Novel Fab-scFv fusion protein and application thereof

Abstract

The invention discloses a Fab-scFv novel fusion protein and application thereof, and belongs to the field of biological medicine. The Fab-scFv fusion protein is obtained by fusing three amino acid sequences, and through mammalian cell expression, purification identification and animal experiments, it is shown that the Fab-scFv fusion protein has potential application value in psoriasis treatment. The Fab-scFv fusion protein exceeds the ability of infliximab to combine with FcRn on keratinocytes, further inhibits proliferation of the keratinocytes, reduces skin tissue neutrophil infiltration, pathogenicity Th1, Th2 and Th22, increases Treg cells and the like, and has the treatment titer exceeding the psoriasis treatment effect of infliximab with the same molar concentration. In addition, the traditional Chinese medicine composition can efficiently interact with keratinocytes which play a key role in psoriasis, so that the effect of more efficiently treating psoriasis is realized.

Description

Technical Field

[0001] The invention relates to a novel Fab-scFv fusion protein and application thereof, and belongs to the field of biomedicine. Background Art

[0002] Psoriasis (Pso) is a chronic, nonspecific autoimmune disease with a long course, easy relapse and variable; its typical clinical features are epidermal erythema, thickening and scaling, which are limited to one place or widely distributed throughout the body. Studies have also found that it is associated with a variety of complications, and special parts that are difficult to treat include the scalp, face, nails, genitals, palms and soles.

[0003] Pso is a complex multifactorial disease, including genetic, environmental, behavioral and immune-related factors, and its pathogenesis has not yet been fully elucidated.

[0004] Existing treatments mainly include corticosteroids, vitamin D analogs, methotrexate and biologics, but they are often accompanied by various side effects, and antibody drugs have limited direct effects on keratinocytes. Infliximab (IFX) is an effective and safe treatment option for moderate to severe psoriasis, but its use has some serious adverse reactions and limitations, such as no response, new onset or worsening of psoriasis, systemic exposure, low skin concentration, and poor tissue permeability due to its high molecular weight, which are the main problems of IFX in treating psoriasis. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, one of the objectives of the present invention is to construct a new Fab-scFv fusion protein, which is formed by fusion of three amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, wherein the amino acid sequence shown in SEQ ID NO: 1 is the heavy chain of the Fab domain of infliximab; the amino acid sequence shown in SEQ ID NO: 2 is the light chain of the Fab domain of infliximab; and the amino acid sequence shown in SEQ ID NO: 3 is the single-chain antibody FnAb8.

[0006] The novel fusion protein is obtained by the following method:

[0007] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 (shown in SEQ ID NO: 4), the nucleotide sequence encoding the amino acid shown in SEQ ID NO: 2 (shown in SEQ ID NO: 5) and the nucleotide sequence encoding the amino acid shown in SEQ ID NO: 3 are connected with (G4S)3, the connected gene sequences are inserted into the expression vector pcDNA3.4, and then the plasmid is transfected into mammalian cells to prepare protein to obtain a novel fusion protein.

[0008] Further preferably, all nucleic acid sequences encoding the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 can be used for vector construction.

[0009] The second purpose of the present invention is to provide an application of the novel Fab-scFv fusion protein in the preparation of a drug for treating psoriasis.

[0010] Beneficial effects of the present invention

[0011] (1) The active substance in the present invention is a novel Fab-scFv fusion protein, the molecular weight of which is about half that of IFX.

[0012] (2) The novel Fab-scFv fusion protein of the present invention has a sustained and significant improvement effect on typical indicators of the mouse psoriasis model (squama, hypertrophy, PASI, etc.) that is superior to IFX of the same molar concentration.

[0013] (3) The novel Fab-scFv fusion protein of the present invention significantly improves skin tissue lesions and damage, inhibits keratinocyte proliferation, and reduces the thickness of skin lesions in mice, and has a better effect on treating psoriasis than IFX of the same molar concentration.

