CN112972651A

CN112972651A - Application of IFITMs in preparation of EBV epithelial infection inhibitor and epithelial tumor prevention and treatment drug

Info

Publication number: CN112972651A
Application number: CN202110197479.2A
Authority: CN
Inventors: 李欣; 杨英桂; 丁腾腾
Original assignee: Individual
Current assignee: Shenzhen Hospital of Southern Medical University
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2021-06-18
Anticipated expiration: 2041-02-22
Also published as: CN112972651B

Abstract

The invention discloses application of IFITMs in preparation of EBV epithelial infection inhibitors and epithelial tumor prevention and treatment medicines, wherein the IFITMs comprise IFITM1, IFITM2 and IFITM 3. The invention constructs an interferon-induced transmembrane protein overexpression and knockdown cell line, and definitely proves that IFITM1 plays an inhibitory role in the EBV infection process for the first time, further discloses the role played by IFITM1 in EBV-positive nasopharyngeal carcinoma and the relation between the IFITM1 and LMP1, and also indicates that IFITM1 can reduce EBV-infected epithelial cells in an EBV exposure state, can reduce the growth rate of tumors, and can be used for developing a prevention and treatment medicine for preventing EBV infection and nasopharyngeal carcinoma.

Description

Application of IFITMs in preparation of EBV epithelial infection inhibitor and epithelial tumor prevention and treatment drug

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to application of IFITMs in preparation of EBV epithelial infection inhibitors and epithelial tumor prevention and treatment medicines.

Background

EB virus (EBV) is one of the members of the gamma herpes virus family, also known as type 4 human herpes virus. The infection rate of EBV is extremely high, and investigation shows that more than 95 percent of people are infected with EBV in the young, and the infected people of EBV are widely distributed, so that diseases can be caused from infants to the old. EBV reaches B lymphocytes mainly through epithelial cells that infect the oropharynx, following vascular transport in epithelial tissues, and then enters the latent infection phase in B lymphocytes. EBV infection can induce a variety of diseases, such as infectious mononucleosis, hemophilus syndrome, oral hairy leukoplakia, chronic active EBV infection and other non-neoplastic diseases, and furthermore, current studies indicate that EBV infection may also induce the development of a variety of malignancies: B/T/NK cell lymphoma such as B/T/NK cell lymphoma in hematological tumor, Burkitt's lymphoma, Hodgkin's lymphoma, etc.; nasopharyngeal carcinoma, gastric cancer, hepatocellular carcinoma, breast cancer, etc. in the non-hematologic tumor. From the eyes and nasopharynx to the skin, hematopoietic system, stomach, liver, lung, and breast. Therefore, EBV infection poses a great threat to human life health.

IFITMs (interferon-induced transmembrane proteins) are ubiquitously expressed in both eukaryotic and prokaryotic animals. The IFITMs can be divided into three categories according to their function: the first of these mainly included human and mouse molecules of IFITM1, IFITM2 and IFITM3 and mouse molecules of IFITM6 and IFITM7, the first three proteins originally designated CD225 (or 9-27), 1-8D and 1-8U, respectively. CD22, 1-8D and 1-8U are also known in mice as fragilis2, fragilis3 and fragilis. All three have strong antiviral activity, so they are also called immune-related IFITMs. The IFITMs have been found to inhibit infection by human pathogenic viruses such as hepatitis virus (HCV), Human Immunodeficiency Virus (HIV), ZIKV and the like, but their roles in EBV infection and related diseases are unknown.

Disclosure of Invention

The invention aims to provide the application of interferon-induced transmembrane protein or an over-expression cell thereof in preparing an EBV infection inhibitor;

the invention also aims to provide the application of the interferon-induced transmembrane protein or the over-expressed cell thereof in preparing the cancer prevention and treatment medicine;

the invention also aims to provide the application of the interferon-induced transmembrane protein or the over-expressed cell thereof in preparing an inhibitor of latent membrane protein LMP 1;

another object of the present invention is to provide the use of an interferon-inducible transmembrane protein or an overexpressed cell thereof for preparing an antagonist of an EBV receptor or a promoter;

it is another object of the present invention to provide a medicament;

another object of the present invention is to provide a cell line.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

the use of interferon-induced transmembrane proteins or cells overexpressing them in the preparation of various EBV infection inhibitors;

wherein the interferon-inducible transmembrane protein comprises IFITM1, IFITM2 and IFITM 3.

