and detecting or analyzing the reporter protein secreted in the culture solution by an appropriate method, the sample is obtained. The infectious titer of HIV-1 can be measured.
[0019]
Although the detection method of the reporter protein secreted in the culture solution is not particularly limited, for example, a method of detecting by a colorimetric reaction, fluorescence emission, chemiluminescence, or the like can be mentioned. The type of secretory reporter protein is not particularly limited, but it is preferable that various enzyme-active substrates are easily available, have high measurement sensitivity, and can simplify the measurement technique, such as alkaline phosphatase, luciferase, or peroxidase. Of these, alkaline phosphatase is particularly preferable.
[0020]
One preferred method for producing animal cells that express a secreted reporter protein by HIV-1 infection according to the present invention includes an expression vector having a gene encoding the secreted reporter protein downstream of the HIV-1 LTR sequence. A method for transforming animal cells can be mentioned. The expression vector used in the above method preferably contains a drug resistance gene for selecting a desired cell.
[0021]
For example, a secreted reporter protein is expressed against cells expressing CXCR4, CD4, and CCR5 that are resistant to a total of four types of selective drugs such as G418, Hygromycin, Puromycin and Blastcitidin as described herein above. The animal cell of the present invention can be prepared by transforming an expression vector having a gene to be encoded. In this case, the drug resistance gene that can be incorporated into an expression vector having a gene encoding a secretory reporter protein may be other than the above four drug resistance genes, and examples thereof include a Zeocin resistance gene.
[0022]
More specifically, an expression vector having a gene encoding a secreted reporter protein is transfected into a cell having HIV-1 virus receptor CD4 and HIV-1 virus co-receptor and selected with a drug to obtain a surviving colony be able to. Cloned cells in which these have been propagated are plated twice on a microplate, one infected with HIV-1 and the other as an uninfected control. Two days after infection, the culture supernatant is collected and treated at 65 ° C. for 15 minutes to inactivate endogenous alkaline phosphatase and HIV. SEAP secreted into the culture supernatant can be measured and selected, for example, by a colorimetric reaction using p-Nitrophenol phosphate as a substrate. SEAP activity is low in non-HIV infection, and target cells can be established by selecting clones that show high SEAP activity due to HIV infection.
An example of a cell that can be established by the above-described method is a cell having a deposit number of FERMP-18078.
[0023]
The animal cell culture method and culture conditions of the present invention can be appropriately selected according to the type and nature of the cells. For example, in the case of cell lines derived from HeLa cells, 5% CO2 at 37 ° C in Dulbecco's Modified Minimum Essential Medium (High glucose; Gibco) containing antibiotics (eg, Kanamycin etc.) and supplemented with 5% FCS₂Can be cultured.
[0024]
The animal cells of the present invention can be used for the measurement of HIV-1 infectivity. Specifically, the animal cell of the present invention is inoculated on a microplate, and a sample containing HIV-1 is diluted stepwise with a medium and added to each well. Appropriate culture conditions (eg 5% CO at 37 ° C)₂), And the supernatant of each well is collected and heated at 65 ° C. for 15 minutes to inactivate endogenous reporter protein and HIV. Using this supernatant, a reporter protein can be assayed according to a conventional method. When the reporter protein is alkaline phosphatase, for example, a colorimetric reaction using p-nitrophenol phosphate as a substrate, fluorescence emission using MUP as a substrate, and chemiluminescence measurement system using CSPD as a substrate can be used. .
[0025]
Furthermore, the animal cells of the present invention can be used for HIV drug resistance tests. Currently available anti-HIV drugs include reverse transcriptase inhibitors and protease inhibitors. The animal cells of the present invention are useful for examining HIV drug resistance to these anti-HIV drugs.