[0014] (4) The novel Fab-scFv fusion protein of the present invention retains the pharmacological activity of IFX and further significantly reduces neutrophil infiltration in skin tissue, regulates immune cell subsets such as reducing pathogenic Th1, Th2, Th22 and increasing Treg cells, etc., thus having an immunomodulatory effect superior to that of IFX of the same molar concentration in the treatment of psoriasis.

[0015] (5) The novel Fab-scFv fusion protein of the present invention can efficiently interact with keratinocytes, which play a key role in psoriasis, and has an ability to bind to FcRn on / in keratinocytes that exceeds that of IFX, thereby achieving a more efficient treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of SDS-PAGE detection of the new Fab-scFv fusion protein.

[0017] Figure 2 This is the result of WB detection of the new Fab-scFv fusion protein.

[0018] Figure 3 High-content cell imaging was used to track the dynamic process of the interaction between the new Fab-scFv fusion protein and keratinocytes HaCaT: representative fluorescence images of the three groups of IFX, Fab, and Fab-scFv at different time periods.

[0019] Figure 4 Dynamic statistical results of the interaction between the novel Fab-scFv fusion protein and keratinocytes HaCaT were tracked by high-content cell imaging: fluorescence intensity statistics of the three groups of IFX, Fab, and Fab-scFv at different time periods.

[0020] Figure 5 This is a graph showing the weight change trend of psoriasis model mice after 10 days of drug administration.

[0021] Figure 6 Erythema scores were calculated for psoriasis model mice and mice treated with medication for 10 days.

[0022] Figure 7 Scoring of scales in psoriasis model mice and mice treated with medication for 10 days.

[0023] Figure 8 Hypertrophy scores were scored for psoriasis model mice and mice treated for 10 days.

[0024] Fig. 9 Total PASI scores of psoriasis model mice and mice given medication for 10 days.

[0025] Fig.10 Representative pictures of the skin of each group of psoriasis model mice.

[0026] Fig.11 Representative images of H&E staining of each group of psoriasis model mice and schematic diagrams of the maximum and minimum values ​​of mouse epidermal thickness.

[0027] Fig.12 This is a statistical chart of the maximum and minimum epidermal thickness of each group of psoriasis model mice stained with H&E.

[0028] Fig.13 Representative figures of immunohistochemistry Ki67 results of each group of psoriasis model mice.

[0029] Fig.14 This is a statistical chart of the immunohistochemistry Ki67 results of each group of psoriasis model mice.

[0030] Fig.15 Representative flow cytometry images of Th1, Th2, Th22, and Treg cells in spleen tissue of each group of psoriasis model mice.

[0031] Fig.16 This is a statistical chart of the proportion of Th1 and Th2 cells in the spleen tissue of each group of psoriasis model mice.

[0032] Fig.17 This is a statistical chart of the proportion of Th22 and Treg cells in the spleen tissue of each group of psoriasis model mice.

[0033] Fig.18 Representative images of neutrophil infiltration in skin tissue of each group of psoriasis model mice.

[0034] Fig.19 This is a statistical chart of neutrophils in the skin tissue of each group of psoriasis model mice. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0036] Unless otherwise specified, the methods used in the present invention are methods well known to those skilled in the art, such as the preparation of fusion protein Fab-scFv.

[0037] Unless otherwise specified, the reagents and experimental materials used in the present invention can be purchased from conventional commercial channels.

[0038] Example 1

[0039] Preparation process of novel Fab-scFv fusion protein

[0040] The heavy chain nucleotide sequence (shown in SEQ ID NO:4) and light chain nucleotide sequence (shown in SEQ ID NO:5) of the Fab expressing infliximab were connected to the 5' end of the nucleotide sequence of FnAb8 using a (G4S)3 connecting sequence. The gene sequence was synthesized and inserted into the expression vector pcDNA3.4 (Baiying), and then the plasmid was transfected into mammalian cells to prepare a new Fab-scFv fusion protein.

