Nematode in which cholesterol transporter expressed in intestine is replaced with human transporter

Abstract

To conduct a test simulating cholesterol absorption in a human intestine easily and rapidly, at a low cost.SOLUTION: A nematode with NPC1L1, a cholesterol transporter expressed in a human intestine, expressed in its intestine instead of NCR-1, a cholesterol transporter expressed in a nematode's intestine, the method for manufacturing such a nematode, and the method for testing for the cholesterol absorption inhibition capacity of a sample by using such a nematode are provided.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a genetically modified nematode. In particular, the present invention relates to a nematode in which a cholesterol transporter expressed in the human intestine is expressed in the intestine. More particularly, the present invention relates to a nematode in which a cholesterol transporter expressed in the human intestine, NPC1L1, is expressed in the intestine instead of the cholesterol transporter NCR-1 expressed in the intestine of the nematode. Furthermore, the present invention relates to a method for producing the nematode, and a method for evaluating the cholesterol absorption inhibitory ability of a sample using the nematode. [Background technology]

[0002] In Japan, which is an ultra-aging society, the number of patients with hyperlipidemia (2.2 million people), which is a significant hindrance to the quality of life in old age, is rapidly increasing. In the field of functional foods as well as medicine and pharmacy, the function of "inhibiting cholesterol absorption into the body" is recognized as one of the important target areas, and there are many related research examples (for example, see Patent Document 1 and Non-Patent Document 1). On the other hand, in the health food market, functional foods have an average annual growth rate of more than 10%, but the number of products that claim to lower blood cholesterol has not increased. The reasons for this are that existing services do not have an evaluation system for cholesterol absorption inhibition / lowering effect at the individual level, and similar functional evaluation (fat accumulation test) services have drawbacks such as long test periods (about 3 months) and high fees (about 1 million yen / sample), which hinder the development of new functional foods and medicines. The sociological significance of developing an evaluation system that can easily, cheaply and quickly examine the cholesterol absorption inhibition function is extremely great, in order to contribute not only to the development of functional foods and medicines, but also to the development of preventive and therapeutic methods for lifestyle-related diseases that impede healthy lifespan. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Patent Publication WO2005 / 069900 Pamphlet [Non-patent literature]

[0004] [Non-Patent Document 1] S. Nashimoto et al., BBA - Biomembranes 1863 (2021) 183508 A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes https: / / doi.org / 10.1016 / j.bbamem.2020.183508 Received 18 April 2020; Received in revised form 17 October 2020; Accepted 8 November 2020 Available online 11 November 2020 Summary of the Invention [Problem to be solved by the invention]

[0005] There has been a demand for the development of a biological device that can easily, inexpensively, and quickly evaluate the ability of a sample to inhibit cholesterol absorption into the body. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and have created a nematode in which a cholesterol transporter expressed in the human intestine has been incorporated instead of a cholesterol transporter expressed in the nematode intestine. Surprisingly, they have found that the human cholesterol transporter is localized to the apical side of the nematode intestine by a nematode transport-mediating protein. Based on this finding, the present inventors have completed the present invention.

[0007] That is, the present invention provides the following. (1) C. elegans expressing a cholesterol transporter expressed in the human intestine in place of the cholesterol transporter expressed in the nematode intestine. (2) The nematode according to (1), wherein the cholesterol transporter in the nematode is NCR-1 and the cholesterol transporter in a human is NPC1L1. (3) The nematode described in (2), in which NPC1L1 is expressed on the apical side. (4) The following process: (a) In C. elegans, disruption of cholesterol transporters expressed in the intestine and then (b) A cholesterol transporter expressed in the human intestine is expressed in the nematode intestine. 23. A method for producing a nematode, the nematode having in its intestine a cholesterol transporter expressed in human intestine instead of a cholesterol transporter expressed in the intestine of the nematode, comprising: (5) The method described in (4), wherein the nematode cholesterol transporter is NCR-1 and the human cholesterol transporter is NPC1L1. (6) The method described in (5), wherein NPC1L1 is expressed on the apical side. (7) A method for evaluating the ability of a sample to inhibit cholesterol absorption, comprising contacting the nematode according to any one of (1) to (3) with a sample and cholesterol, and then measuring the amount of cholesterol taken up by the nematode. (8) A kit for evaluating the cholesterol absorption inhibitory ability of a sample, comprising the nematode according to any one of (1) to (3). Effect of the Invention

