A recombinant fusion protease capable of clearing immunoglobulin A in vivo, a preparation method thereof, and application thereof in treating IgA nephropathy

Abstract

The present invention provides a recombinant fusion protein based on AK183 protease and its application in treating IgA nephropathy and other related diseases mediated by IgA complex deposition, wherein the recombinant fusion protein structure comprises a human commensal bacteria protease AK183 active region and a human immunoglobulin fragment; the human immunoglobulin fragment is located at the N-terminus of the protease AK183 active region. The present invention also provides a method for preparing the recombinant fusion protein. The recombinant fusion protein of the present invention has the comprehensive advantages of prolonged plasma half-life, effective clearance of circulating and renal IgA complexes, and greatly reducing the immunogenic side effects of the protease on the human body, by recombinantly fusing the natural AK183 protease with the human immunoglobulin fragment, and is suitable for specific treatment of IgA nephropathy and other related diseases mediated by IgA complex deposition.

Description

Technical Field

[0001] The present invention relates to the field of fusion protein recombinant synthesis, and in particular to an artificially synthesized polypeptide with protease activity, a synthesis method thereof, and a method for treating IgA nephropathy and other diseases caused by polymerized IgA complexes by utilizing the pharmaceutical enzymatic activity of the polypeptide. Background Art

[0002] IgA nephropathy is one of the most common primary glomerular diseases in the world. Especially in East Asia, it accounts for 40%-50% of primary glomerulonephritis diagnosed by renal biopsy. More than 30%-40% of patients progress to end-stage renal disease (uremia) 20 to 30 years after onset. 1,2 , which brings a heavy burden to patients and society. There is currently a lack of specific treatment for IgA nephropathy. In clinical practice, supportive treatment based on RAS blockers is often used to slow down the deterioration of renal function. For patients who are ineffective with supportive treatment, combined hormone immunosuppressant treatment is given. However, the long-term efficacy of hormone immunosuppressants is poor and brings serious side effects to patients. 3,4 .

[0003] In recent years, the focus of drug research has been on developing effective therapeutic drugs with low side effects. The main directions include the development of drugs that reduce and specifically clear renal IgA deposition. These include treatments that systemically reduce immune responses and IgA production, as well as reduce IgA-induced complement activation. IgA nephropathy is caused by the deposition of circulating IgA1 complexes in the glomeruli. The human body and its pathogens or symbiotic microorganisms have biological mechanisms to clear IgA. IgA1 consists of two heavy chains and two light chains. There is a hinge region (HR) between the Fab and Fc segments. 6 , which is the site of action for microorganisms to cut IgA1. Many pathogens and symbiotic microorganisms have this type of protease to fight human immunity. Among them, AK183 is an IgA protease derived from Clostridium, a symbiotic bacterium in the human intestine. This invention aims to use the human body's excellent tolerance to this symbiotic bacterium to develop a drug based on its AK183 protease, and to further optimize the drug half-life through recombinant fusion protein technology to effectively clear circulating IgA and its renal deposits. This fusion protein drug is expected to have the characteristics of low immunogenicity, high specificity and long-term effectiveness, and is used to quickly control progressive IgA nephropathy to stabilize renal function. Summary of the invention

[0004] In view of the above background, the present invention intends to utilize biorecombinant technology to modify IgA enzymes derived from commensal bacteria to achieve excellent pharmacological activity and pharmacokinetic properties, as well as methods for the specific treatment of IgA nephropathy (including primary and secondary IgA nephropathy) and a wide range of diseases caused by the deposition of pathogenic polymeric IgA complexes.

[0005] One aspect of the present invention is to provide a fusion protease that can clear pathogenic IgA1 complexes, so as to achieve an ultra-long blood drug half-life of the protease, thereby prolonging the efficacy of clearing pathogenic IgA1 complexes in the body.

[0006] Another object of the present invention is to provide a method for treating IgA nephropathy (including primary and secondary IgA nephropathy) and a wide range of diseases caused by pathogenic polymeric IgA complexes using recombinant fusion proteins.

[0007] Another object of the present invention is to provide a method for preparing the fusion protease capable of clearing pathogenic IgA1 complexes.

[0008] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0009] First, the first aspect of the present invention provides a recombinant fusion protein, the structure of which includes the active region of human symbiotic bacteria protease AK183 and a human immunoglobulin fragment; the human immunoglobulin fragment is located at the N-terminus of the active region of the protease AK183, and the amino acid sequence of the human immunoglobulin fragment is at least 90% identical to the sequence shown in SEQ ID No.1, preferably at least 95% identical, and most preferably 100% identical; the active region of the protease AK183 is from an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID No.2, preferably at least 95% identical, and most preferably 100% identical. The recombinant fusion protein of the present invention is named "Fc-AK183". In the recombinant fusion protein of the present invention, the active region of AK183 can provide a drug active center to degrade IgA, and the human immunoglobulin fragment can enhance the compatibility of the drug with the human body and prolong the half-life of the drug.

[0010] In the process of proposing the present invention, in order to determine the region of the wild AK183 enzyme active fragment (protease AK183 active region), we first constructed a series of truncated AK183 fragments. By verifying the in vitro enzyme activity of the series of truncated AK183 fragments, we found that the inactive regulatory region of the C segment inhibited the enzyme activity, and that the wild-type AK183 precursor protease needed to complete the C-terminal self-cutting and then mature. Thus, we carried the human immunoglobulin fragment to the amino terminus of the protease AK183 active region, so that the target recombinant fusion protein Fc-AK83 can always retain the human immunoglobulin fusion fragment during its process from precursor to maturity.

[0011] The most preferred recombinant fusion protein of the present invention has an amino acid sequence as shown in SEQ ID No.5.