[0026]
Specifically, the animal cell of the present invention is inoculated on a microplate, and a sample containing HIV-1 is diluted stepwise with a medium and added to each well. Furthermore, an appropriate dilution series of anti-HIV drug is added, and appropriate culture conditions (for example, 5% CO at 37 ° C.₂), And the supernatant of each well is collected. Thereafter, in the same manner as in the measurement of HIV-1 infectious titer, the drug resistance of HIV against an anti-HIV drug can be examined by assaying the reporter protein.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
[0027]
【Example】
1. Method
All HeLa cell-derived cell lines contain 50 μg / ml Kanamycin as an antibiotic and Dulbecco's Modified Minimum Essential Medium (High glucose; Gibco) supplemented with 5% FCS at 37 ° C with 5% CO2.₂In culture.
The CCR5-expressing MAGI cell line MAGIC-5A already established by the present inventor expresses CD4 by a mouse retroviral vector carrying the Neo resistance gene on the original parental HeLa cell, and the LTR-βGal expression unit is combined with the Hygromycin resistance gene. The MAGI cell line, a cell line selected by FACS (Fluorescent Activated Cell Sorter), is further added to the expression vector pEFBOSpacCD4 (to give CD4 expression and puromycin resistance to Transformant), and the expression vector for CCR5 expression. A cell line transformed with pEFBOSbsr (confers CCR5 expression and Blastocitidine resistance to Transformant), cloned and established by the limiting dilution method, was resistant to a total of four selective drugs: G418, Hygromycin, Puromycin and Blastcitidin.
[0028]
Construction of an expression vector that incorporates secreted alkaline phosphatase (SEAP) as a reporter gene together with Transcriptional Blocker from pSEAP2-Basic (Clontech) downstream of LTR derived from HIV-1 subtype B, and further loaded with Zeocin resistance gene as a new selection gene (FIG. 1). This expression vector was transfected into the MAGIC-5A cell line and then selectively cultured with Zeocin to obtain 165 surviving colonies. These expanded clonal cells were double seeded in 96-well microplates, one infected with HIV-1 and the other as a non-infected control. Two days after infection, 100 μl of culture supernatant was collected, treated for 15 minutes at 65 ° C. for inactivation of endogenous alkaline phosphatase and HIV, and stored at −20 ° C. until measurement. The secreted SEAP in the specimen was selected by colorimetric reaction using p-nitrophenol phosphate with L-Homoarginine as an endogenous alkaline phosphatase inhibitor as a substrate (Berger J. et al., Secreted placental alkaline phosphatase : a powerull new quantitative indicator of gene expression in eukaryotic cells. Gene 66: 1-10, 1988). A clone with low SEAP activity in non-HIV infection and high SEAP activity in HIV infection was selected, and MAGIC-5 / SEAP clone 3-1-1 was established by serial limiting dilution in the absence of a selective drug. did.
[0029]
The HIV sensitivity of this established cell line was measured by the following methods using a colorimetric reaction using p-Nitrophenol phosphate as a substrate, fluorescence emission using MUP as a substrate (Clonetech), and chemiluminescence measurement system using CSPD as a substrate (Toyobo Co Ltd.) and compared with the Blue Cell Count of MAGIC-5 cells for which data has already been accumulated.
[0030]
(Method of chemiluminescence assay)
One million cells of MAGIC-5 / SEAP indicator cells were inoculated into each well of a 96-well microplate the day before infection. R5 macrophage-directed HIV-1, Indie-C1 (10^Fourbcc / ml) or X4T cell line-directed HIV-1 NL432 (2 × 10^Fourbcc / ml) is diluted stepwise with medium, added to each well, and 5% CO at 37 ° C.₂Incubated with. After 48 hours of incubation, the supernatant of each well was collected, heated at 65 ° C. for 15 minutes, and chemiluminescent assay was performed using Reporter Assay Kit-SEAP- (Toyobo) according to the instruction manual.
