UMOD antibody, sialidase NEU3 fusion protein and application

Abstract

The invention discloses a UMOD antibody, a sialidase NEU3 fusion protein and application thereof.The UMOD antibody is invented firstly, VL-CL and VH-CH1 nucleotide sequences of a Fab fragment of the UMOD antibody and a nucleotide sequence of a sialidase NEU3 functional area peptide fragment are used for constructing a recombinant vector, then the recombinant vector is expressed and purified to obtain the fusion protein, and the fusion protein is directly administrated through a bladder to treat bladder cancer. The sialidase NEU3 can identify the UMOD antigen, can play a role in degrading sialic acid of the sialidase NEU3, can significantly inhibit the growth of bladder cancer, and has the advantages of simple operation and good treatment effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical drug synthesis, and particularly relates to a UMOD antibody, a sialidase NEU3 fusion protein and application. BACKGROUND

[0002] Antibody fusion protein drugs are prepared by fusing antibodies with other active proteins, have multiple biological functions, and have been widely used in immune diagnosis, immunotherapy, antibody purification and quantitative analysis of antibodies and antigens, and can be particularly used for the preparation of immune-guided drugs. Most antibody fusion proteins are constructed by chemical cross-linking or genetic engineering methods. The proteins constructed by the chemical cross-linking method are not uniform in composition, unstable in performance, large in molecule, low in penetration ability and high in immunogenicity. The antibody fragments constructed by the genetic engineering method are small in molecule, strong in function, high in stability and easy to be genetically fused.

[0003] Abnormal sialylation modification is a common feature of bladder cancer, plays an important role in bladder cancer cell proliferation, apoptosis, metastasis and EMT, and the change in sialylation modification level is a key driving factor of these processes. UMOD (uromodulin) protein is a glycoprotein mainly synthesized by renal tubular epithelial cells of the kidney and secreted into urine, and is one of the most abundant proteins in urine. The invention patent with the Chinese patent number 201310501366.2 discloses a "kit for detecting uroepithelial cancer based on LIP identification of modified UMOD Neu5Gc in urine", which is based on the discovery that the sialylation modified UMOD protein in the urine of bladder cancer patients is significantly increased, and then the sialic acid modified UMOD is used as an epithelial cancer marker. At present, the reported UMOD antibody is a single antigen specific antibody specifically targeting UMOD, which is mainly used for the detection of UMOD, and has no effect on the treatment of bladder cancer. Sialidase is a class of enzymes that can catalyze the removal of sialic acid residues on the surface of glycoproteins, glycolipids and other molecules. It has been reported in the literature that overexpression of sialidase NEU3 can effectively inhibit the occurrence of colon cancer, but sialidase NEU3 has no effect on the treatment of bladder cancer.

[0004] So far, there is no record of a drug for treating bladder cancer by targeting sialylated UMOD. SUMMARY

[0005] The present application is to solve the above technical problems existing in the prior art, and provides a UMOD antibody, a sialidase NEU3 fusion protein and application.

[0006] The technical solution of the present application is: a UMOD antibody, the amino acid sequence of the heavy chain variable region and constant region VH-CH1 of the Fab fragment of the UMOD antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region and constant region VL-CL of the Fab fragment of the UMOD antibody is shown in SEQ ID NO. 2.

[0007] A UMOD antibody and sialidase NEU3 fusion protein are constructed by constructing a recombinant vector from the VL-CL and VH-CH1 nucleotide sequences of the Fab fragment of the UMOD antibody and the nucleotide sequence of the functional region peptide segment of the sialidase NEU3, and then expressing and purifying the fusion protein, and the amino acid sequence of the functional region peptide segment of the sialidase NEU3 is shown in SEQ ID NO. 3.

[0008] The UMOD antibody and sialidase NEU3 fusion protein are preferably prepared according to the following steps: Step 1. Synthesize genes by using overlapping PCR technology according to the VL-CL and VH-CH1 nucleotide sequences of the Fab fragment of the UMOD antibody, the nucleotide sequence of the connecting peptide, and the nucleotide sequence of the sialidase NEU3, and then cut the synthesized genes and connect them to vectors, respectively. Step 2. Infect the constructed recombinant vector into E. coli, and purify to obtain the UMOD antibody and sialidase NEU3 fusion protein.

[0009] The UMOD antibody and sialidase NEU3 fusion protein are preferably prepared according to the following steps:

[0010] The present application first invents a UMOD antibody, constructs a recombinant vector from the VL-CL and VH-CH1 nucleotide sequences of the Fab fragment of the UMOD antibody and the nucleotide sequence of the functional region peptide segment of the sialidase NEU3, and then expresses and purifies the fusion protein, and the fusion protein is directly administered to the bladder to treat bladder cancer, which can recognize the UMOD antigen and also has the function of degrading sialic acid by sialidase NEU3, can significantly inhibit the growth of bladder cancer, and has the advantages of simple operation and good treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is the SDS-PAGE denaturing electrophoresis diagram of the UMOD antibody and sialidase NEU3 fusion protein of the embodiment of the present application.

[0012] Figure 2 is the SDS-PAGE non-denaturing electrophoresis diagram of the UMOD antibody and sialidase NEU3 fusion protein of the embodiment of the present application.

[0013] Figure 3Figure is a WB result diagram of UMOD antibody and sialidase NEU3 fusion protein of the embodiment of the present application recognizing UMOD antigen.