[0012] Given that the etiology of IgA nephropathy is caused by chronic deposition of circulating polymeric IgA complexes in the glomerular mesangial region, polymeric IgA complexes can also cause a variety of diseases with different clinical manifestations, resulting in involvement of different organs. Broadly speaking, all such IgA complex deposition-mediated related diseases, whether or not involving the kidneys, can be used as indications for the drug of the present invention. Therefore, the second aspect of the present invention provides a method for treating related diseases mediated by IgA complex deposition using a recombinant fusion protein based on AK183 protease; that is, the recombinant fusion protein based on AK183 protease is used to prepare a drug for treating related diseases mediated by IgA complex deposition; the recombinant fusion protein structure based on AK183 protease comprises a human commensal bacteria protease AK183 active region and a human immunoglobulin fragment; the human immunoglobulin fragment is located at the N-terminus of the protease AK183 active region, and the amino acid sequence of the human immunoglobulin fragment is at least 90% identical to the sequence shown in SEQ ID No.1, preferably at least 95% identical, and most preferably 100% identical; the protease AK183 active region is derived from an amino acid sequence that is at least 90% identical to the sequence shown in SEQ ID No.2, preferably at least 95% identical, and most preferably 100% identical; the most preferred amino acid sequence of the recombinant fusion protein is as shown in SEQ ID No.5.

[0013] Furthermore, our research and development team used in vitro experiments and in vivo animal models to verify the efficacy of the recombinant fusion protein of the present invention as a new experimental drug in disease models. In our in vitro drug validation, Fc-AK183 can cut IgA from patients in a variety of diseases, such as samples of patients with IgA nephropathy, purpuric nephritis or Kawasaki disease with different clinical pathological manifestations. Therefore, in the preferred application of the present invention, the related diseases mediated by the deposition of IgA complexes may include: IgA nephropathy, IgA vasculitis renal damage (or Henoch-Schönlein purpuric nephritis) or other secondary IgA nephropathy, or other diseases caused by IgA complexes such as Kawasaki disease, IgA rheumatoid factor-positive rheumatoid arthritis, IgA anti-GBM disease or IgA ANCA-associated vasculitis.

[0014] In the application described in the present invention, based on the fact that the key to treatment is to clear the polymeric IgA complexes in tissues (kidneys) or circulation, we use immunoglobulin-derived fusion fragments to achieve a long-acting blood circulation half-life to achieve the effect of systemic clearance of pathological deposits. Therefore, in a preferred embodiment, the recombinant fusion protein based on AK183 protease is prepared into a liquid preparation for administration to mammals suffering from related diseases mediated by the deposition of the IgA complex; it is further preferably prepared into an injection and administered by intravenous injection or infusion.

[0015] In the application of the present invention, the mammal can be a human or other mammals (eg, a transgenic mammal model that can be used for drug testing).

[0016] Our research and development team has used a variety of humanized mouse models to verify the efficacy of Fc-AK183 and obtain pharmacokinetic parameters. Our experimental results suggest that when the mammal is an animal model for drug testing, the animal model can be a passive human IgA injection mouse model, a human IgA1 transgenic mouse model or a primate model, and the intravenous administration regimen includes: injection once every 5-10 days at a dose of 5-10 mg / kg; preferably injection once every 5 days at a dose of 5 mg / kg.

[0017] Since the immunoglobulin fragment we use is derived from a human sequence, it has the best match with the human-related receptor (FcRn), and previous data suggest that the half-life of this type of fusion drug in humans is significantly longer than that in mice. Therefore, we speculate that when the mammal is a human, the dosing interval can be longer than that in mice. The estimated intravenous dosing regimen for humans is approximately: 5-10 mg / kg once every 10-15 days; preferably, a dose of 10-15 mg / kg is used as the initial injection dose, and then changed to a maintenance dose of 5-10 mg / kg once every 10 days. According to our experimental pharmacokinetic data, this dosing regimen can achieve complete clearance of circulating IgA in the blood within a few weeks; during this period, the deposits of existing IgA in the kidneys can be fully cleared, allowing partial repair of glomerular damage, thereby achieving the transition of the kidney from a progressive injury period to a stable repair period.

[0018] In a third aspect, the present invention provides a recombinant fusion protein gene expression vector, which is a plasmid composed of a recombinant fusion of a carrier and a target gene, wherein the target gene is a recombinant fusion gene, including a gene sequence encoding a human immunoglobulin fragment (human IgG hinge region CH2 and CH3) and a gene sequence encoding the active region of protease AK183; the gene sequence encoding the human immunoglobulin fragment has a nucleotide sequence that is at least 90% identical to the sequence shown in SEQ ID No.3, preferably at least 95% identical, and most preferably 100% identical; the gene sequence encoding the active region of protease AK183 is at least 90% identical to the sequence shown in SEQ ID No.4, more preferably at least 95% identical, and most preferably 100% identical.

[0019] In the preferred recombinant fusion protein gene expression vector of the present invention, the vector is a vector suitable for a prokaryotic expression system, further preferably a vector matching an Escherichia coli expression system, more preferably a vector with a purification tag, and most preferably a pET30a plasmid.

[0020] In a most preferred embodiment of the present invention, the recombinant fusion protein gene expression vector is a plasmid composed of a carrier and a target gene recombined and fused, wherein the carrier is an Escherichia coli expression vector pET30a plasmid; the target gene is a recombinant fusion gene, which sequentially includes, from its 5' end, a gene sequence encoding a human immunoglobulin fragment, whose nucleotide sequence is shown in SEQ ID No.3, and a gene sequence encoding a protease AK183 active region, whose nucleotide sequence is shown in SEQ ID No.4.

[0021] In a fourth aspect, the present invention provides a method for constructing a recombinant fusion protein gene expression vector, comprising: selecting an expression system, and synthesizing two cDNA sequences suitable for expression by the expression system according to the amino acid sequence containing the protease AK183 active region and the amino acid sequence of a human immunoglobulin fragment, respectively, taking the two cDNA sequences as target genes, respectively performing DNA recombination with the expression system to obtain two recombinant plasmids, and finally recombining and fusing the target genes in the two recombinant plasmids in the same recombinant plasmid, in the recombinant fusion, the cDNA sequence of the human immunoglobulin fragment is inserted into the 5' end of the cDNA sequence of the protease AK183 active region to obtain the recombinant fusion protein gene expression vector; the amino acid sequence of the human immunoglobulin fragment is at least 90% identical to the sequence shown in SEQ ID No.1, preferably at least 95% identical, and most preferably 100% identical.

