Anti-inflammatory CD209 polypeptide fragment and its application in anti-inflammatory of non-alcoholic fatty liver disease

Abstract

The present invention discloses the construction of a CD209 active polypeptide and its activity against fatty liver disease inflammation; wherein, a construction sequence of a CD209 active polypeptide includes an amino acid sequence and a nucleic acid sequence, the amino acid sequence is as shown in SEQ ID NO:1, and the nucleotide sequence is as shown in SEQ ID NO:2; a method for constructing a CD209 active polypeptide expression vector includes synthesizing the cDNA sequence encoding the CD209 active polypeptide by gene synthesis, and ligating it with the linearized pcDNA-Flag eukaryotic expression vector using T4 DNA ligase to obtain a recombinant plasmid; transfecting the recombinant plasmid into macrophages isolated from the liver. The present invention uses a variety of functional means to clarify the antagonistic effect of the CD209 active polypeptide on the pro-inflammatory activity of macrophages, and elucidates that its mechanism is related to the inhibition of the expression of TLR4 and downstream pro-inflammatory factors. At the cell biology level, it is demonstrated that this CD209 active polypeptide has the therapeutic value of antagonizing the inflammatory activity of macrophages and inhibiting the inflammatory damage of fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and relates to the construction of an active polypeptide fragment of CD209 protein and its activity against fatty liver disease inflammation and injury. Specifically, it is to clarify the expression of an intracellular polypeptide fragment of CD209 and its application in the targeted treatment of fatty liver disease. Background Art

[0002] Metabolic associated fatty liver disease (referred to as fatty liver disease) is the largest chronic liver disease in China, accounting for 50% of all chronic liver diseases. It mainly includes simple fatty liver and fatty hepatitis. In the late stage of the disease, liver cell injury and death can be caused by liver inflammation, which can further lead to liver tissue fibrosis and the occurrence of liver cancer, seriously threatening the health of patients. Fatty liver disease is highly prevalent in China, with the total number of patients exceeding 200 million, and it is still growing rapidly with the improvement of the national economy and nutritional level. The pathogenesis of fatty liver disease is complex, and chronic inflammation plays a core role in promoting liver fat accumulation and inflammatory injury. During the occurrence of fatty liver disease, innate immune cells such as macrophages and Kupffer cells can be activated, and the activation of the Toll-like receptor (TLR) pathway on their surface can be promoted, thereby releasing pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, which can change the lipid and glucose metabolism in the liver and trigger liver cell injury, thus mediating the occurrence of liver fat accumulation and fatty hepatitis. Therefore, inhibiting the occurrence of chronic inflammation in the liver can improve the pathological process of fatty liver disease and prevent the occurrence of liver inflammatory lesions, liver fibrosis, and liver cancer.

[0003] During the development of fatty liver disease, macrophages are a crucial type of inflammatory regulatory cells. Under normal conditions, macrophages have both functions of promoting tissue inflammation and immune responses, and can also exert immunosuppressive and tolerogenic activities, which is closely related to their activated surface receptors and signal pathways. We recently found through research that the anti-inflammatory receptor molecule CD209 is specifically expressed in liver macrophages, and its expression is significantly down-regulated during the development of fatty liver disease. The expression of CD209 can significantly inhibit the expression of the pro-inflammatory immune receptor TLR4 in macrophages, thereby inhibiting the pro-inflammatory function of macrophages and enhancing their immunosuppressive activity. Summary of the Invention

[0004] The present invention constructs an active polypeptide fragment of CD209 using genetic recombination technology. This polypeptide fragment can effectively eliminate the expression of the pro-inflammatory receptor TLR4 in macrophages, thereby inhibiting the secretion of various pro-inflammatory cytokines in macrophages, and further reducing the fat accumulation and inflammatory death of liver cells. The present invention can provide an effective targeted therapeutic polypeptide for the anti-inflammatory treatment of fatty liver disease and a new biological targeted drug for the clinical treatment of fatty liver disease.

[0005] The present invention provides the following technical solutions: A construction sequence of an active polypeptide fragment of CD209, and the amino acid sequence of the expressed polypeptide:

[0006]

[0007]

[0008] The cDNA sequence encoding:

[0009]

[0010] As another aspect of the present invention, the present invention provides a method for expressing an active polypeptide fragment of CD209, which includes synthesizing the cDNA sequence encoding the active polypeptide of CD209 by using the total gene synthesis technology, and ligating it with the linearized pcDNA-Flag eukaryotic expression vector using T4 ligase after restriction enzyme digestion to obtain a recombinant CD209 active polypeptide expression plasmid; transfecting the macrophages isolated from the liver with the obtained recombinant plasmid.