[0041] Example 2

[0042] Coomassie brilliant blue staining (SDS-PAGE) and protein immunoblotting (Western blotting, WB) experiments verified the molecular weight of the novel Fab-scFv fusion protein prepared in Example 1.

[0043] SDS-PAGE: prepare 15% precast gel, add Fab-scFv new fusion protein, Fab, IFX, 2μg / 20μL to each well at a loading volume of 20μL, run SDS-PAGE at 120V for 1h, then take out the gel and incubate it with Coomassie Brilliant Blue for 30min, ultrapure water for 10min / time, wash 3 times, dehydrate with ultrapure water at room temperature overnight, and take pictures for observation.

[0044] WB: Prepare 15% precast gel, add Fab-scFv new fusion protein, Fab, IFX, 2μg / 20μL per well at a loading volume of 20μL, run SDS-PAGE at 120V for 1.5h, then transfer to PVDF membrane at 300mA for 2h, then block the membrane with 5% skim milk at room temperature for 30min, and then incubate at 4℃ with primary antibody (Goat anti-Human IgG F(ab) 2 Antibody 1:1000) overnight, and then the membrane was incubated with HRP-conjugated secondary antibody (1:3000) at room temperature for 1 h, and the image was observed using a developer.

[0045] The results of SDS-PAGE and WB detection of the new Fab-scFv fusion protein showed that the molecular weight of the non-reduced Fab-scFv new fusion protein was about 76kDa, and the molecular weights of the two bands of the reduced protein were 53kDa (Fab fragment, heavy chain and light chain) and 23kDa (molecular weight of FnAb8). Under the same conditions, the molecular weight of the non-reduced Fab was about 48kd, and the two molecular weights of the reduced version overlapped at 25kda (heavy chain of Fab) and 23kd (light chain of Fab). The molecular weight of the non-reduced IFX was about 144kd, and the two bands of the reduced version were about 55kd and 25kd, respectively. The results are as follows Figure 1 , Figure 2 shown.

[0046] Example 3

[0047] High-content cell imaging was used to track the dynamic process of the interaction between the novel Fab-scFv fusion protein and keratinocytes HaCaT, and the experimental results were explained in detail with the accompanying figures.

[0048] HaCaT keratinocytes in the logarithmic growth phase were taken and counted with a cell counting plate. 1x10 4The cell suspension of each well was placed under a microscope to observe whether it was uniform. After being cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours, different drug-containing culture media (Fab-scFv, IFX, Fab) were added to each well and cultured in a constant temperature incubator at 37°C and 5% CO2 for different time periods (0.5h, 1h, 2h, 4h, 6h, 12h, 24h). The supernatant was discarded, 100μL of 4% PFA was added to each well for fixation for 20 minutes, the supernatant was discarded, 100μL of permeabilization agent was added to each well, permeabilized for 20 minutes, and then washed with PBS. 100μL of 1.5% BSA was added to each well for blocking for 40 minutes, and the blocking solution was discarded. 50μL of primary antibody (Goat anti-Human IgG F(ab)) was added to each well. 2 Antibody), incubated at 4°C overnight, and washed with 1xTBST. 50 μL of secondary antibody (Alexa FluorTM488 Donkey anti-Goat IgG) was added to each well, incubated in the dark for 1 hour, washed with TBST, incubated with phalloidin in the dark for 1 hour at room temperature, washed with 1xTBST, incubated with DAPI in the dark for 10 minutes at room temperature, and photographed with a high-content cell imaging analysis system to observe whether the new Fab-scFv fusion protein enters keratinocytes.