[0022] In a preferred embodiment of the present invention, the method for constructing a recombinant fusion protein gene expression vector comprises the following steps:

[0023] (I) based on the amino acid sequence of the active region of protease AK183 from Clostridium difficile naturally commensal in the human intestine, after codon optimization, a cDNA sequence I' suitable for expression in E. coli was synthesized; based on the amino acid sequence of a human immunoglobulin fragment that is at least 90% identical to the sequence shown in SEQ ID No. 1, after codon optimization, a cDNA sequence II' suitable for expression in E. coli was synthesized;

[0024] (II) using the cDNA sequence I' obtained in (I) as the target gene, and carrying out DNA recombination with Escherichia coli to obtain a subcloning plasmid I'; using the cDNA sequence II' obtained in (I) as the target gene, and carrying out DNA recombination with Escherichia coli to obtain a subcloning plasmid II';

[0025] (III) cutting the subcloning plasmid II' obtained in (II) to obtain a cDNA sequence II' fragment with a sticky end; cutting the subcloning plasmid I' obtained in (II) to form a gap with a sticky end upstream of the cDNA sequence I';

[0026] (IV) performing DNA recombination on the cDNA sequence II' fragment obtained in (III) and the cut subclone plasmid I', wherein the cDNA sequence II' is inserted into the 5' end of the cDNA sequence I' to obtain the recombinant fusion protein gene expression vector.

[0027] In a further preferred embodiment of the present invention, the cDNA sequence II' in step (I) is a fragment with a base added before the codon of the sequence shown in SEQ ID No. 1 to prevent frameshift and a GGGGGGGS connecting peptide added after the fragment to protect the activity of the protease from being affected by the fusion IgG Fc fragment.

[0028] In a further preferred embodiment of the present invention, in the step (IV), during the DNA recombination of the cDNA sequence II' fragment obtained in (III) and the subclone plasmid I' after cutting, the N-terminus of the cDNA sequence II' fragment is fused with the His tag attached to the vector of the subclone plasmid I'.

[0029] In a fifth aspect, the present invention also provides a method for expressing and purifying the recombinant fusion protein, comprising: transfecting the recombinant fusion protein gene expression vector described in the third aspect of the present invention into Escherichia coli (BL21-DE3) competent cells, expressing the protein under the induction of an inducer to obtain the recombinant fusion protein; the inducer is preferably isopropyl-β-D-thiogalactoside (IPGT) at a concentration of 0.1-0.5 mM; the protein expression temperature is preferably 15-18°C; the protein expression time is preferably 20-30 hours; after the expression is completed, the Escherichia coli cell bodies are treated by ultrasonic fragmentation according to a conventional method, and then the supernatant is retained by high-speed centrifugation, and then affinity chromatography and molecular sieve purification are used to obtain the recombinant fusion protein.

[0030] In a further preferred expression and purification method of the present invention, the inducing agent is IPGT with a concentration of 0.3 mM; the protein expression temperature is 16-18° C.; and the protein expression time is 24 hours.

[0031] In a further preferred expression purification method of the present invention, the affinity chromatography and molecular sieve purification are performed at 4 degrees Celsius, and a buffer containing 0.8 mM EDTA is used to protect the protease activity to the maximum extent. Finally, the purified protein obtained is frozen in a neutral phosphate buffer.

[0032] In a sixth aspect, the present invention also provides a pharmaceutical composition comprising the recombinant fusion protein described in the first aspect of the present invention.

[0033] In summary, the construction strategy of the recombinant fusion protein described in the present invention aims to maximize the pharmacological advantages: the potent IgA hydrolase (AK183) from bacteria is used as the core part of the pharmacological effect, and combined with the long-acting biopharmacological advantages of human immunoglobulin fragments to achieve the best therapeutic effect. Compared with the natural AK183 protease in the prior art, the recombinant fusion protein of the present invention has improved pharmacological effects in the following aspects:

[0034] 1. Prolonged plasma half-life

[0035] The recombinant fusion protein of the present invention, as a protease based on AK183 after being fused with the human immunoglobulin fragment, has a sufficiently long plasma half-life in mice compared with the previous natural protease AK183 (see Experimental Example 1 for details).

[0036] 2. Effective removal of circulating and renal IgA complexes

[0037] Since the drug's half-life in vivo is significantly prolonged, the efficacy of clearing circulating IgA complexes and renal IgA deposits is significantly enhanced. In vitro experiments have shown that the fusion protease of the present invention can effectively cleave the blood-derived poly-IgA1 complexes of patients with IgA nephropathy, purpura nephritis and Kawasaki disease (effect reference Fig.15 , 16 and 17 are described in detail in the Examples section). Through experimental treatments on two different humanized IgA mouse models, the recombinant fusion protease of the present invention can not only clear human IgA in the mouse circulation, but also clear the IgA1 complex that has been deposited in the glomerulus (Experimental Example 2).

[0038] 3. No neutralizing antibodies against AK183 protease are produced

[0039] In order to greatly reduce the human body's immune resistance to heterologous protein sequences after multiple administrations, we specifically selected IgA proteases from human intestinal commensal bacteria. Most of the bacteria with IgA protease activity reported in the prior art are pathogenic bacteria, including meningococci, Haemophilus influenzae, Streptococcus pneumoniae, Neisseria gonorrhoeae, etc. Such pathogenic microorganisms use the function of cutting the hinge region of human IgA to resist the host's immune protection. We noticed that a few human intestinal commensal bacteria have also acquired the ability to resist the host's IgA-mediated immune response in their evolution, thereby acquiring the ability to coexist. At the same time, humans are also highly tolerant to their commensal bacteria and their secretory proteins. Therefore, we specifically selected IgA protease AK183 from intestinal Clostridium as a drug template.

[0040] Preliminary animal experiments on mice have shown that after repeated injections of the recombinant fusion protease described in the present invention, no neutralizing antibodies that significantly affect the activity of AK183 protease were produced in the mice (Experimental Example 3). Therefore, the recombinant fusion protease described in the present invention still has AK183 protease activity after repeated injections into the body, and still has the effect of quickly clearing the IgA1 complex.