Further, the amino acid sequence of the IFITM1 protein is:

MHKEEHEVAVLGPPPSTILPRSTVINIHSETSVPDHVVWSLFNTLFLNWCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTIGFILLLVFGSVTVYHIMLQIIQEKRGY(SEQ ID NO.1)。

further, the amino acid sequence of the IFITM2 protein is:

MNHIVQTFSPVNSGQPPNYEMLKEEQEVAMLGVPHNPAPPMSTVIHIRSETSVPDHVVWSLFNTLFMNTCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGIFMTILLIIIPVLVVQAQR(SEQ ID NO.2)。

further, the amino acid sequence of the IFITM3 protein is:

MNHTVQTFFSPVNSGQPPNYEMLKEEHEVAVLGAPHNPAPPTSTVIHIRSETSVPDHVVWSLFNTLFMNPCCLGFIAFAYSVKSRDRKMVGDVTGAQAYASTAKCLNIWALILGILMTILLIVIPVLIFQAYG(SEQ ID NO.3)。

still further, the interferon-inducible transmembrane protein further comprises: the amino acid sequence shown by the SEQ ID NO.1 or the SEQ ID NO.2 or the SEQ ID NO.3 is subjected to substitution, deletion and/or addition of one or more amino acids and/or terminal modification, and has the amino acid sequence which can inhibit fusion of the EB virus and a host cell, entry of the EB virus into the host cell, replication of the EB virus in the host cell and the like.

Further, the interferon-inducible transmembrane proteins are IFITM1, IFITM2 and IFITM 3.

In the present embodiment, the interferon-inducible transmembrane protein is IFITM 1.

EBV primarily infects B cells and epithelial cells. The inventors found that EBV enters epithelial cells by a different mechanism than B cells. And whether the IFITMs are used as EBV receptor-associated proteins or competitive binding proteins to influence the EBV infection, particularly the infection process in epithelial cells and the mechanism thereof is researched, so that the IFITMs are favorable for breaking the barrier of prevention and treatment of EBV-associated diseases, particularly malignant epithelial tumors, and bring huge benefits to human health.

Further, the above EBV infection inhibitor acts on epithelial cells.

Further, the above-mentioned epithelial cells include normal epithelial cells (NP69 cells, NP460 cells) and epithelial tumor cells (HK1 cells).

According to the actual use requirement, the invention comprises other EBV possibly infected epithelial cells.

In a second aspect of the present invention, there is provided:

the use of interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of a medicament for the prevention and treatment of cancer; wherein the interferon-inducible transmembrane protein comprises IFITM1, IFITM2 and IFITM 3.

Further, the above cancers include nasopharyngeal carcinoma and other epithelial tumors associated with EBV.

The examples in the invention prove that the IFITM1 recombinant protein can reduce the growth rate of tumors, so that a new prevention and treatment means for nasopharyngeal carcinoma can be developed based on IFITM 1.

Further, the dosage forms of the cancer prevention and treatment drugs include, but are not limited to, tablets, capsules, injections, granules, solutions and powders; of course, the skilled in the art can reasonably adopt any dosage form to prepare the cancer prevention and treatment medicine according to the actual use requirement.

In a third aspect of the present invention, there is provided:

the use of interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of an inhibitor of latent membrane protein LMP 1;

The inventor finds that after EBV infects nasopharyngeal epithelial cells and develops nasopharyngeal carcinoma, IFITM1 can inhibit nasopharyngeal carcinoma proliferation and delay tumor development by reducing expression of latent stage key oncoprotein LMP 1.

Further, the above-mentioned latent membrane protein LMP1 inhibitor may be in the form of, but not limited to, tablets, capsules, injections, granules, solutions and powders; of course, the latent membrane protein LMP1 inhibitor can be prepared by any dosage form reasonably according to the actual use requirement by the skilled in the art.

In a fourth aspect of the present invention, there is provided:

the use of an interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of an EBV receptor or a promoter antagonist; wherein the interferon-inducible transmembrane protein comprises IFITM1, IFITM2 and IFITM 3.

Further, the above EBV receptors or promoters include MYH9, EGFR, ITGAV, and EphA 2.

Further, the EBV receptor or enhancer is EphA 2.

EphA2 is a transmembrane tyrosine kinase receptor and belongs to one of the members of the superfamily of tyrosine kinase Receptors (RTKs). EphA2 is the primary host-derived receptor for entry of EBV into epithelial cells by binding to the EBV envelope glycoproteins (gH/gL, gB) and promoting EBV infection. In exerting antiviral effects of IFITMs, members of the same family may exert different effects in different host cells and therefore IFITM1 may co-act to influence the progress of viral infection by interacting with host proteins.