[0031]
(Method of colorimetric assay)
One million cells of MAGIC-5 / SEAP indicator cells were inoculated into each well of a 96-well microplate the day before infection. R5 macrophage-directed HIV-1, Indie-C1 (10⁶bcc / ml) or X4T cell line-directed HIV-1 NL432 (2 × 10⁶bcc / ml) is diluted stepwise with medium, added to each well, and 5% CO at 37 ° C.₂Incubated with. HIV-1 NL432 (1 × 10⁶bcc / ml) was also incubated in the presence of the RT inhibitor AZT serially diluted at a starting concentration of 1 mM. After 48 hours of incubation, the supernatant of each well was collected, heated at 65 ° C. for 15 minutes, and a colorimetric assay was performed according to the method of Cullen, B.R. and Malim, M.M. Methods in Enzymology 216: 362-368, 1992.
[0032]
2. result
A total of 162 surviving colonies were obtained by selective culture in the presence of 200 μg / ml Zeocin. However, most surviving colonies secreted high SEAP with or without HIV infection. Clone MAGIC that showed stable HIV-1 susceptibility by two limiting dilutions from a group of cells that only detected Backgroud in the absence of HIV, and secreted a large amount of SEAP due to HIV infection, from a few colonies -5 / SEAP 3-1-1 was established.
MAGIC-5 / SEAP 3-1-1 is FERM P-18078, dated October 16, 2000, Institute of Biotechnology, Institute of Industrial Technology, 1-3 1-3 Higashi, Tsukuba City, Ibaraki, Japan ( Deposited in postal code 305-8566).
[0033]
FIG. 2 shows the results of the chemiluminescence assay for HIV-1 against MAGIC-5 / SEAP, and FIG. 3 shows the results of the colorimetric assay for HIV-1 against MAGIC-5 / SEAP.
MAGIC-5 / SEAP was highly correlated with MAGIC-5A in HIV-1 sensitivity. As far as this cell line was examined to date, it was sensitive to all HIV-1 isolated in peripheral blood lymphocyte cultures and was detectable regardless of the Tropism of the virus. In addition, in an experiment using infectious molecular clones X4 HIV-1 NL432 and R5 HIV-1 Indie-C1 in terms of detection sensitivity, 10 bcu (Blue Cell Unit; MAGIC-5A cells were added to 10,000 MAGIC-5 / SEAP cells. The chemiluminescent substrate was so sensitive that significant SEAP activity was detected in the culture supernatant 2 days after HIV-1 infection. In addition, since this cell line does not require fixed staining like MAGIC-5A X-Gal staining to measure its infectious titer, culture can be continued and MAGIC-5A cells can proliferate HIV-1. Furthermore, HIV-1 with a lower titer can be detected by continuing the culture. This measurement system was quick and simple by simply adding a reaction substrate to the culture supernatant, and was able to detect a wide range of HIV infections with high sensitivity. In addition, the MAGIC-5 / SEAP cell line uses the fact that the nuclei of infected cells are blue-stained in the same manner as the parent MAGIC-5A. It can be expressed as the amount of chemiluminescence of SEAP transcribed and expressed by the Tat molecule produced by, and it is considered possible to compare the transcriptional activity of Tat protein of isolated viruses.
[0034]
3. Consideration
In the present invention, MAGIC-, which incorporates, as a reporter, SEAP secreted into the culture supernatant by HIV-1 Tat protein further infected with MAGIC-5A cells recognized as useful as cells for measuring HIV-1 infectivity. 5 / SEAP cell line was established. In the process of establishing this cell line, the majority of colony cell groups resistant to the selective drug Zeocin in MAGIC-5A cells incorporating the SEAP expression unit are significantly higher than the background despite being non-infected with HIV-1. It was found that SEAP activity was secreted into the culture supernatant. This indicates that although HIV-1 LTR is weak, it shows Basal promoter activity and that there is a region that promotes significant trans transcriptional activity in the vicinity of the genome of the integrated HeLa cell. The cell group of Zeocin-resistant colonies that initially incorporated SEAP expression units that did not incorporate Transcriptional Blocker (TB) downstream of the LTR into MAGIC-5A showed higher background values than the cell groups of expression units that incorporated TB. However (data not shown), it was considered that when a sensitive reporter gene was used, the probability of obtaining a cell line that could actually be used for infectivity titration was not high.