[0049] All experimental results that required statistics were processed using Prism 8.0, and comparisons among multiple groups were performed using two-way ANOVA. P<0.05 indicated statistical significance, and vice versa. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0050] The results of high-content cell imaging analysis showed that under a 63x field of view, the normal groups of the three drugs had no green (his) fluorescence. 0.5h after administration of the Fab-scFv group, a large amount of green fluorescence could be observed by incubation with the primary antibody Fab for color development, while there was almost no green fluorescence in the IFX group and Fab group 0.5h to 4h after administration. The results are shown in Figure 3 The fluorescence intensity of the new fusion protein Fab-scFv (green) was significantly higher than that of IFX and Fab after 0.5h (P<0.0001), about 5 times that of Fab and 10 times that of IFX. Figure 4 The experimental results show that the new fusion protein Fab-scFv can efficiently interact with keratinocytes, which play a key role in psoriasis, and has the ability to bind to FcRn on / in keratinocytes that exceeds that of IFX and Fab, and continuously enriches and exerts its effect, achieving a more efficient treatment of psoriasis.

[0051] Example 4

[0052] The experimental process of the new fusion protein Fab-scFv in model mice is described in detail with the accompanying figures.

[0053] 1. Experimental Materials

[0054] 1.1 Experimental animals

[0055] 8-10 weeks old healthy SPF double transgenic hTNF-α / hFcRn C57BL / 6 male mice were randomly selected, separated into cages, and marked. After being adapted to the IVC system (20-25°C, 50%-60% RH) for 1 week, they were allowed to drink water and eat freely.

[0056] 1.2 Test drugs

[0057] Modeling and drug administration reagents: 5% imiquimod cream (IMQ), Sichuan Mingxin Pharmaceutical Co., Ltd.

[0058] Test solution for the control group: IFX (infliximab), Cilag AG, Switzerland.

[0059] Drug group test solution: The fusion protein Fab-scFv was obtained in Example 1 above.

[0060] 2. Experimental methods

[0061] 2.1 Animal grouping and model establishment

[0062] 2.1.1 Animal grouping

[0063] Before the experiment, the mice were weighed and marked, and randomly divided into five groups according to their body weight: normal group, model group, IFX group, Fab-scFv-1 group, and Fab-scFv-2 group, with six mice in each group.

[0064] 2.1.2 Model establishment

[0065] The mice had their backs shaved for 3 consecutive days, and IMQ was administered to the back skin of all groups except the normal group every day for 9 days to induce a psoriasis model; the normal group drank purified water until the end of the experiment, for a total of 10 days.

[0066] 2.1.3 Administration and dosage

[0067] The test drug was injected subcutaneously into the mice once every two days. The IFX group was injected subcutaneously at a dose of 10 mg / kg; the Fab-scFv-1 group was injected subcutaneously at a dose of 5 mg / kg; and the Fab-scFv-2 group was injected subcutaneously at a dose of 2.5 mg / kg.

[0068] 2.2 Psoriasis Area and Severity Index (PASI) score

[0069] The weight and skin condition of the mice were recorded every day, and three different parameters (i.e., erythema, scaling, and thickness) were scored from 0 to 4 (0 for none, 1 for mild, 2 for moderate, 3 for severe, and 4 for very severe), and then added together to obtain the total severity score during the entire treatment process. Weight change: weight loss (%) = (weight on day 0 - weight on the day) × 100%. P < 0.05 indicates statistical significance, otherwise it is not statistically significant. # indicates comparison with the model group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. + indicates comparison with IFX, +P < 0.05, ++P < 0.01, +++P < 0.001, ++++P < 0.0001.

[0070] 2.3 Collection of materials

[0071] After the experiment, spleen tissue was removed, ground with PBS and placed in a 50 mL centrifuge tube for use with a FACS Calibor flow cytometer to measure changes in the proportion of immune cell populations; skin tissue was removed, rinsed with PBS, and fixed in 4% paraformaldehyde for H&E staining and immunohistochemical analysis.