The examples in the invention show that on the basis that IFITM1 can inhibit EBV infection of epithelium, the action mechanism of IFITM1 on EphA2 is as follows: IFITM1 inhibits or impairs the binding between the latter and EBV glycoproteins (gH/gL, gB) by binding to EphA2, thereby reducing the ability of EBV to infect epithelial cells.

In a fifth aspect of the present invention, there is provided:

a medicament comprising an interferon-inducible transmembrane protein or an overexpressed cell thereof; wherein the interferon-inducible transmembrane protein comprises IFITM1, IFITM2 and IFITM3, and the medicament is used for preventing EBV infection.

Furthermore, the medicine also contains other pharmaceutically acceptable auxiliary materials.

Further, the dosage forms of the above drugs include, but are not limited to, tablets, capsules, injections, granules, solutions, powders, and exosome preparations, AAV preparations.

In a sixth aspect of the present invention, there is provided:

a cell line expressing one or more of IFITM1, IFITM2 or IFITM 3.

The over-expression IFITM1 cell line constructed by the invention can effectively and stably express IFITM 1.

The invention has the beneficial effects that:

1. the invention has the advantages that the IFITM1 is proved to play an inhibitory role in the EBV infection process for the first time by constructing the cell line of the interferon-induced transmembrane protein overexpression and knockdown, and the inhibitory role exists in normal epithelia (NP69 and HEK293) and tumor cells (HK 1). Meanwhile, the IFITM2 and IFITM3 possibly play an inhibitory role in the EBV infection process, and provide a theoretical basis for the development of anti-EBV infection medicines.

2. The invention also discloses the function of the IFITM1 in EBV positive nasopharyngeal carcinoma and the relation between the IFITM1 and LMP1, and confirms that after EBV infects nasopharyngeal epithelial cells and develops the nasopharyngeal carcinoma, the IFITM1 can inhibit the proliferation of the nasopharyngeal carcinoma and delay the tumor development by reducing the expression of latent stage key oncoprotein LMP 1.

3. The invention also indicates that IFITM1 can reduce EBV infection of epithelial cells in an EBV exposed state, and a means for effectively preventing EBV infection can be developed based on IFITM 1; the IFITM1 recombinant protein can reduce the growth rate of tumors, and a new prevention and treatment means for nasopharyngeal carcinoma can be developed based on IFITM 1.

Drawings

FIG. 1 is a heat map plotted against the difference in the expression of EBV-infected epithelial cell receptors or promoters and IFITMs;

FIG. 2 shows the stable IFITM1 expression of NP69(A), HEK293(B) and HK1(C) overexpressing IFITM1 cell lines;

FIG. 3 shows IFITM1 expression of NP69(A), HEK293(B) and HK1(C) cell lines after knocking down IFITM 1;

FIG. 4 is a flow chart of EBV-GFP infection efficiency of NP69, HEK293 and HK1 cell lines stably overexpressing IFITM1 (A, B and C) and a representative fluorescent photograph (D);

FIG. 5 is a flow chart of EBV-GFP infection efficiency of NP69, HEK293 and HK1 cell lines of IFITM1 (A, B and C) and a representative fluorescent photograph (D);

FIG. 6 shows fluorescence co-localization experiments (6A) and double co-immunoprecipitation (6B) of IFITM1 with EphA 2;

FIG. 7 is a flow chart (A) showing the efficiency of infection by EBV of the cell line overexpressing IFITM1 with EphA2 in the HEK293 cell line and a representative fluorescence photograph (B);

FIG. 8 is a graph of IFITM1 in vitro and in vivo experiments on the inhibition of EBV + tumor proliferation, wherein A is a comparison of IFITM1 in vitro inhibition of tumor proliferation; b is a picture of the change of the tumor volume in the in vivo experiment; c is the tumor volume growth curve in vivo experiments;

figure 9 is an immunoblot map of the effect of IFIMT1 overexpression (a) and knockdown (B) on LMP1 expression.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

Detection of IFITMs expression in epithelial cells

In this example, a B cell line (Daudi), a nasopharyngeal carcinoma cell line (HK1), an EBV positive cell line (HK1-EBV and C666-1) for nasopharyngeal carcinoma, a human embryonic kidney 239 cell (HEK293), an immortalized normal nasopharyngeal epithelial cell line (NP69), an immortalized normal nasopharyngeal epithelial cell line (NP460) and an EBV positive nasopharyngeal epithelial cell line (NP460-EBV) were selected as test subjects, and the expression of IFITMs in each test subject was tested.