[0035]
The MAGIC-5 / SEAP cell line of the present invention has all the advantages of the parental MAGIC-5A cell line. In other words, the PBMC culture method currently used to measure HIV-1 isolation and its proliferation and infection process generally requires several weeks of culture for virus isolation, but MAGIC-5A and MAGIC-5 / In SEAP, it depends on the amount of infectious virus contained in the patient sample.^FourVirus separation is possible from 2 days to 7 days from specimens of copy / ml or more, and there is an advantage that variation in the separation rate by donors is not observed as in the PBMC method. Furthermore, in many HIV-1 viruses, confirmation of separation by syncytium formation by growth is easier compared to PBMC culture, and it is possible to estimate Coreceptor Usage of HIV-1 separated by sharing with MAGI cells, and p24 gaga ELISA method Infection can be confirmed by more rapid and simple X-Gal staining or SEAP activity measurement such as RT and RT measurement. Furthermore, as with MAGIC-5A cells, it was also found that the cell line has a broad spectrum capable of measuring the infectious titer of HIV-2 and SIV, which are other Prime Lentiviruses other than HIV-1.
[0036]
The rationale for selecting SEAP as a new reporter protein in attempting to modify the MAGIC-5A cell line for measuring HIV-1 infection this time is as follows.
(1) Since SEAP is secreted into the culture supernatant outside the cell, cell lysis is not necessary, unlike the luciferase used in normal high-sensitivity reporter assays, and not only sample preparation is easy, SEAP expression, that is, changes in HIV-1 over time can be continuously monitored in the same culture while the cells remain intact.
(2) SEAP is derived from placental alkaline phosphatase, has higher thermal stability than endogenous alkaline phosphatase produced by many cells, and has different sensitivities to reagents, so that SEAP activity can be measured selectively. .
[0037]
(3) Colorimetric reaction, fluorescence reaction, chemiluminescence reaction as a substrate for SEAP, and flexibility in selection depending on the equipment equipped in the laboratory.
(4) From the standpoint of detection sensitivity, SEAP chemiluminescence detection shows sensitivity that is not inferior to that of Luciferase, which is currently the most sensitive, and can detect a very small amount of SEAP of about 10 fg and is quantitative. The linearity has a wide measuring range up to 5 digits.
[0038]
In particular, the MAGIC-5A cell line has the sensitivity to detect only one infectious HIV-1, and the simple operation of measuring the infectious titer by simply adding an enzyme substrate to the culture supernatant is a 96-well microplate. And Liminometer can be used to quickly and easily measure multiple specimens, making it easy to robotize the measurement process, and high throughput in various HIV-1 infection measurement systems such as drug resistance tests and anti-HIV drug screening tests. It is considered to provide basic technology that can be expected to be applied to the construction of measurement systems.
[0039]
【The invention's effect】
The present invention is a cell suitable for measuring the infectious titer of HIV-1 virus itself, and can be applied to many clinical isolates including not only T-cell-directed HIV-1 but also macrophage-directed HIV-1. In addition, a cell that is rapid, simple and economical and enables rapid processing of multiple specimens has been provided.
[Brief description of the drawings]
[Fig. 1] Fig. 1 shows pSEAP2-Basic (Clontech) downstream of HIV-1 subtype B-derived LTR and Transcriptional Blocker together with secreted alkaline phosphatase (SEAP) as a reporter gene, and Zeocin resistance gene as a new selection gene It is a figure which shows the structure of expression vector pHIV-LTRbleoSEAP which carried | supported. The size of the plasmid is 5618 bp.
FIG. 2 shows the results of an HIV-1 chemiluminescence assay against MAGIC-5 / SEAP.
FIG. 3 shows the results of a colorimetric assay for HIV-1 against MAGIC-5 / SEAP.

Claims (2)

  Animal cell with deposit number FERM P-18078.   The animal cell according to claim 1, which is used for measurement of HIV-1 infectivity titer.

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