[0008] The nematode of the present invention expresses the human cholesterol transporter NPC1L1 in the intestine, specifically on the apical side, and therefore allows tests simulating cholesterol absorption in the human intestine to be carried out simply, inexpensively and quickly. [Brief description of the drawings]

[0009] [Figure 1] Figure 1 is a schematic diagram showing the construction of a plasmid expressing the human cholesterol transporter NPC1L1 in the C. elegans intestine. [Diagram 2]Figure 2 shows the results of examining the cholesterol transport ability of the produced nematodes. The upper panel shows the results of observing lipid droplets (Lipi-Green stained) in the nematode body using a laser scanning microscope. WT shows an image of the wild-type strain, ncr-1 KO shows an image of the ncr-1 gene-disrupted strain, ncr-1 KO; ges-1p::npc1l1 cDNA shows an image of the cholesterol transporter rescued strain, and ges-1p::npc1l1 cDNA shows an image of the cholesterol transporter overexpressing strain. The bars in the lower panel show, from the left, the diapause rate of the wild-type strain, the ncr-1 gene-disrupted strain, the cholesterol transporter rescued strain, and the cholesterol transporter overexpressing strain. [Diagram 3] Figure 3 shows confocal laser scanning micrographs showing the effect of ezetimibe on the human cholesterol transporter expressed in the C. elegans intestine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In a first aspect, the present invention provides a nematode in which a cholesterol transporter expressed in the intestine of a human is expressed in place of a cholesterol transporter expressed in the intestine of the nematode.

[0011] "Expressing a cholesterol transporter expressed in the human intestine in the intestine instead of a cholesterol transporter expressed in the nematode intestine" means expressing a cholesterol transporter expressed in the human intestine in the intestine of a nematode in which the cholesterol transporter expressed in the nematode intestine has been destroyed. For the destruction of the cholesterol transporter, see the explanation of step (a) of the method for producing a nematode of the present invention described below.

[0012] Nematodes are animals belonging to the phylum Nematoda, and many species live in soil or water. There are non-parasitic and parasitic nematodes. In the present invention, the nematode may be of any type, but is typically Caenorhabditis elegans (C. elegans). C. elegans is a non-parasitic nematode, and is the most well-known and most researched of all nematodes. In the following description, unless otherwise specified, nematodes refer to C. elegans.

[0013] Nematodes are characterized by being easy to breed, having a short life cycle (about 3 days), a life span of about 4 weeks (useful for research on aging and life span), being monoclonal and essentially free of individual differences, being a simple organism (about 1000 cells), having a transparent body, having established nervous, digestive, muscular, and reproductive systems, being well studied genetically, having known entire gene sequences, and being capable of gene function suppression / gene disruption / gene introduction. Therefore, the nematode of the present invention can be easily obtained. A method for culturing the nematode has also been established, and the nematode of the present invention can be obtained in large quantities. By using the nematode of the present invention, the cholesterol absorption inhibitory function can be examined inexpensively, simply, and quickly, and highly reliable data can be obtained with little variation in the results.

[0014] NCR-1 is a cholesterol transporter expressed in the intestine of C. elegans, where it is localized to the apical side of the intestine (the lumen side of the intestine) by the transporter protein RAB-18.

[0015] NPC1L1 is a cholesterol transporter expressed in the human intestine. Its cholesterol transport activity is reduced by ezetimibe.

[0016] The nematode of the present invention typically expresses NPC1L1 in the intestine instead of NCR-1.

[0017] In the nematode of the present invention, NPC1L1 is typically expressed on the apical side of the intestine.

[0018] The nematode of the present invention expresses a human cholesterol transporter in its intestine instead of the native cholesterol transporter, and is therefore useful as a model for cholesterol absorption in the human intestine. Moreover, as described below, the nematode of the present invention can be easily produced.