(1) Cell culture:

the B cell line (Daudi), nasopharyngeal carcinoma cell line (HK1) and nasopharyngeal carcinoma EBV positive cell line (HK1-EBV and C666-1) were cultured in RPMI1640 medium containing 10% fetal bovine serum.

Human embryonic kidney 239 cells (HEK293) were cultured in DMEM medium containing 10% fetal bovine serum.

Immortalized normal human nasopharyngeal epithelial cell line (NP69) was cultured in serum-free Defined keratinocyte-SFM medium (DKSFM) supplemented with epithelial growth factors during culture.

The immortalized normal human nasopharyngeal epithelial cell line (NP460) and EBV positive nasopharyngeal epithelial cell line (NP460-EBV) both adopt mixed culture medium (1:1 mixed DKSFM culture medium and serum-free

Culture medium) and supplemented with epithelial growth factors according to the respective growth conditions.

All the above cells were cultured in the presence of 1% penicillin and streptomycin at 37 deg.C in 5% carbon dioxide.

(2) RNA extraction and quality identification:

RNA extraction adopts phenol-chloroform extraction method, Nanodrop^TMThe RNA concentration was determined by spectrophotometer, the purity by agarose gel electrophoresis and the RNA integrity by Agilent 2100 bioanalyzer.

(3) Transcriptome sequencing and library building:

transcriptome sequencing and library construction

The UltraTM RNA Library Prep Kit, methods of use refer to Kit instructions. And after the database is built and qualified, performing transcriptome sequencing.

(4) Differential expression analysis of IFITMs

NP69, NP460, HK1, and HEK293 cells were used as EBV (-) epithelial cell group (EBV.N), and NP460-EBV, HK1-EBV, and C666-1 were used as EBV (+) epithelial cell group (EBV.P). The groups were screened for differentially expressed genes from the B cell line group (B _ Cells), respectively (3 technical replicates per group).

After calculating the average value of the expression levels of each group of genes, logarithms of Log10 are taken, and heat maps of IFITMs (IFITM1, IFITM2 and IFITM3) and EBV infection-related receptors or promoting factors such as MYH9, EGFR, ITGAV, EphA2 and the like are drawn (figure 1), and IFITM1, IFITM2 and IFITM3 are found to have lower expression levels in B cells and show a tendency of basically reverse expression with the EBV infection-related receptors or promoting factors in EBV.N and EBV.P, which indicates that IFITM1, IFITM2 and IFITM3 can play specific inhibitory roles in the process of EBV infection of epithelial cells. Among them, IFITM1 is most pronounced in ebv.n and ebv.p. Indicating that IFITM1, IFITM2, IFITM3 (especially IFTM1) may be associated with inhibiting EBV infection of epithelial cells.

Construction of IFITM1 overexpression epithelial cell line

3 lines of cells were selected for construction of an over-expression cell line of IFITM 1. Among them, NP69, HEK293 and HK1 were selected as test subjects. The reason is that the HEK293 epithelial cells are common model cells in virus infection research; NP69 is an immortalized normal human nasopharyngeal epithelial cell line used to demonstrate whether IFITM1 exerts a protective effect on normal epithelial cells before it has been exposed to EBV; HK1 is a human nasopharyngeal carcinoma cell line, a nasopharyngeal epithelial tumor, whose occurrence and progression are highly correlated with EBV infection. This example illustrates whether IFITM1 protects tumor cells after epithelial cells have undergone malignant transformation by HK 1.

(1) Cell culture:

HEK293T cells are selected to carry out lentivirus construction and amplification, and the culture method is the same as that of the HEK293 cells. NP69, HEK293, HK1 were cultured as described in the above examples.

(2) Construction of an IFITM1 overexpression plasmid and packaging of lentiviruses:

IFITM1 sequence was obtained from normal nasopharyngeal cells by PCR amplification, the obtained IFITM1 sequence was cloned into lentiviral pCW57.RFP-P2A-MCS plasmid (purchased from AddGene), HEK293T cells were transfected with lentiviral packaging plasmid VSVG and psPAX2 (purchased from AddGene) after the plasmid was confirmed by sequencing, and IFITM1 high expression virus and control virus without IFITM1 cloned in the cells were collected and concentrated for cell infection or stored at-80 ℃ for use.