[0041] In summary, the recombinant fusion protein of the present invention is suitable for preparing a drug for the specific treatment of IgA nephropathy, and its indications may include induction remission treatment of chronic progressive IgA nephropathy, and rapid control treatment of patients with rapid clinical progression such as crescentic IgA nephropathy. By using the drug prepared by the recombinant fusion protein of the present invention, the circulating IgA complex can be reduced in a timely and effective manner, thereby improving the prognosis of the disease. In addition, the recombinant fusion protein of the present invention can also be used to treat Henoch-Schönlein purpura, Kawasaki disease, ANCA-associated vasculitis with IgA as the main pathogenic antibody, anti-glomerular basement membrane disease and IgA1 heavy chain myeloma, and is expected to replace plasma exchange to quickly and efficiently remove pathogenic IgA antibodies, thereby improving patient survival and disease prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A The structure embodies Example 1 in which a base is added before the human immunoglobulin fragment to prevent frameshift, and GGGGGGS is added as a linker.

[0043] Figure 1B and Figure 1C The position of restriction endonuclease cutting in Example 1 is reflected.

[0044] Figure 2 It is a schematic diagram of the construction process of the recombinant fusion protein gene expression vector described in Example 1.

[0045] Figure 3 This is a schematic diagram of the functional domain structure of the natural protease AK183.

[0046] Figure 4 It is a schematic diagram of the functional region structure of the recombinant fusion protein expressed by the Escherichia coli cells described in Example 2.

[0047] Figure 5 . is the elution curve after purification by Superdex S200 molecular sieve described in Example 2.

[0048] Figure 6 . is an immunoblot image of protein peaks F1, F2 and F3 after purification by Superdex S200 molecular sieve described in Example 2.

[0049] Figure 7It shows the protein peaks F1, F2 and F3 obtained by purification as described in Example 2, and their in vitro activity verification.

[0050] Figure 8 It reflects the enzymatic activity of the protein peak F1 purified in Example 2 after being added to purified poly-IgA1 samples from the circulation of patients with IgA nephropathy in different proportions in vitro.

[0051] Fig. 9 This shows the activity of the purified protein peak F1 described in Example 2 in cleaving IgA1 in a serum environment.

[0052] Fig.10 This is a schematic diagram of the functional region structure of the fusion protein constructed by the method described in Example 3.

[0053] Fig.11 This is a schematic diagram of the functional region structure of the fusion protein constructed by the method described in Example 1.

[0054] Fig.12 This shows that the human immunoglobulin fragment of the fusion protein constructed by the method described in Comparative Example 1 is self-cleaved after expression.

[0055] Fig.13 This is a curve chart showing the change in the concentration of fusion protease in mouse serum over time after the fusion protease was injected into mice at a dose of 5 mg / kg in Experimental Example 1.

[0056] Fig.14 This is a graph showing the concentration of native IgA protease in mouse plasma over time from a study by Lechner et al. (J Am Soc Nephrol. 2016 Sep; 27(9): 2622-9).

[0057] Fig.15 The results show that the fusion protease prepared in Example 2 has enzymatic activity in vitro on polymeric IgA1 purified from plasma of 9 patients with IgA nephropathy.

[0058] Fig.16 The results show that the fusion protease prepared in Example 2 has enzymatic activity in vitro on polymeric IgA1 purified from plasma of 4 patients with purpura nephritis.

[0059] Fig.17 The results show that the fusion protease prepared in Example 2 has enzymatic activity against IgA1 purified from the plasma of a child with Kawasaki disease in vitro.

[0060] Fig.18 The fusion protease described in Example 3 has enzymatic activity against IgA1 of IgA nephropathy patients and healthy controls in vitro in a serum environment.

[0061] Fig.19This demonstrates the passive human IgA1 mouse model described in Experimental Example 1, as well as the efficacy of rapidly and effectively clearing IgA1 after administration through different routes.

[0062] Fig. 20 The immunoblotting method described in Experimental Example 1 was used to detect the efficacy of the fusion protease in the passive IgA1 mouse model.

[0063] Fig.21 As described in Experimental Example 2, after the fusion protease was intravenously injected into wild BALB / C mice, the fusion protease was metabolized in vivo and the concentration decreased. The enzymatic cleavage ability of passively injected human IgA1 at different time points indicated that the protease has the ability to remove IgA1 in the body for a long time.

[0064] Fig. 22 The AK183 protease without Fc tag described in Experimental Example 2 had no enzymatic activity in mice after 24 hours.

[0065] Fig.23 This is a curve chart of body weight change in the passive IgA1 mouse model described in Experimental Example 1 after injection of fusion protease.

[0066] Fig.24 It is the humanized IgA1-α1KI described in Experimental Example 1 + / WT In the mouse model, after injection of the fusion protease, circulating IgA1 levels rapidly decreased to undetectable levels within 1 hour and persisted for 5-7 days.

[0067] Fig.25 It is the humanized IgA1-α1KI described in Experimental Example 1 + / WT Body weight change curve of mouse model after intravenous injection of fusion protease.

[0068] Fig.26 The passive human IgA deposition model in mouse kidney was established.

[0069] Fig. 27 It is shown in Experimental Example 3 that compared with mice not injected with fusion protease, the fusion protease in the drug-treated group can clear the IgA1 deposited in the glomeruli in the passive human IgA1 renal deposition mouse model, and the Fc fragment of hIgA1 after enzymatic cleavage is reabsorbed by the renal tubules through glomerular filtration.

[0070] Fig.28 The humanized IgA1-α1KI in Experimental Example 3 was injected with the recombinant fusion protease and then raised in a normal environment. + / WT IgA1 that chronically accumulates and deposits in the mouse kidneys can be cleared by proteases.

[0071] Fig.29 This shows that the injection of recombinant fusion protease does not affect the humanized IgA1-α1KI + / WTImmune barrier function of secretory IgA in the intestinal mucosa of mice.

[0072] Fig.30 As described in Experimental Example 3, no obvious antibodies against AK183 were produced in the mice 30 days after the injection of the fusion protease.