[0019] In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: (a) In C. elegans, disruption of cholesterol transporters expressed in the intestine and then (b) A cholesterol transporter expressed in the human intestine is expressed in the nematode intestine. The present invention provides a method for producing a nematode in which a cholesterol transporter expressed in the intestine of a human is expressed in place of a cholesterol transporter expressed in the intestine of the nematode, comprising:

[0020] Process (a) Destruction of the cholesterol transporter expressed in the intestine of the nematode means sufficiently suppressing or eliminating the expression of the gene of the cholesterol transporter in the intestine of the nematode, or sufficiently reducing or eliminating the amount of the cholesterol transporter in the intestine of the nematode. It is preferable to eliminate the expression of the gene of the cholesterol transporter in the intestine of the nematode. It is preferable to eliminate the cholesterol transporter in the intestine of the nematode. Sufficiently suppressing the expression of the gene of the cholesterol transporter means, for example, reducing the normal expression level of the gene to 10% or less, preferably 5% or less, more preferably 3% or less, and even more preferably 1% or less. Eliminating the expression of the gene of the cholesterol transporter means making the expression of the gene of the cholesterol transporter in the nematode undetectable by a known method such as the PCR method. Sufficiently reducing the amount of the cholesterol transporter means, for example, reducing the normal amount of the transporter to 10% or less, preferably 5% or less, more preferably 3% or less, and even more preferably 1% or less. Eliminating the cholesterol transporter means making the presence of the cholesterol transporter in the nematode undetectable by a known method such as the ELISA method.

[0021] Preferably, the cholesterol transporter expressed in the intestine of the nematode is disrupted to eliminate the expression of the gene for the cholesterol transporter expressed in the intestine of the nematode, or to eliminate the cholesterol transporter, thereby reducing the variability of the results in the evaluation of the cholesterol absorption inhibitory function using the nematode of the present invention.

[0022] The cholesterol transporter expressed in the intestine of a nematode can be disrupted by a known method, for example, a gene knockout or genome editing technique.

[0023] Process (b) Then, the cholesterol transporter expressed in the human intestine is expressed in the intestine of the nematode. This operation can be performed using known gene recombination techniques. For example, a gene encoding the cholesterol transporter expressed in the human intestine may be inserted into a vector and injected into the nematode by a method such as microinjection.

[0024] Here, we have found that human NPC1L1 is localized to the apical side of the nematode intestine by C. elegans RAB-18, i.e., C. elegans RAB-18 is compatible with human RAB-18, a finding that is unexpected and surprising.

[0025] Since human NPC1L1 is localized to the apical side of the nematode intestine by nematode RAB-18, when expressing human NPC1L1 in the nematode intestine, there is no need to introduce human RAB-18 into the nematode, thereby simplifying the production process of the nematode of the present invention.

[0026] The nematodes obtained by the above-mentioned production method typically express human NPC1L1 in place of native NCR-1 in the intestine, specifically in the apical side, and can be used as a biological device that mimics cholesterol absorption in the human intestine.

[0027] In a further aspect, the present invention provides a method for evaluating the cholesterol absorption inhibition ability of a sample, comprising contacting the nematode of the first aspect with a sample and cholesterol, and then measuring the amount of cholesterol taken up by the nematode.Using this method, the test that mimics the cholesterol absorption in human intestine can be carried out simply, cheaply and quickly.

[0028] The above method may be used to assess the ability of a sample to enhance cholesterol absorption.

[0029] In the above method, the amount of cholesterol taken up when no contact with the sample (but contact with cholesterol) can be used as a control. The evaluation may be quantitative or qualitative.

[0030] The sample may be of any type, for example, food and drink, agricultural products, marine products, candidate antihyperlipidemic drugs, etc. Using the nematode and method of the present invention, for example, data relating to the development of functional foods including functional food products, functional evaluation and high added value of agricultural and marine products, screening and development of new drugs, etc. may be performed.

[0031] Those skilled in the art can use appropriate means and methods to bring the nematode into contact with the sample and cholesterol and to incorporate the sample and cholesterol into the nematode's body. For example, the nematode may be brought into contact with the sample and cholesterol by culturing the nematode in a medium containing the sample and cholesterol.