(3) IFITM1 overexpresses lentivirus-infected cells:

after optimizing the infection efficiency of the MSCV-IFITM1-ires-hCD2 and the control virus MSCV-ires-hCD2 obtained in the above step, NP69, HEK293 and HK1 cells cultured in the above step are infected respectively. Fluorescence expression intensity and ratio were observed 72 hours after infection, and uninfected cells were further knocked out with 700. mu.g/mL puromycin (or infected cells were sorted by flow cytometry). When the fluorescence ratio is above 90%, the over-expression effect is detected by using a fluorescence quantitative Polymerase Chain Reaction (PCR) method.

As a result:

compared with the IFITM1 expression of cells infected by control viruses, the IFITM1 stable over-expression cell line effectively expresses IFITM1, which indicates that the IFITM1 stable over-expression cell line is successfully constructed (as shown in FIG. 2).

Construction of IFITM1 knockdown epithelial cell line

NP69, HEK293, HK1 cells were selected for construction of knock-down cell lines of IFITM1 for the same reasons.

(1) Cell culture:

(2) IFITM1 knockdown plasmid constructs and lentiviral packaging:

short hairpin RNAs (shRNAs) 870-hushiFITM1 (SEQ ID NO: 5'-TGTCTACAGTGTCATTCAAT-3' (SEQ ID NO: 4)) and 642-hushiFITM1 (SEQ ID NO: 5'-TGTGACAGTCTACCATATTA-3' (SEQ ID NO: 5)) were designed to target IFITM 1. The shRNA was cloned into a Tet-pLKO-puro vector (purchased from AddGene) and then HEK293T cells were co-transfected with psPAX2 and pMD2 (purchased from AddGene), and 72 hours later cell supernatants containing the assembled lentiviruses were collected and concentrated for cell infection or stored at-80 ℃ for use.

(3) IFITM1 knockdown lentivirus infected cells:

after the infection efficiency of the 642-shIFITM 1-containing virus, 870-shIFITM 1-containing virus and the control lentivirus sh-LacZ is optimized, NP69, HEK293 and HK1 cells are respectively infected; changing the solution after 24 hours, continuously culturing for 1-2 days, adding 1 microgram/mL puromycin (puro) for screening for 2 days, and then screening for 2-3 days by using 3 microgram/mL puro to obtain the puromycin. The resulting infected cells were either stored at-80 for use or subsequently induced for 48 hours with the addition of 2.5. mu.g/mL doxycycline (Dox) for IFITM1 knockdown for low effect testing.

As a result:

compared with the IFITM1 expression level of the cells infected by the control lentivirus sh-LacZ, the IFITM1 expression levels of the virus containing 642-shIFITM1 and the virus containing 870-shIFITM1 are obviously reduced, which indicates that the IFITM1 knockdown cell line construction is successful (figure 3).

Verification that overexpression of IFITM1 inhibits EBV infection of epithelial cells

(1) Preparation of EBV-GFP:

EBV-GFP specifically refers to EBV carrying GFP fluorescent labeling sequence, and the preparation method refers to Zhang H, et, al.nat Microbiol.2018Feb; 3(2):1-8.

(2) Infection of cells overexpressing IFITM 1:

cells overexpressing IFITM1 (NP69, HEK293, HK1 cells) constructed in the above example were seeded at an appropriate density in 6cm dishes and incubated overnight. When the cells grew to about 50% confluence, the medium was replaced with fresh medium and incubated for 1 hour. EBV-GFP prepared by the above procedure was used in accordance with the corresponding multiplicity of infection (MOI 10)³～2.5×10³) Adding virus for infection, removing original culture medium after 24 hr, washing with PBS 2 times to remove unbound virus particles, replacing fresh culture medium, adding 0.8% (v/v) goat anti-human IgG antibody, and culturing at 37 deg.CAfter 6 hours of incubation, residual antibody was washed away, cells (GFP +) were observed for fluorescence under a fluorescence microscope, and the ratio of GFP + cells was measured by flow cytometry.

As shown in FIG. 4, after the IFITM1 is over-expressed in epithelial cells, the infection efficiency of EBV-GFP is reduced by about 50% compared with that of the control group, which indicates that the EBV infection can be effectively inhibited by over-expressing IFITM 1.