[0073] Fig.31 There was no significant difference in the antibody titer recognizing AK183 in the serum of mice injected with the fusion protease described in Experimental Example 3 30 days after injection compared with the non-injected control mice.

[0074] Fig.32 The serum of mice injected with the fusion protease at 30 days in Experimental Example 3 had no significant effect on the enzymatic activity of the fusion protease after incubation with the fusion protease in vitro.

[0075] Fig.33 As described in Experimental Example 3, after the fusion protease was injected, the mice were intravenously administered the same dose of fusion protease again on the 23rd day and were still able to quickly clear the human IgA1 passively injected into the mice.

[0076] Fig.34 It is the humanized IgA1-α1KI described in Experimental Example 3 + / WT In the mouse model, 30 days after intravenous injection of fusion protease, the level of circulating IgA1 still decreased rapidly after another intravenous injection of the same dose of fusion protease and remained below the baseline level 7 days later. DETAILED DESCRIPTION

[0077] The technical solution of the present invention will be further described in detail below by enumerating embodiments, but the scope of the present invention is not limited to the enumerated embodiments.

[0078] Example 1. Construction of fusion protein expression plasmid

[0079] like Figure 2 As shown, the steps for constructing the fusion protein expression plasmid are as follows:

[0080] ① Subcloning plasmid construction

[0081] The AK183 protease from the natural symbiotic Clostridium in the intestine was selected as the natural enzyme to be fused, and its gene structure is as follows Figure 3 As shown, it contains a signal peptide protease active region and a transmembrane anchor region. According to previous studies ( Fig.11 , Fig.12 ), the self-cleavage site of AK183 is located downstream of the C-terminal transmembrane anchor region of its amino acid sequence.

[0082] The amino acid sequence of natural AK183 protease (as shown in SEQ ID No. 2) and the amino acid sequence of human immunoglobulin were obtained by searching the NCBI database. The corresponding cDNA sequence suitable for Escherichia coli expression was obtained by codon optimization. Different restriction endonucleases were added at both ends. Bases were added before the codon of the human immunoglobulin fragment to prevent frameshift, and GGGGGGS was added after it as a linker (as shown in Figure 1A The above cDNA sequences (shown in SEQ ID No.3 and SEQ ID No.4, where Figure 1B and Figure 1C The cDNA fragments were linked into the PET30a plasmid vector using DNA ligase to obtain the subclone PET30a-AK183 and the plasmid containing SEQ ID No.3.

[0083] ② Construction of fusion protein plasmid

[0084] The plasmid containing SEQ ID No.3 was cut with restriction endonuclease, and SEQ ID No.3 was recovered and purified by Figure 1B Fragment I after being cut at the position shown, the same restriction endonuclease cuts the PET30a-AK183 plasmid (cutting position see Figure 1C ), forming a sticky end that can be spliced, DNA ligase linking fragment I into the upstream (N-terminus) of the AK183 sequence away from its self-cleavage site and active center, forming a recombinant fusion vector, and at the same time, the His tag attached to the N-terminal fusion vector is convenient for later protein purification. The His tag is before the Enterokinase in the PET30a plasmid, and the His tag of the purified protease can be removed by Enterokinase in the later stage to avoid its influence on the protease activity.

[0085] Example 2. Expression and purification of recombinant fusion protein

[0086] 1) Conditions for expression of recombinant fusion protease in E. coli

[0087] The recombinant fusion vector obtained in Example 1 was transfected into BL21-DE3 Escherichia coli competent cells, and the cells were expressed for 24 hours under the induction of 0.3 mM IPGT at 16°C to obtain bacteria expressing the recombinant fusion protein. The gene structure of the recombinant fusion protein expressed in the bacteria is as follows: Figure 4 shown.

[0088] 2) Isolation and purification of recombinant fusion protein

[0089] After adding high salt solution (500 mM sodium chloride, 20 mM sodium dihydrogen phosphate, 30 mM imidazole, 0.8 mM EDTA) and lysozyme to the collected cells and ultrasonically lysing the DNA, the recombinant fusion protease with a His tag was purified by Ni column affinity chromatography and Superdex S200 molecular sieve to obtain four protein peaks F1, F2, F3 and F4 ( Figure 5 ), the F1 protein peak was identified as fully expressed ( Figure 6 ). The three protein peaks F1, F2 and F3 were added to the serum sample of the same IgA nephropathy patient at 37°C overnight. After Jacalin purification of IgA1, Western blot verification was performed. It was found that the F1 and F2 protein peaks had enzymatic cleavage activity of IgA1 in the serum sample of the IgA nephropathy patient. ( Figure 7 ), and the F1 protein peak has higher protease activity in vitro, and can still cleave IgA1 well when the mass ratio with the substrate IgA1 is 1:100 ( Figure 8 ), further experiments found that the recombinant fusion protein in the F1 protein peak can efficiently cut IgA1 in human serum samples in a very short time (1 hr) at 37°C. Fig. 9 ). From the above test results, it can be seen that the F1 protein peak obtained in this example fully expresses the recombinant fusion protein, the single chain molecular weight is about 150kDa, the dimer is ~300KD, and the expressed recombinant fusion protein has efficient in vitro enzyme activity.

[0090] Example 3

[0091] Select eukaryotic expression system HEK293 cells, construct a fusion protein expression vector suitable for HEK293 cells, select the signal peptide of human IL-2, and construct a fusion protein with the same human immunoglobulin fragment and AK183 as in Example 1. The human immunoglobulin fragment is fused to the N-terminus of the AK183 protein to form a plasmid to transfect HEK293 cells for expression and separation and purification. The fully expressed recombinant fusion protein (gene structure as shown in FIG. 1 ) can be obtained from the cell culture fluid and cell body. Fig.10 shown).

[0092] Comparative Example 1.

[0093] Referring to the construction method described in Example 3, HEK293 was selected as the expression system to construct an expression plasmid (gene structure as shown in Fig.11 The structure of the fusion protein in Example 3 is different in that DNA ligase links the human immunoglobulin fragment into the C-terminus (downstream of the transmembrane anchor region) of the natural enzyme sequence of AK183 to form a recombinant fusion vector.