[0032] Since the body of the nematode is transparent, the amount of cholesterol taken up can be evaluated, for example, by using fluorescently labeled cholesterol. The amount of cholesterol taken up can be evaluated by a known method. For example, the fluorescence may be observed visually using a fluorescence microscope. Also, for example, the fluorescence may be quantified by computer processing of images obtained by a confocal laser scanning microscope.

[0033] In a further aspect, the present invention provides a kit for evaluating the cholesterol absorption inhibitory ability of a sample, comprising the nematode of the first aspect. The kit may also comprise fluorescently labeled cholesterol. Usually, an instruction manual is attached to the kit.

[0034] Unless otherwise specified, the terms used in this specification are to be interpreted as having the meanings commonly understood in the fields of biology, biochemistry, chemistry, medicine, pharmacology, food, and the like.

[0035] The present invention will be described in more detail and specifically below by showing examples, but the examples should not be construed as limiting the present invention. EXAMPLES

[0036] biological materials Generation of ncr-1 gene knockout nematodes We obtained strain JT10800, ncr-2(nr2023)III; ncr-1(nr2022)X, from the Caenorabditis Genetics Center (CGC). The ncr-2(nr2023) mutation was eliminated by backcrossing with the wild-type strain, and a nematode with only the ncr-1(nr2022) mutation (ncr-1 gene disruptant (NCR-1 disruptant)) was generated.

[0037] Preparation of expression plasmids A cDNA was designed based on the amino acid sequence of human NPC1L1 (GenBank Accession No. AAS56939, SEQ ID NO: 1). In order to achieve high expression in nematodes, the codon usage frequency was adjusted to that of nematodes. The cDNA designed in this way (SEQ ID NO: 2) was obtained by custom synthesis. Using nematode genomic DNA as a template, the ges-1 promoter region (sequence number: 3) (to ensure intestine-specific expression) and the unc-54 gene 3'-untranslated region (sequence number: 4) (to ensure mRNA stability) were amplified. The above three fragments were ligated to the Eco RV site of the cloning vector pBluescript II by the in fusion method (using a TakaraBio kit) to obtain a plasmid that expresses the human cholesterol transporter NPC1L1 in the intestine of the nematode. A schematic diagram of the obtained plasmid is shown in Figure 1.

[0038] Production of transgenic nematodes The expression plasmid was microinjected into C. elegans carrying only the ncr-1(nr2022) mutation (ncr-1 gene knockout strain) and into wild-type strains to generate cholesterol transporter rescue strains and cholesterol transporter overexpression strains, respectively. The diapause rate of the produced strains was measured, lipid droplets were detected (Lipi-Green staining), and fluorescent cholesterol uptake (with or without ezetimibe) was examined. The dormancy rate was measured by a conventional method.

[0039] Lipi-Green staining Reagents etc. Lipi-Green was purchased from Dojindo Laboratories. method E. coli OP50 strain, which serves as food for the nematodes, was cultured overnight in liquid medium. The cells were precipitated by centrifugation, the supernatant was removed, and the cells were suspended in M9 medium. This procedure was repeated three times (removal of cholesterol in the medium). Lipi-Green was added to the final E. coli suspension to a final concentration of 0.25 mM, and this solution was applied to the nematode growth medium. Parent nematodes were allowed to lay eggs in the above medium (approximately 4 hours), and the parent nematodes were removed. 40 hours after the nematodes hatched from the eggs and became second-instar larvae, green fluorescence was observed using a confocal laser scanning microscope. result The results are shown in Figure 2. In the ncr-1 gene knockout strain (NCR-1 knockout strain), lipid droplets were clearly reduced compared to the wild type (WT) (second image from the left in the upper panel of Figure 2), and the dormancy rate increased (growth was inhibited) (second bar from the left in the lower panel of Figure 2). In the cholesterol transporter rescue strain (a strain expressing NPC1L1 instead of NCR-1), lipid droplets were restored (second image from the right in the upper panel of Figure 2), and the dormancy rate decreased (growth was restored) (second bar from the right in the lower panel of Figure 2). In the cholesterol transporter overexpression strain (a strain expressing NPC1L1 in addition to NCR-1), more lipid droplets were observed (rightmost image in the upper panel of Figure 2), and growth was promoted (dormancy rate was almost 0). These results confirmed that nematodes expressing NPC1L1 instead of NCR-1 had been produced, and that the produced nematodes had cholesterol transport ability. In addition, lipid droplets were observed on the apical (luminal) side of the intestine in the nematodes produced, confirming that NPC1L1 is expressed on the apical (luminal) side of the intestine, similar to NCR-1. The nematodes used in the above experiments did not exchange the transport-assisting protein RAB-18 with that of humans, and therefore these results indicate that it is not necessary to exchange the transport-assisting protein with that of humans in the nematodes of the present invention.