Verification of inhibition of EBV infection of epithelial cells by knocking down IFITM1

The procedure for preparing EBV-GFP and the procedure for infecting the cells were the same as in the above examples.

The result is shown in FIG. 5, after the IFITM1 is knocked down in epithelial cells, the infection efficiency of EBV-GFP is increased by 63% -119% compared with that of the control group, and the result shows that IFITM1 can effectively inhibit the EBV infection.

Effect of IFITM1 on the mechanism of EBV infection

The effect of IFITM1 on the receptor for EBV infection of epithelial cells EphA 2.

(1) Cell culture:

in this example, NP69, HEK293, and HK1 cells were selected as the subjects, and the cell culture method was as described in the above examples.

(2) The cultured NP69, HEK293 and HK1 cells are respectively cultured in a 35cm confocal dish, when the cells grow to 50-60%, the cells are fixed for 10 minutes by adding 4% paraformaldehyde after washing once with PBS, then the fixing solution is washed away, and 0.2% TritonX-100 is added for permeabilization for 10 minutes.

(3) Blocking with 5% goat serum at room temperature for 1 hour, incubating overnight with the addition of primary antibodies to IFITM1 and EphA2, washing off the primary antibody the next day, and adding a fluorescent secondary antibody (Alexa)

488 goat anti-mouse IgG H&L and Alexa

647 goat anti-rabbit IgG H&L) incubation for 1 hour at room temperature in the dark, followed by 10 min staining with the nuclear dye DAPI. Staining was observed using a CQ1 rotary confocal high content quantitative analysis system.

As a result:

after staining, the IFITM1 showed green fluorescence and EphA2 showed red fluorescence, and the overlap of the two showed yellow (i.e., indicating co-localization of IFITM1 and EphA 2). As can be seen from fig. 6A (showing only NP69 results), there was significant co-localization of IFITM1 and EphA2 at the cell membrane site.

Interaction of IFITM1 with EphA2

The cell culture method in the above examples was used to prepare NP69, HEK293, and HK1 cells, and to prepare protein lysates of NP69, HEK293, and HK1 cells.

1 mg of each of the NP69, HEK293, and HK1 groove-milling protein lysates prepared in the above procedures were added with IFITM1, and an equal amount of non-specifically immunized homologous antibody (IgG) was used as a control, and gently mixed overnight at 4 ℃. mu.L of Protein A and 5. mu.L of Protein G were added according to the instructions of the co-immunoprecipitation kit (Aibitin, abs955), and gently mixed at 4 ℃ for 3 hours to obtain an immunocomplex precipitate.

And (3) cleaning the immune complex precipitate, adding a loading buffer solution, heating and denaturing at 95 ℃ for 5 minutes, then carrying out instantaneous centrifugation at 14000g for 1 minute, and taking a proper amount (10-30 mu L) for carrying out Western blot detection.

The results are shown in figure 6B (only NP69 results are shown) and western blot detection results show that IFITM1 is able to specifically precipitate EphA2 and vice versa compared to the non-specific immune IgG control group. Indicating that there is an interaction between the two.

Partial reversal of the promoting Effect of highly expressed EphA2 on EBV infection by IFITM1

EphA2 lentivirus was constructed by the IFITM1 lentivirus construction method of the above example using a commercially available EphA2 overexpression plasmid (in this example, EphA2 overexpression plasmid is from the great Janus group of Zhongshan university).

The prepared EphA2 lentivirus is used for infecting NP69, HEK293 and HK1 cells respectively, and the influence of EphA2 overexpression on EBV infection efficiency is detected.

As a result, as shown in FIG. 7, the EBV infection efficiency can be remarkably improved by over-expressing EphA2, thereby reversely verifying the conclusion that IFITM1 can inhibit EBV infection.

In summary, based on the finding that IFITM1 can inhibit EBV infection of epithelium in the above examples, this example further reveals the mechanism: IFITM1 inhibits or impairs the binding between the latter and EBV glycoproteins (gH/gL, gB) by binding to EphA2, thereby reducing the ability of EBV to infect epithelial cells.

Inhibition of EBV-positive epithelial tumor proliferation by IFITM1

(1) Cell culture:

c666-1 cells are selected as an experimental object, and the C666-1 cells are an EBV (Epiphenylene nasopharyngeal carcinoma cell line) natural positive epithelial cell line. The cultivation method is as described in the above examples.

Meanwhile, IFITM1 was constructed to overexpress C666-1 cell line (C666-1-IFITM1-OE) by the method described in the above example.