[0011] As a preferred embodiment of the method for expressing the active polypeptide fragment of CD209 according to the present invention, wherein: the restriction enzyme includes one or more of BamHI and XhoI.

[0012] As a preferred embodiment of the method for expressing the active polypeptide fragment of CD209 according to the present invention, wherein: the macrophages are macrophages isolated from human peripheral blood.

[0013] As a preferred embodiment of the method for expressing the active polypeptide fragment of CD209 according to the present invention, wherein: for the transfection, 2 μg of the CD209 polypeptide fragment vector is mixed with 4 μl of Fugene liposome reagent and 100 μl of opti-mem medium and incubated for 15 minutes, and then added to 6 1×10 macrophages, and transfected by centrifugation at 2000 rpm for 60 min. After 36 hours of transfection of the CDC42 polypeptide expression vector, the polypeptide expression is detected.

[0014] The beneficial effects of the present invention:

[0015] The present invention utilizes biotechnology to clone the cDNA coding sequence corresponding to a polypeptide into the pcDNA-Flag eukaryotic expression vector. The successful construction of the recombinant clone was demonstrated by PCR and DNA sequencing analysis. This eukaryotic expression vector was then transfected into macrophages, and the expression of the polypeptide was confirmed by immunoblotting, demonstrating that the polypeptide can function in macrophages. Furthermore, various functional methods were used to analyze its inhibitory effect on macrophage-mediated liver inflammation. At the cellular biology level, it was demonstrated that this polypeptide has important activities of antagonizing macrophage-mediated inflammation and inhibiting lipid accumulation and inflammatory death of hepatocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings. Among them:

[0017] Figure 1 CD209 expression was significantly downregulated in fatty liver disease tissues compared to normal liver tissues;

[0018] Figure 2 CD209 was mainly expressed in macrophages in liver tissues;

[0019] Figure 3 Transfection of the CD209 active polypeptide fragment in macrophages;

[0020] Figure 4 The CD209 active polypeptide fragment inhibits TLR4 expression in macrophages;

[0021] Figure 5 The CD209 active polypeptide fragment inhibits the expression of pro-inflammatory cytokines in macrophages (**, P < 0.01, compared to the control group);

[0022] Figure 6 Transduction of the CD209 active polypeptide fragment can inhibit lipid accumulation and inflammatory death of hepatocytes (**, P < 0.01, compared to the control group). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] As Figure 1 shown, CD209 expression was significantly downregulated in fatty liver disease tissues compared to normal liver tissues.

[0024] 100 mg of normal and fatty liver disease tissues obtained from clinical biopsies were each added with 1 ml of tissue lysate and homogenized thoroughly in a quartz homogenizer. Subsequently, the homogenized samples were carefully transferred to 1.5-ml EP tubes and centrifuged at 13,000 g and 4 °C for 15 min. The supernatant was quantitatively analyzed for protein using the BCA method, and an equal amount of total protein sample was taken and added with 5× loading buffer and boiled for 5 min. 40 μg of each of the boiled samples was loaded onto a 12% SDS-PAGE for separation. After the separation, the protein samples were transferred to PVDF membranes, blocked with TBST containing 5% skim milk for 1 hour, and then incubated overnight with anti-CD209 and anti-housekeeping protein GAPDH antibodies at a dilution of 1:1000. They were washed 3 times with TBST, 5 minutes each time; incubated with a horseradish peroxidase-labeled goat anti-mouse secondary antibody at a dilution of 1:10,000 for 30 minutes, and developed using the ECL method. The results showed that CD209 was significantly downregulated in fatty liver disease tissues ( Figure 1 ).

[0025] As Figure 2 shown, CD209 was mainly expressed in macrophages in liver tissues.

[0026] Fatty liver disease tissues obtained from clinical biopsies were washed 3 times with physiological saline to remove blood stains in the tissues. Subsequently, under sterile conditions, the tissues were cut into tissue blocks of 1-2 mm 3 using surgical scissors, and then the tissues were suspended in RPMI-1640 medium and digested at 37 °C for 2-3 hours with the addition of collagenase at a final sterile concentration of 1 mg / mL, DNase I at 0.002%, and hyaluronidase at 0.01%. Subsequently, a single-cell suspension was obtained by filtering through a 70-μm filter. The cell suspension was resuspended in staining buffer and incubated with flow cytometry-fluorescently labeled CD209 and CD68 antibodies at room temperature for 30 minutes, and the co-expression of CD68 and CD209 in the cells was analyzed using a BD flow cytometer. The results showed that CD209 was mainly expressed in CD68-positive macrophages ( Figure 2 ).