[0072] 2.4 Detection of immune cell populations

[0073] On the day of killing the mouse model, the spleen of each mouse was taken, ground and filtered through a 100-mesh sieve to prepare a single-cell suspension, centrifuged (1200rpm, 5min, 4°C), the supernatant was discarded, and red blood cell lysis buffer was added for 5min. The digestion was terminated by RPMI1640 complete medium, centrifuged (1200rpm, 5min, 4°C), the supernatant was discarded, and the complete medium was added for resuspending. The cell suspension was taken into a 96-well plate, and Th1\Th2\Th22\Treg were stimulated by BD cocktail stimulator in a cell culture incubator for 6-10h, and then the following staining was performed. The changes in the proportion of immune cell populations in the spleen tissue of each group were detected by FACS Calibor flow cytometer;

[0074] ①Th1 / Th2:CD3+\CD4+\IFN-γ\IL-4

[0075] ②Th22:CD3+\CD4+\IL22

[0076] ③Treg:CD4+\CD25+\Foxp3

[0077] 2.5 Observation of skin tissue structure (H&E)

[0078] The skin tissue fixed with 4% paraformaldehyde was embedded and sliced, and the tissue slices were placed at 60℃ for 4h. Dewaxing and rehydration: xylene I, II, III for 10min each, anhydrous ethanol I, II, 90% ethanol, 85% ethanol, 75% ethanol for 5min each, and running water for 10min. Hematoxylin was used to stain the nucleus for 2min, and running water was used to rinse for 10min. The nuclear staining was observed under a microscope. 1% hydrochloric acid alcohol was used for differentiation for 2s, and running water was used for 10min. Eosin was used for staining for 2min, and running water was used for 1min. 90% ethanol, 95% ethanol, and 100% ethanol were used for 1s each, and xylene was used for 2min. The slides were sealed with neutral gum, and then observed and photographed under a microscope.

[0079] 2.6 Immunohistochemistry (Ki67)

[0080] Bake the slides for 30 minutes, dewax, let stand at room temperature for 20 minutes with 3% hydrogen peroxide, perform antigen repair with citrate buffer, fix with immunostaining fixative for 5 minutes, and add immunostaining blocking solution for 60 minutes. Incubate with primary antibody Ki67 at 37°C for 1.5 hours, and secondary antibody at room temperature for 1 hour. Incubate with DAB colorimetric solution at room temperature in the dark for 5 to 10 minutes, observe the color change, and terminate the color development when it shows brown-yellow color. Wash with pure water 3 times, 5 minutes each time. Stain with hematoxylin for 5 minutes. Wash with water, return to blue with PBS for 3 minutes, and wash with water. 70%, 85% for 5 minutes each, 100% alcohol twice, 5 minutes each; xylene twice, 10 minutes each. Dry and seal with neutral gum. Observe and photograph the sections under a 20x field of view using an optical microscope. Count the number of positive cells in each photo and take the average value for evaluation.

[0081] 2.7 Statistical analysis

[0082] All experimental results that required statistics were processed using Prism 8. One-way ANOVA was used for comparisons among multiple groups, and independent sample t-test was used for two samples. P<0.05 indicated statistical significance, otherwise it was not statistically significant. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0083] 3. Experimental results

[0084] 3.1 Therapeutic effect of the novel fusion protein Fab-scFv on psoriasis model mice

[0085] During the establishment of the psoriasis model and the administration period, the backs of the mice were photographed and recorded every day. Compared with the normal group, the back skin of the mice in the model group had significant psoriasis-like lesions, such as white scales, erythema and raised inflammatory infiltration. Fig.10As shown, compared with the model group, the control group and the two drug groups were relieved to varying degrees on the sixth day of the experiment, with less scaling on the back skin of the mice, less erythema, and a smoother epidermis. The results showed that both the IFX group and the Fab-scFv group had a certain effect on alleviating psoriasis-like skin symptoms induced by imiquimod.