(2) IFITM1 inhibition of tumor proliferation assay:

in vitro experiment of ifitmm 1 inhibition of tumor proliferation:

the IFITM1 in-vitro experiment for inhibiting tumor proliferation adopts an MTT proliferation method, and comprises the following specific steps:

control cells (C666-1-vector) and C666-1-IFITM1-OE overexpressing IFITM1 in log phase growth were counted separately and plated into wells at 1000 cells per well (100. mu.L). Cell proliferation assays were performed on days 1-7, respectively (1/10 volumes of MTT were added, supernatants were discarded after 4h, absorbance values were measured after 10 min shaking with dimethyl sulfoxide (DMSO)), and proliferation curves were plotted.

In vivo experiment of ifitmm 1 inhibition of tumor proliferation:

IFITM1 in vivo experiment for inhibiting tumor proliferation adopts nude mouse tumorigenesis method, and comprises the following steps:

the number of C666-1-vector and C666-1-IFITM1-OE digestions in the logarithmic growth phase was counted, washed with pre-cooled PBS and then 5X 10⁶The amount per mouse (volume about 200. mu.L) was inoculated subcutaneously into the posterior part of the axilla of the nude mouse. After approximately 2 weeks of feeding, tumor volumes were measured every 3 days and volume growth curves were plotted.

As a result:

the results are shown in fig. 8, and the over-expression of IFITM1 was effective in inhibiting tumor proliferation in both MTT cell proliferation experiments (in vitro) and nude mouse tumorigenesis experiments (in vitro).

IFITM1 downregulation of EBV latent membrane protein LMP1

(1) Cell culture:

c666-1 cells were selected as the test subjects, and the culture method was as shown in the above example.

Meanwhile, IFITM1 overexpression C666-1 cell line and IFITM1 knock-down C666-1 cell line were constructed as shown in the above examples.

(2) The expression level of IFITM1 in the C666-1 cell strain over-expressed by IFITM1 and the C666-1 cell strain knocked down by IFITM1 is detected by an immunoblotting method.

Immunoblotting detection procedures were as described in the above examples.

The results are shown in fig. 9, the overexpression of IFITM1 significantly reduced the expression of LMP1, and the knock-down of IFITM1 significantly increased the expression of LMP 1.

In conclusion, after EBV infects nasopharyngeal epithelial cells and develops nasopharyngeal carcinoma, IFITM1 can inhibit nasopharyngeal carcinoma proliferation and delay tumor progression by reducing expression of latent stage key oncoprotein LMP 1.

Prevention effect of IFITM1 on EBV infection of nasopharyngeal epithelial cells

Nude mice were randomly divided into two groups of 6 mice each, and were intraperitoneally injected with IFITM1 recombinant protein (experimental group) and physiological saline (control group), respectively, once every 3 days. After 3 injections, HEK293, NP69, HK1 cells overexpressing EphA2 were subcutaneously inoculated into two groups of nude mice, respectively, and then EBV-GFP carrying a fluorescent marker (prepared as shown in the above example) was inoculated into the same site, and every 12 hours after inoculation, a photograph was taken with a small animal imaging device, and the amount of accumulated fluorescence at the inoculated site was recorded. Nude mice were sacrificed after continuous observation for 7 days, and the inoculated tissue was taken out to prepare a cell homogenate for flow cytometry for EBV infection efficiency analysis and comparison.

As a result, the fluorescence accumulation amount and infection efficiency of the inoculation site of the nude mice intraperitoneally injected with the IFITM1 recombinant protein are obviously lower than those of the mice injected with physiological saline. Indicating that IFITM1 confers cells with the ability to fight EBV infection.

Therapeutic effect of IFITM1 on nasopharyngeal carcinoma

Nude mice were randomly divided into two groups of 6 mice each, and simultaneously inoculated subcutaneously with C666-1 cells. On day 14 after inoculation, physiological saline (control group) and IFITM1 recombinant protein (experimental group) were intraperitoneally injected every 3 days, respectively, after which tumors were measured every 3 days

As a result, compared with a normal saline group, the IFITM1 recombinant protein injected into the abdominal cavity obviously delays the growth rate of subcutaneous tumors of nude mice.