[0094] After purification, it was found that due to the C-terminal self-cleavage of AK183 during maturation, the human immunoglobulin fragment at the C-terminus of the fusion protein was self-cleaved (see Fig.12).

[0095] Experimental Example 1. In vivo pharmacokinetics of recombinant fusion protein

[0096] The in vivo pharmacokinetics of the recombinant fusion protease prepared in Example 2 were evaluated using wild-type BALB / c mice.

[0097] 5 mg / kg body weight of recombinant fusion protease with His tag was injected into the tail vein of 3 wild BALB / c mice, and tail vein plasma was collected at 5min, 1.5h, 4h, 1d, 2d, 3d, 4d, 6d, 8d, and 11d after intravenous injection. The concentration of recombinant fusion protease in plasma at different time points was detected by ELISA method with anti-His tag monoclonal antibody as coating antibody and HRP-labeled anti-human IgG Fc as detection antibody, and pharmacokinetic parameters were calculated using 2Distribution phase. The detection and calculation results are shown in the figure. Fig.13 As shown in Figure 1, after intravenous injection of the recombinant fusion protease of the present invention into mice at a dose of 5 mg / kg body weight, the recombinant fusion protease can still be detected in the plasma on the 11th day, with an AUC (area under the curve) of 93063 and a half-life of 62.5 hours. In contrast, the study by Lechner et al. (J Am Soc Nephrol. 2016 Sep; 27(9): 2622-9) showed that after injecting mice with natural AK183 protease at a dose of 10 mg / kg body weight for 24 hours, AK183 protease was completely undetectable in the mouse plasma (see Fig.14 ). It can be seen that the recombinant fusion protease prepared in Example 2 of the present invention has a significantly extended half-life and can maintain stable activity.

[0098] Experimental Example 2. In vitro and in vivo functional verification of recombinant fusion protease

[0099] The in vitro activity of the recombinant fusion protease prepared in Example 2 was verified by the following method:

[0100] This experiment was conducted using plasma or plasma-purified IgA complexes from patients with primary IgA nephropathy confirmed by clinical renal puncture biopsy. The diagnostic criteria for IgA nephropathy are defined in accordance with the KIDIGO guidelines as renal biopsy pathology with IgA deposition in the mesangial area and / or capillary loops as the main feature, with or without complement C3 deposition, and clinical manifestations of hematuria and / or varying degrees of proteinuria and renal function impairment.

[0101] Fig.15The activity of in vitro recombinant fusion protease was detected using plasma purified poly IgA complexes from 9 patients diagnosed with IgA nephropathy (numbered PT.1, PT.3, PT.6, PT.7, PT.9, PT.12, PT.19, PT.22 and PT.26, among which PT.1, PT.3, PT.6 and PT.9 were patients with crescentic IgA nephropathy). The numbers of the purified poly IgA and the clinical and pathological data of the patients are shown in Table 1.

[0102] Table 1. Clinical data of patients with IgA nephropathy during renal biopsy

[0103]

[0104] The poly-IgA complex samples with corresponding numbers in Table 1 were used as the experimental group, and the recombinant fusion protease prepared in Example 2 was added to the 9 purified poly-IgA complex samples respectively; the poly-IgA complex sample numbered PT.1 without the addition of the recombinant fusion protease was used as the control group; all the samples of the experimental group and the control group were kept at 37°C overnight, and the size of the IgA1 heavy chain fragment was detected by immunoblotting with anti-human IgAalpha chain antibody to verify the activity of the recombinant fusion protease added to the above experimental groups. The results are shown in Fig.15 As shown, the recombinant fusion protease of Example 2 can 100% cleave the poly-IgA1 complex purified from the circulation of IgA nephropathy patients at 37°C overnight. Compared with the complex in the control group, the IgA1 heavy chain of the excised IgA1 complex is cleaved into Fc fragments with smaller molecular weight that can be recognized by anti-human IgA alpha antibodies.

[0105] Fig.16 The activity of the in vitro recombinant fusion protease was detected using the plasma purified poly-IgA complexes from four patients (numbered HSPN1, HSPN2, HSPN3, and HSPN4) diagnosed with purpuric nephritis. The purified poly-IgA numbers and the clinical and pathological data of the patients are shown in Table 2.

[0106] Table 2. Clinical data of patients with purpura nephritis

[0107]

[0108]

[0109] Fig.16The poly-IgA complex samples with corresponding numbers in Table 2 were used as the experimental group, and the recombinant fusion protease prepared in Example 2 was added to the four purified poly-IgA complex samples respectively; the poly-IgA complex sample numbered HSPN.1 without the addition of the recombinant fusion protease was used as the control group; all the samples of the experimental group and the control group were kept at 37°C overnight, and the size of the IgA1 heavy chain fragment was detected by immunoblotting with anti-human IgAalpha chain antibody to verify the activity of the recombinant fusion protease added to the above experimental groups. The results are shown in Fig.16 As shown, the recombinant fusion protease of Example 2 can 100% cleave the poly-IgA1 complex purified from the circulation of patients with purpura nephritis at 37°C overnight. Compared with the complex in the control group, the IgA1 heavy chain of the excised IgA1 complex is cleaved into Fc fragments with smaller molecular weight that can be recognized by anti-human IgA alpha antibodies.

[0110] Fig.17 IgA1 purified from the plasma of a child diagnosed with Kawasaki disease was used as the substrate of the experimental group, and the recombinant fusion protease prepared in Example 2 was used as the control group. The same poly-IgA complex sample without the addition of the recombinant fusion protease was used as the control group. The samples of the experimental group and the control group were subjected to immunoblotting detection at 37°C overnight, and the results showed that the fusion protease could significantly remove IgA1 in the circulation of children with Kawasaki disease.