[0040] Ezetimibe Addition: Absorption of Cholesterol Reagents etc. BODIPY-Cholesterol (manufactured by Cayman Chemical Company) was purchased from Funakoshi Co., Ltd. Ezetimibe was purchased from Tokyo Chemical Industry Co., Ltd. method The cultured 4th instar nematodes were washed three times with M9 buffer (to remove excess E. coli and cholesterol in the medium) and the nematodes were collected. Ezetimibe was dissolved in M9 medium to a final concentration of 10 nM to prepare an ezetimibe solution. 100 μL of ezetimibe solution was added to 10 mg of nematodes and treated at 20°C for 1 hour. After treatment with the ezetimibe solution, the nematodes were washed five times with M9 buffer and collected. BODIPY-Cholesterol (green fluorescent cholesterol) was dissolved in a small amount of ethanol, and a green fluorescent cholesterol solution with a final concentration of 10 μg / mL was prepared in M9 buffer. 100μL of green fluorescent cholesterol solution was added to 10mg of nematodes and treated at 20℃ for 1.5 hours. After treatment with the green fluorescent cholesterol solution, the nematodes were washed five times with M9 buffer and collected. The collected nematodes were immediately observed for green fluorescence using a confocal laser scanning microscope. result The results are shown in Figure 3. Cholesterol absorption was observed in the ezetimibe-free system, but not in the ezetimibe-added system. This result confirmed that the human cholesterol transporter NPC1L1 expressed in the nematode intestine maintained its properties (ezetimibe sensitization). [Industrial Applicability]

[0041] By using the nematode of the present invention, it is possible to screen and evaluate the efficacy of cholesterol absorption inhibitor drugs, and to evaluate the cholesterol absorption inhibitory function of functional foods. Therefore, the present invention can be used in the development of functional foods and pharmaceuticals. [Sequence List Free Text]

[0042] SEQ ID NO: 1 shows the amino acid sequence of human NPC1L1. SEQ ID NO: 2 shows the nucleotide sequence of the designed cDNA encoding human NPC1L1. SEQ ID NO:3 shows the nucleotide sequence of the ges-1 promoter region. Sequence number: 4 shows the nucleotide sequence of the unc-54 gene 3'-untranslated region.

Claims

1. A nematode in which a cholesterol transporter expressed in the human intestine was expressed in the intestine instead of the cholesterol transporter expressed in the nematode intestine.

2. 2. The nematode of claim 1, wherein the nematode cholesterol transporter is NCR-1 and the human cholesterol transporter is NPC1L1.

3. The nematode of claim 2 , wherein NPC1L1 is expressed on the apical side.

4. The following steps: (a) disrupting the cholesterol transporter expressed in the intestine in C. elegans, and then (b) A cholesterol transporter expressed in the human intestine is expressed in the nematode intestine.

23. A method for producing a nematode, the nematode having in its intestine a cholesterol transporter expressed in human intestine instead of a cholesterol transporter expressed in the intestine of the nematode, comprising:

5. 5. The method of claim 4, wherein the nematode cholesterol transporter is NCR-1 and the human cholesterol transporter is NPC1L1.

6. The method of claim 5, wherein NPC1L1 is expressed on the apical side.

7. A method for evaluating a cholesterol absorption inhibitory ability of a sample, comprising contacting the nematode according to any one of claims 1 to 3 with a sample and cholesterol, and then measuring the amount of cholesterol taken up by the nematode.

8. A kit for evaluating the cholesterol absorption inhibitory activity of a sample, comprising the nematode according to any one of claims 1 to 3.

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