[0027] As Figures 3 - 6 shown, the steps for the construction and application of the CD209 active polypeptide fragment include:

[0028] The first step is the construction of the CD209 active polypeptide fragment expression vector;

[0029] Synthesize the cDNA fragment encoding the CD209 active polypeptide fragment using whole-genome synthesis technology, and subject the synthesized cDNA fragment to digestion treatment by incubating with BamHI and XhoI restriction endonucleases at 37 °C for 4 hours. After the digestion treatment, recover the DNA using a DNA recovery kit, and mix it with the pcDNA-Flag vector that has also been digested with BamHI and XhoI restriction endonucleases at a molar ratio of 7:1. Add 10% volume of 10×T4 DNA ligase buffer and 1 μl of T4 DNA ligase, and incubate at room temperature for 1 hour. Transform the ligated sample into DH5α competent bacteria, pick monoclonal bacteria for shaking culture, and identify positive clones by PCR. Sequence verification is performed to confirm the correctness of the cloned sequence.

[0030] Step 2: Transfection and expression detection of the CD209 active polypeptide fragment;

[0031] First, isolate CD68 + / CD45 + macrophages from fatty liver disease tissues. According to the method in Figure 2 , first obtain a single-cell suspension of fatty liver disease tissues, incubate with flow cytometry fluorescently labeled CD68 and CD45 antibodies at room temperature for 30 minutes, and use a BD FACSAria flow cytometer to sort out CD68 + / CD45 + macrophages.

[0032] Sequentially add 2 μg of the CD209 polypeptide fragment expression vector or the empty pcDNA-Flag vector and 4 μl of Fugene liposome reagent to 100 μl of opti-mem medium, incubate at room temperature for 15 minutes, and then add 1×10 6 CD68 + / CD45 + macrophages isolated from fatty liver disease tissues. Transfer the sample to a 6-well plate and place it in a centrifuge. Centrifuge at 2000 rpm for 60 min for transfection. After transfection, continue to culture the monocytes for 36 hours, collect the cell samples for immunoblotting to detect the expression of the CD209 active polypeptide. The results show that compared with the group transfected with the empty vector, the CD209 active polypeptide fragment was successfully expressed in the macrophages of the group transfected with the CD209 active polypeptide vector ( Figure 3 ).

[0033] Step 3: The CD209 active polypeptide fragment inhibits the expression of TLR4 and its downstream pro-inflammatory cytokines in macrophages;

[0034] Example 1: The CD209 active polypeptide fragment inhibits the expression of TLR4 in macrophages;

[0035] In the macrophages transfected with empty vector and CD209 active polypeptide fragment respectively above, incubate with TLR4 antibody labeled by flow cytometry fluorescence at room temperature for 30 minutes, and then analyze the expression difference of TLR4 in the above two groups by using BD FACSAria flow cytometer. The results show that, compared with the empty vector group, the expression of TLR4 in the group transfected with CD209 active polypeptide fragment significantly decreases ( Figure 4 ).

[0036] Example 2: CD209 active polypeptide fragment inhibits the expression of pro-inflammatory cytokines in macrophages;

[0037] Collect the macrophages transfected with empty vector and CD209 active polypeptide fragment respectively above, lyse the cells with RNAzol and extract the total cellular RNA. Reverse transcribe 1 μg of total RNA into cDNA by using RevertAid reverse transcription kit according to the instruction manual, and then perform real-time quantitative PCR detection by using Sybr Green PcrMaster Mix of Takara company. Analyze the samples in Roche LightCycler480 real-time quantitative PCR instrument, and use △△ Ct method to analyze the effect of CD209 active polypeptide fragment expression on the expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in macrophages. The results show that CD209 active polypeptide fragment can significantly inhibit the expression of TNF-α, IL-1β and IL-6 in macrophages ( Figure 5 ).

[0038] Step 4: Transduction of CD209 active polypeptide fragment can inhibit lipid accumulation and inflammatory death of hepatocytes;

[0039] In the macrophages transfected with empty vector and CD209 active polypeptide fragment, culture for 36 hours and collect the supernatant conditioned medium of the above cells respectively, and add it to the cultured HepG2 hepatocytes. After culturing for 48 hours, collect the cells and analyze the effect of control and CD209 active polypeptide macrophage conditioned medium on lipid accumulation and death of hepatocytes by using total triglyceride detection kit and cell apoptosis kit respectively. The results show that after transfection with CD209 active polypeptide fragment, the ability of macrophages to induce lipid accumulation and inflammatory death of hepatocytes significantly decreases ( Figure 6 ).