[0111] In order to further verify the enzymatic activity of the recombinant fusion protease of the present invention in the serum environment, the recombinant fusion protease prepared in Example 2 and human IgA1 were added to the serum of different IgA nephropathy patients and healthy people (the IgA nephropathy patients numbered PT.3 and PT.6 and the healthy people numbered HC.1 and HC.2) at a mass ratio of 1:50 in vitro, and the serum sample numbered PT.3 without the addition of the recombinant fusion protease was used as the control group; all the experimental group and control group samples were kept at 37°C overnight, and the size of the IgA1 heavy chain fragment was detected by immunoblotting to verify the activity of the recombinant fusion protease added to the above experimental groups. The results are as follows Fig.18 As shown, the recombinant fusion protease of Example 2 can 100% cleave the multimeric IgA1 complexes in the circulation of IgA nephropathy patients and healthy people in a serum environment at 37°C overnight in vitro. Compared with the complexes in the control group, the IgA1 heavy chains of the excised IgA1 complexes are cleaved into IgA-Fc fragments with smaller molecular weights that can be recognized by anti-human IgAalpha antibodies.

[0112] The activity of the recombinant fusion protease prepared in Example 2 in the humanized mouse model was verified according to the following method:

[0113] The in vivo protease activity of the recombinant fusion protease obtained in Example 2 was verified in a passive human IgA injection wild-type BALB / c mouse model and a humanized IgA1 alpha chain knock-in (α1KI-Tg) C57BL / 6 mouse model.

[0114] All wild-type passive model BALB / c mice were randomly divided into administration group A (intravenous administration), administration group B (intraperitoneal administration) and control group, with 3 mice in each group. The verification scheme A of the passive model is: first, 25 mg / kg of human IgA1 is injected into the tail vein of the BALB / c mice of the administration group A and the control group, so that the human IgA1 level in the blood is rapidly increased, and a passive human IgA injection wild-type BALB / c mouse model is obtained. After 5 minutes, the recombinant fusion protease prepared in Example 2 is injected into the administration group A mice at a dose of 5 mg / kg body weight through the tail vein, and the control group is injected with PBS through the tail vein. The tails are cut and plasma is collected at 3min, 15min, 45min, 75min, 105min and 135min after injection, respectively, and EDTA anticoagulation is added to the collection EP tube in advance to avoid the in vitro enzyme cleavage reaction from continuing.

[0115] In order to prove that intraperitoneal administration can still be well absorbed into the blood, the verification scheme B of the passive model is: the recombinant fusion protease prepared in Example 2 is intraperitoneally injected into the BALB / c mice in the administration group B. According to previous experience, the blood concentration of the recombinant fusion protein can reach a peak in about 2 hours after intraperitoneal injection. Therefore, 25 mg / kg human IgA1 is injected into the tail vein 1.5 hours later. The tail is cut to collect plasma 3min, 30min, 90min, and 150min after the injection. EDTA anticoagulation is added to the collection EP tube in advance to prevent the in vitro enzymatic cleavage reaction from continuing.

[0116] The ELISA method was used to coat the plate with anti-human IgA Fc antibody and HRP-labeled anti-human Ig Fab antibody as the detection antibody to detect the concentration of intact IgA in the plasma samples collected at different time points in the above verification schemes (5mM EDTA was added to the plasma diluent to prevent the recombinant fusion protease in the blood from continuing to cleave hIgA1 during the experiment). The results were Fig.19 As shown, the initial blood IgA1 concentration of all mouse models injected with human IgA1 was similar to that of human blood IgA1, and it was metabolized slowly in mice. The circulating human IgA1 concentration of the PBS control group decreased by 35% at 2.25 hours compared with the initial 3 minutes (reflecting the natural clearance of non-protease-mediated heterologous IgA in mice), suggesting that this passive model can be used for in vivo recombinant fusion protease activity detection. Compared with the PBS treatment group, the tail vein administration ( Fig.19 A) and intraperitoneal injection of recombinant fusion protease ( Fig.19B) Both can quickly clear passively injected human IgA1 in mice, and the blood IgA level of the intravenous recombinant fusion protease administration group was significantly lower than that of the PBS control group starting from 3 minutes. After 1.75 hours, only ~5% complete human IgA1 remained in the blood of the administration group. The intravenous administration method can make the blood concentration of the recombinant fusion protease quickly reach the peak value and can clear the human IgA1 in the blood more quickly.

[0117] Jacalin beads were used to purify human IgA1 in plasma collected at different time points in the above-mentioned group A (6 μl of plasma was added to 100 μl of PBS containing 5 mM EDTA), and the collected human IgA1 heavy chain fragments were detected by immunoblotting with anti-human IgA alpha antibody. Fig. 20 As shown in A, consistent with the above ELISA results, the IgA1Fc (molecular weight ~65KD) fragments in the plasma of Group A after intravenous administration of the recombinant fusion protease of Example 2 were significantly reduced at 3 minutes, and some IgA1 Fc fragments were cleaved into smaller molecular weight Fd fragments (theoretical molecular weight 27KD, and a protein band at ~37KD can be seen in the reduced state). No IgA1 Fc band was seen in Group A after 1.75 hours of administration. Fig. 20 B is Fig. 20 Grayscale quantitative results of the complete band of human IgA1 Fc in A. The results confirmed that recombinant Fc-AK183 has significant efficacy in cleaving IgA1 in mice.

[0118] In addition, in order to detect the process of drug activity attenuation of the recombinant fusion protease of the present invention in vivo, we injected the recombinant fusion protease prepared in Example 2 into the tail vein at a dose of 5 mg / kg into 12 wild BALB / c mice at 0 o'clock. After that, the experiment was divided into 4 groups, each with 3 mice, and 25 mg / kg human IgA1 was injected into the tail vein on the 1st, 3rd, 7th and 10th day after the injection of the recombinant fusion protease. The plasma of each group was collected at 0.05h, 0.5h, 1.5h, 3h, 5h and 7h after the injection. At the same time, 3 mice (control group) of PBS and human IgA1 were intravenously injected into the control group. Fig.21 A). The intact human IgA1 in plasma was detected by ELISA (the specific method is the same as that described in Experimental Example 1). The results were compared with the PBS control treatment group. The blood IgA level of each mouse at 3 minutes was taken as the baseline 100%, and the curve of the rate of decrease of the human IgA1 level in the mouse blood over time was used as the enzyme activity evaluation. The experimental group of mice receiving the recombinant fusion protease of Example 2 intravenous injection of human IgA1 on days 1, 3, 7, and 10 showed that the rate of decrease of IgA1 concentration within 7 hours was faster than that of the PBS group, and the decrease on day 1 was faster than that on days 3, 7, and 10, indicating that the enzymatic activity of the fusion protease in vivo will slowly decrease with the extension of time (such as Fig.21 B).