[0014] Figure 4 Figure is a body weight monitoring result diagram of UMOD antibody and sialidase NEU3 fusion protein of the embodiment of the present application treating tumor-bearing mice.

[0015] Figure 5 Figure is a tumor growth curve result diagram of UMOD antibody and sialidase NEU3 fusion protein of the embodiment of the present application treating bladder cancer. DETAILED DESCRIPTION Embodiment 1

[0016] Take 1 mL of UMOD protein separated from the urine of bladder cancer patients with a mass concentration of 0.5 mg / mL to immunize BALB / c mice for 5 times to obtain mouse-derived UMOD antibody. After amino acid sequence sequencing and optimization of humanized antibody sequence, the UMOD antibody is obtained. The amino acid sequence of the heavy chain variable region and constant region VH-CH1 of the UMOD antibody Fab fragment is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region and constant region VL-CL of the UMOD antibody Fab fragment is shown in SEQ ID NO. 2.

[0017] Embodiment 2: Preparation of UMOD antibody and sialidase NEU3 fusion protein Step 1. According to the VL-CL and VH-CH1 nucleotide sequences of the UMOD antibody Fab fragment, the connecting peptide nucleotide sequence, and the nucleotide sequence of sialidase NEU3, the gene is synthesized by using the overlapping PCR technology. After synthesis, the gene is cut and connected to the vector respectively; Step 2. The constructed recombinant vector is infected into E. coli, and the E. coli lysate is added to the nickel affinity column for purification. The low-concentration 5 mM imidazole is used for impurity removal, and the high-concentration 300 mM imidazole is used for elution of the fusion protein drug. The SDS-PAGE denaturing electrophoresis diagram of this process is shown in Figure 1 , Figure 1 wherein M lane is marker, 1 lane is E. coli lysate, 2 lane is 5 mM imidazole elution component, and 3 lane is 300 mM imidazole elution component.

[0018] The eluate is placed in a dialysis bag, and the PBS liquid is changed at 4℃ overnight. The concentrated tube is used to concentrate the dialyzed liquid to obtain the UMOD antibody and sialidase NEU3 fusion protein. The SDS-PAGE non-denaturing electrophoresis diagram is shown in Figure 2 , Figure 2 wherein M lane is marker, 1 lane is pre-concentration component, and 2 lane is post-concentration component.

[0019] Experiment: Determination of antigen recognition of UMOD antibody and sialidase NEU3 fusion protein Purify urine protein of normal volunteers and bladder cancer patients respectively, 3 cases for each, and detect the recognition of UMOD antibody and sialidase NEU3 fusion protein to UMOD antigen by WB, and the results are shown in Figure 3 As shown, the UMOD antibody and sialidase NEU3 fusion protein prepared in the application has good UMOD antigen recognition activity.

[0020] 2. In vivo experiment of UMOD antibody and sialidase NEU3 fusion protein on treatment of mouse bladder cancer Grouping is as follows: 20 normal control mice; 20 bladder cancer modeling mice, bladder cancer modeling + drug treatment low concentration (5 mg / kg) 20 mice; bladder cancer modeling + drug treatment high concentration (10 mg / kg) 20 mice; bladder cancer modeling + UMOD antibody alone treatment high concentration (10 mg / kg) 10 mice, the drug is UMOD antibody and sialidase NEU3 fusion protein. The mouse weight monitoring and bladder cancer growth curve are shown in Figure 4 , Figure 5 respectively. Figure 4 It is shown that the body weight of mice in each group has no obvious difference; Figure 5 It is shown that the low concentration and high concentration groups of the drug of the application can significantly inhibit the growth of bladder cancer, and the effect is significantly better than that of the UMOD protein alone group. The results show that the UMOD antibody and sialidase NEU3 fusion protein of the application has good bladder cancer treatment effect.

Claims

1. A UMOD antibody, characterized in that: The heavy chain of the UMOD antibody Fab fragment can The amino acid sequences of the variable and constant regions VH-CH1 are shown in SEQ ID NO. 1, and the amino acid sequences of the light chain variable and constant regions VL-CL of the UMOD antibody Fab fragment are shown in SEQ ID NO.

2.

2. A fusion protein of UMOD antibody and sialidase NEU3 as described in claim 1, characterized in that: A recombinant vector was constructed by combining the VL-CL and VH-CH1 nucleotide sequences of the UMOD antibody Fab fragment with the nucleotide sequence of the NEU3 functional region peptide of sialidase, and then the purified fusion protein was expressed. The amino acid sequence of the NEU3 functional region peptide of sialidase is shown in SEQ ID NO.

3.

3. The UMOD antibody and sialidase NEU3 fusion protein according to claim 2, characterized in that... It is prepared according to the following steps: Step 1. Based on the VL-CL and VH-CH1 nucleotide sequences, linker peptide nucleotide sequence, and sialidase NEU3 nucleotide sequence of the UMOD antibody Fab fragment, the gene was synthesized using overlap PCR technology. After synthesis, the gene was digested with enzymes and ligated into vectors respectively. Step 2. Infect E. coli with the constructed recombinant vector and purify to obtain the UMOD antibody and sialidase NEU3 fusion protein.

4. The use of the UMOD antibody and sialidase NEU3 fusion protein as described in claim 2 or 3 in the preparation of a drug for treating bladder cancer.

Images