[0040] Based on the fact that macrophage-mediated liver inflammation is a key mechanism triggering hepatocyte fat accumulation and death in fatty liver disease, the present invention invented an inflammation-inhibiting polypeptide that is targeted to be expressed in macrophages, thereby inhibiting the expression of the key pro-inflammatory receptor molecule TLR4 in macrophages, significantly inhibiting the pro-inflammatory activity of macrophages, and thus protecting hepatocytes from inflammation-induced fat accumulation and death. Through a series of molecular and cellular studies, we found that the CD209 active polypeptide fragment can significantly inhibit the expression of pro-inflammatory receptors and their downstream pro-inflammatory cytokines, thereby slowing down macrophage-mediated chronic inflammation and alleviating inflammation-mediated fat accumulation and liver death in fatty liver disease. The present invention provides preclinical targeted treatment of fatty liver disease by specifically modifying macrophages with the CD209 active polypeptide, which has important significance for the clinical treatment of fatty liver disease and the development of biological targeted drugs.

[0041] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention. Sequence Listing <110> Nantong First Geriatric Hospital (Nantong Hospital Affiliated to Shanghai University, Nantong Sixth People's Hospital, Nantong Pulmonary Hospital) <120> Anti-inflammatory CD209 Polypeptide Fragment and Its Application in Anti-inflammatory of Fatty Liver Disease <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 120 <212> PRT <213> Homo sapiens <400> 1 Gly Pro Leu Val Leu Gln Leu Leu Ser Phe Thr Leu Leu Ala Gly Leu Leu 1 5 10 15 Val Gln Val Ser Lys Val Pro Ser Ser Ile Ser Gln Glu Gln Ser Arg Gln 20 25 30 Asp Ala Ile Tyr Gln Asn Leu Thr Gln Leu Lys Ala Ala Val Gly Glu Leu 35 40 45 50 Ser Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln Glu Leu Thr Gln Leu Lys 55 60 65 Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys Leu Gln Glu Ile Tyr Gln 70 75 80 85 Glu Leu Thr Arg Leu Lys Ala Ala Val Gly Glu Leu Pro Glu Lys Ser Lys 90 95 100 Leu Gln Glu Ile Tyr Gln Glu Leu Thr Trp Leu Lys Ala Ala Val Gly Glu 105 110 115 Leu 120 <210> 2 <211> 360 <212> DNA <213> Artificial sequence <400> 2 gggccgctcg ttttgcaact gctcagtttt accttgttgg ctgggcttct tgttcaggtc 60 agtaaagtac ctagtagcat aagtcaagag cagagtaggc aagacgctat ctatcaaaat 120 cttactcagc tcaaagcggc agtgggcgag ttgtctgaaa agtccaaact gcaagaaatt 180 taccaggaat tgactcagct caaggccgct gttggcgagc ttccagagaa gtctaaactg 240 caggaaatat accaagaact cacgaggctc aaggctgccg tgggggaact tccagaaaag 300 tctaaacttc aagaaattta ccaagaactg acttggctga aagcggctgt gggggagctt 360

Claims

1. A CD209 polypeptide, characterized in that, Its amino acid sequence is SEQ ID NO.

1.

2. The expression method of a CD209 polypeptide according to claim 1, characterized in that, It includes synthesizing the cDNA sequence encoding the CD209 polypeptide using the total gene synthesis technique, and ligating it with the linearized pcDNA-Flag eukaryotic expression vector using T4 ligase after digestion with restriction enzymes to obtain a recombinant CD209 active polypeptide expression plasmid; transfecting the macrophages isolated from the liver with the recombinant plasmid obtained above, and the cDNA sequence is SEQ ID NO.

2.

3. The expression method of a CD209 polypeptide according to claim 2, wherein The restriction enzymes include one or more of BamHI and XhoI.

4. The expression method of a CD209 polypeptide according to claim 2, wherein, The macrophages are macrophages isolated from human peripheral blood.

5. The expression method of a CD209 polypeptide according to claim 2, wherein For the transfection, mix the CD209 polypeptide fragment vector with Fugene liposome reagent and opti-mem medium and incubate, then add it to the macrophages, and perform centrifugal transfection. After transfecting the polypeptide expression vector for several hours, detect the polypeptide expression.

6. The expression method of a CD209 polypeptide according to claim 5, characterized in that, Mix 2 μg of the CD209 polypeptide fragment vector with 4 μl of Fugene liposome reagent and 100 μl of opti-mem medium and incubate for 15 minutes, then add it to 1×10 6 macrophages and transfect by centrifugation at 2000 rpm for 60 min. After 36 hours of transfection of the polypeptide expression vector, detect the polypeptide expression.

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