[0086] Starting from the first day after modeling, the weight of normal mice increased steadily, while that of mice in all groups decreased. However, on the fourth day after modeling, the weight loss of the drug group was alleviated compared with the model group. The weight changes of each group were as follows: Figure 5 As shown. PASI scores were performed on the skin area and severity of mice to analyze the treatment effects of each group. The back skin of mice in the normal group did not change during the experiment. In the first 6 days, the back skin lesions of mice in the model group gradually worsened as the days went by. From the analysis of erythema scores, there was no significant change between the IFX group and the two drug groups and the model group, as shown in Figure 2. Figure 6 As shown in the figure, from the analysis of scale score, Fab-scFv (5 mg / kg) showed a downward trend from the 5th day of modeling, and there was a statistical difference between the IFX group and the model group on the 5th and 7th days (P = 0.0446, P = 0.0199), while there was no statistical difference between the IFX group and the model group. Figure 7 As shown in the figure, from the analysis of hypertrophy scores, Fab-scFv (5 mg / kg) showed a downward trend from the 5th day of modeling, and on the 5th day, there was a statistical difference between the model group and the IFX group (P = 0.0078, P = 0.0017), while there was no statistical difference between the IFX group and the model group. Figure 8 As shown in the analysis of PASI total score, Fab-scFv (5 mg / kg) began to decrease from the 5th day of modeling, and on the 5th day, there was a statistical difference between the model group and the IFX group (P = 0.0072, P = 0.0162), while there was no statistical difference between the IFX group and the model group. Fig. 9 The above results indicate that the novel Fab-scFv fusion protein can improve the macroscopic manifestations of the IMQ-induced Pso mouse model more significantly than IFX.

[0087] 3.2 Improvement of the back lesions of psoriasis model mice by the novel fusion protein Fab-scFv

[0088] The skin lesions on the back of the mice were taken for H&E staining. Fig.11The results showed that under a 20x field of view, the epidermis of mice in the normal group was thinner, the skin tissue structure was intact, there was no subcutaneous hemorrhage, and there was no obvious inflammatory cell infiltration in the epidermis. Compared with the normal group, the model group showed poor keratinization, subcutaneous hemorrhage, increased epidermal thickness, inflammatory cell infiltration, etc., indicating that the psoriasis model was successfully established. Compared with the normal group, the skin tissue of the model group was severely infiltrated by neutrophils, and the neutrophil infiltration of the skin tissue of Fab-scFv (5 mg / kg) and Fab-scFv (2.5 mg / kg) was significantly lower than that of the model group (P=0.0005, P=0.0003) and IFX group (P=0.041, P=0.0256), while there was no statistical difference between the IFX and model groups. The results are as follows Fig.19 As shown. Compared with the normal group, the thickness of the skin lesions in the model group was significantly thickened (P<0.0001). From the maximum value of the mouse epidermal thickness, there was a statistical difference between the Fab-scFv (5 mg / kg) and IFX groups (P=0.0139), and there were statistical differences between the Fab-scFv (2.5 mg / kg) and the model group and IFX group (P=0.0168, P=0.003). The thickness of the mouse skin lesions was significantly thinner, while there was no statistical difference between the IFX and model groups; from the minimum value of the mouse epidermal thickness, Fab-scFv (5 mg / kg) and Fab-scFv (2.5 mg / kg) were significantly lower than the model group (P=0.0325, P=0.0034), while there was no statistical difference between the IFX and model groups. The results are shown in Fig.12 The experimental results show that the novel fusion protein Fab-scFv has a better therapeutic effect than IFX.

[0089] Ki67 is a key indicator for evaluating psoriasis, reflecting the degree of proliferation of keratinocytes in the epidermis. Immunohistochemical staining methods, such as Fig.13 As shown, the results were observed under a microscope with a 20x field of view. There were a small number of Ki67-positive cells (brown or yellow) in the epidermis of the skin of mice in the normal group. In the thickened epidermis of the model group, a large number of brown cells aggregated and proliferated, indicating that a large number of keratinocytes proliferated in the epidermis of psoriasis-like lesions. Compared with the model group, the number of brown cells in the IFX group was relatively reduced (P=0.0232), indicating that IFX had a certain degree of relief, but the keratinocytes in most lesions still had abnormal proliferation, while the brown cells of Fab-scFv (5 mg / kg) and Fab-scFv (2.5 mg / kg) were significantly lower than those in the model group (P<0.0001, P<0.0001), which significantly inhibited the expression of Ki67 and reduced the abnormal proliferation of a large number of keratinocytes. The results are shown in Fig.14 The experimental results show that the new fusion protein Fab-scFv has a better inhibitory effect on keratinocyte proliferation than IFX.