In summary, the above examples demonstrate that IFITM1 reduces EBV infection of epithelial cells in an EBV-exposed state, and therefore, a means for effectively preventing EBV infection can be developed based on IFITM 1. However, the above examples also demonstrate that the IFITM1 recombinant protein can reduce the growth rate of tumors, and therefore, new therapeutic approaches for nasopharyngeal carcinoma or other EBV-associated epithelial tumors can also be developed based on IFITM 1.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> Lixin

Yang Ying Gui (a Chinese character of' Yang Ying Gui

Ding Tengteng

Application of <120> IFITMs in preparation of EBV epithelial infection inhibitor and epithelial tumor prevention and treatment drug

<130>

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 125

<212> PRT

<213> IFITM1

<400> 1

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Thr Ile Leu Pro Arg Ser Thr Val Ile Asn Ile His Ser Glu Thr Ser

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Val Pro Asp His Val Val Trp Ser Leu Phe Asn Thr Leu Phe Leu Asn

35 40 45

Trp Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr Ser Val Lys Ser Arg

50 55 60

Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala Gln Ala Tyr Ala Ser

65 70 75 80

Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Ile Leu Gly Ile Leu Met

85 90 95

Thr Ile Gly Phe Ile Leu Leu Leu Val Phe Gly Ser Val Thr Val Tyr

100 105 110

His Ile Met Leu Gln Ile Ile Gln Glu Lys Arg Gly Tyr

115 120 125

<210> 2

<211> 132

<212> PRT

<213> IFITM2

<400> 2

Met Asn His Ile Val Gln Thr Phe Ser Pro Val Asn Ser Gly Gln Pro

1 5 10 15

Pro Asn Tyr Glu Met Leu Lys Glu Glu Gln Glu Val Ala Met Leu Gly

20 25 30

Val Pro His Asn Pro Ala Pro Pro Met Ser Thr Val Ile His Ile Arg

35 40 45

Ser Glu Thr Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn Thr

50 55 60

Leu Phe Met Asn Thr Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr Ser

65 70 75 80

Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala Gln

85 90 95

Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Ile Leu

100 105 110

Gly Ile Phe Met Thr Ile Leu Leu Ile Ile Ile Pro Val Leu Val Val

115 120 125

Gln Ala Gln Arg

130

<210> 3

<211> 133

<212> PRT

<213> IFITM3

<400> 3

Met Asn His Thr Val Gln Thr Phe Phe Ser Pro Val Asn Ser Gly Gln

1 5 10 15

Pro Pro Asn Tyr Glu Met Leu Lys Glu Glu His Glu Val Ala Val Leu

20 25 30

Gly Ala Pro His Asn Pro Ala Pro Pro Thr Ser Thr Val Ile His Ile

35 40 45

Arg Ser Glu Thr Ser Val Pro Asp His Val Val Trp Ser Leu Phe Asn

50 55 60

Thr Leu Phe Met Asn Pro Cys Cys Leu Gly Phe Ile Ala Phe Ala Tyr

65 70 75 80

Ser Val Lys Ser Arg Asp Arg Lys Met Val Gly Asp Val Thr Gly Ala

85 90 95

Gln Ala Tyr Ala Ser Thr Ala Lys Cys Leu Asn Ile Trp Ala Leu Ile

100 105 110

Leu Gly Ile Leu Met Thr Ile Leu Leu Ile Val Ile Pro Val Leu Ile

115 120 125

Phe Gln Ala Tyr Gly

130

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

tgtctacagt gtcattcaat 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

tgtgacagtc taccatatta 20

Claims

1. Use of an interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of an EBV infection inhibitor;

2. The use of claim 1, wherein the EBV infection inhibitor acts on epithelial cells.

3. The use of claim 2, wherein the epithelial cells comprise normal epithelial cells and epithelial tumor cells.

4. The use of interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of a medicament for the prevention and treatment of cancer;

5. The use of claim 4, wherein the cancer comprises nasopharyngeal carcinoma and other EBV-associated epithelial tumors.

6. The use of interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of an inhibitor of latent membrane protein LMP 1;

7. The use of an interferon-induced transmembrane protein or an overexpressed cell thereof in the preparation of an EBV receptor or a promoter antagonist;

8. The use of claim 7, wherein the EBV receptor or enhancer comprises MYH9, EGFR, ITGAV, and EphA 2.

9. A medicament comprising an interferon-inducible transmembrane protein or an overexpressed cell thereof;

wherein the interferon-inducible transmembrane protein comprises IFITM1, IFITM2 and IFITM 3;

the medicine is used for preventing EBV infection.

10. A cell line expressing one or more of IFITM1, IFITM2 or IFITM 3.