[0090] In summary, the new fusion protein Fab-scFv can improve the progression of skin lesions and inhibit further development of inflammation better than IFX, such as reducing skin thickness, alleviating inflammatory cell infiltration, and better inhibiting keratinocyte proliferation, indicating that it has significantly better anti-psoriasis effect than IFX.

[0091] 3.3 Novel fusion protein Fab-scFv regulates immune cell populations in psoriasis model mice

[0092] Flow cytometry analysis of mouse spleen tissue showed that compared with the normal group, the Th1 of the model group increased significantly on the 10th day (P = 0.0085); compared with the model group, Fab-scFv (5mg / kg) and Fab-scFv (2.5mg / kg) significantly reduced Th1 (P = 0.0366, P = 0.0234), while there was no significant difference in the IFX group. Compared with the normal group, the Th2 of the model group increased significantly on the 10th day (P = 0.0002); compared with the model group, the IFX group, Fab-scFv (5mg / kg) and Fab-scFv (2.5mg / kg) significantly reduced Th2 (P = 0.0002, P < 0.0001, P < 0.0001). Fig.16 As shown. Compared with the normal group, the model group Th22 was significantly increased (P = 0.0053); compared with the model group, the IFX group, Fab-scFv (5mg / kg) and Fab-scFv (2.5mg / kg) all significantly reduced Th22 (P = 0.0489, P = 0.0224, P = 0.0495). Compared with the normal group, the model group Treg was significantly reduced (P = 0.0093); and compared with the IFX group, Fab-scFv (5mg / kg) significantly increased Treg (P = 0.0478). The results are shown as follows Fig.17 The above flow cytometry results showed that at the end of the experiment on day 10, Fab-scFv (5 mg / kg) increased the proportion of Treg and reduced the proportion of Th1, Th2, and Th22, achieving a better effect in treating Pso than IFX.

[0093] In summary, the novel fusion protein Fab-scFv significantly reduced the scales, hypertrophy, PASI score, and epidermal thickness of mice from the 5th day of modeling, inhibited the proliferation of keratinocytes, and continuously alleviated the degree of psoriasis inflammation and the progression of the disease. IFX only had a certain inhibitory effect on the proliferation of keratinocytes. At the end of the experiment on the 10th day, although there was no significant change in the weight, scales, hypertrophy, and PASI score of the psoriasis model mice, in vivo, Fab-scFv (5 mg / kg) significantly increased the proportion of mouse Treg and reduced the proportion of pathogenic Th1, Th2, and Th22, while the same molar concentration of IFX only reduced Th2 and Th22, without significantly changing Th1 and Treg cells. In summary, the novel fusion protein Fab-scFv, on the basis of retaining the pharmacological activity of infliximab Fab, further inhibited the proliferation of keratinocytes, reduced the infiltration of neutrophils in skin tissue, regulated pathogenic Th1, Th2, and Th22, and increased regulatory Treg cells, achieving a better effect than the same molar concentration of IFX in the treatment of psoriasis.

[0094] Although only specific implementation examples of the present invention are described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these implementations without departing from the principles and essence of the present invention, but these changes or modifications all fall within the protection scope of the present invention.

Claims

1. A novel Fab-scFv fusion protein, characterized in that: The Fab-scFv fusion protein is formed by fusing the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

2. The novel Fab-scFv fusion protein according to claim 1, characterized in that: The amino acid sequence shown in SEQ ID NO:1 is encoded by the nucleotide sequence shown in SEQ ID NO:4; the amino acid sequence shown in SEQ ID NO:2 is encoded by the nucleotide sequence shown in SEQ ID NO:5; and the amino acid sequence shown in SEQ ID NO:3 is encoded by the nucleotide sequence shown in SEQ ID NO:

6.

3. Use of the novel Fab-scFv fusion protein according to claim 1 in the preparation of a drug for treating psoriasis.

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