[0119] In order to more clearly verify that the recombinant fusion protein of the present invention can function for a long time, the recombinant AK183 protease without Fc tag was expressed by Escherichia coli by conventional methods as a control protease, and BALB / c mice were intravenously injected with the above control protease at an equal dose of 5 mg / kg. One day later, 25 mg / kg human IgA1 was also injected into the tail vein. After that, plasma was collected at 0.05h, 0.5h, 1.5h, and 3h, and the human IgA1 concentration was detected by ELISA. Compared with the recombinant fusion protease of Example 2, the concentration curve of human IgA1 changing with time showed that the control protease group did not significantly accelerate the degradation trend of IgA1, and it coincided with the IgA1 metabolic curve of the PBS group ( Fig. 22 ). This indicates that the control protease has been metabolized and cleared from the mouse body after 1 day and has no protease activity, which is consistent with the research results of Lechner et al.

[0120] The body weight of the 6 passive IgA1 mice in the above-mentioned administration group A and administration group B injected with the recombinant fusion protease of Example 2 was measured every day within 10 days after injection, and no weight loss trend was found (such as Fig.23 The mice behaved and responded normally, indicating that the injection of the recombinant fusion protease of the present invention would not cause adverse effects such as weight loss on the mice.

[0121] In order to solve the problem of the lack of stable levels of IgA1 in the circulation of passive IgA1 mouse model, humanized IgA1 transgenic animal model mice (IgA1-α1KI + / WT Mouse Model)( Fig.24 A). This heterozygote expresses stable levels of human IgA1 and mouse IgA (see immunoblot Fig.24 C (Baseline time point). In this experiment, mouse IgA cannot be cleaved by Fc-AK183 because it has no hinge region sequence, so it is used as a control for drug-specific cleavage of human IgA1. The recombinant fusion protease of Example 2 was intravenously injected at 5 mg / kg into three IgA1-α1KI + / WT Mice, EDTA anticoagulated plasma (such as Fig.24 B), the level of complete IgA-H (IgA heavy chain) in plasma at each time point was detected by immunoblotting with anti-human IgA1alpha antibody, and the level of mouse plasma IgA at the above time points was detected by immunoblotting with anti-mouse IgA antibody. The results showed that after injection of the recombinant fusion protease of Example 2, the circulating IgA1 level rapidly decreased to no obvious band within 1 hour and lasted for 5-7 days, then slowly increased and returned to the baseline level after 14 days. During this period, there was no significant change in the mouse IgA level ( Fig.24C). Fig.24 D is a semi-quantitative analysis of human IgA immunoblot bands at different time points in three mice in 24A. At the same time, these three mice were observed for 14 days without significant weight loss ( Fig.25 ). This revealed that when the recombinant fusion protease of the present invention is administered to a passive IgA1 mouse model, a dosage of 5 mg / kg injected once every 5-7 days is effective.

[0122] In addition, we established a passive IgA1 deposition mouse model and a normal environment-fed IgA1-α1KI mouse model. + / WT The passive IgA1 deposition model was established in mice to study the drug clearance ability of the recombinant fusion protease of the present invention on IgA1 deposited in the kidney. The research scheme of the passive IgA1 deposition model can be found in Fig.26 Previous studies have found that immunofluorescence staining can reveal significant IgA1 deposition in the glomeruli about 2.5 hours after a single injection of human IgA1 at a dose of ~25 mg / kg, mainly in the capillary loops and mesangial areas. This deposition pattern is similar to renal IgA1 deposition in human IgA nephropathy ( Fig.26 In this experiment, 25 mg / kg human IgA1 was injected into the tail vein of BABL / c mice twice at an interval of 1 hour (scheme as Fig. 27 A), 2.5 hours after the last IgA1 injection in the PBS control group, 4 μm sections of frozen kidney tissue embedded in OCT were stained with anti-human IgA1 Fc FITC-labeled antibody for immunofluorescence, and it was found that IgA1 was only deposited in the glomerular mesangial area and some capillary loops, proving that the model was successfully established (see Fig. 27 B lower left, lower right). Compared with the PBS control treatment group, 5 mg / kg of the recombinant fusion protease of Example 2 was injected into the tail vein 1 hour after the last IgA1 injection. After 1.5 hours of in vivo reaction, no IgA1 deposition was found in the glomeruli (see Fig. 27 B upper left), and IgA1 positive signals can be seen in renal tubular epithelial cells (see Fig. 27 B upper right), indicating that the IgA1 deposited in the kidney can be rapidly cleaved by the recombinant fusion protease of Example 2 within 1.5 hours, and the IgA1-Fc fragment after cleavage can be filtered and cleared and then metabolized by the renal tubular epithelial cells because its molecular weight is lower than the mechanical filtration barrier of the kidney.

[0123] In addition, IgA1-α1KI + / WT In order to more accurately verify the efficacy of Fc-AK183 in clearing renal deposited IgA1, the two kidneys of the same mouse were removed before and after administration. After removing one kidney, 5 mg / kg of the recombinant fusion protease of Example 2 of the present invention was intravenously injected. After 3.5 hours, the contralateral kidney was removed ( Fig.28 A). Immunofluorescence staining with FITC-labeled anti-human IgA antibody was then performed to detect renal IgA1 deposition. The results showed that the kidneys removed before administration had obvious human IgA deposition (2+ to 3+) in the glomerular mesangial area. However, the IgA1 in the contralateral kidney removed 2 hours after administration was significantly weakened (+- to -) (see Fig.28 B), and then proved the efficacy of Fc-AK183 in clearing chronic cumulative IgA1 deposition. At the same time, in the staining of small intestinal tissue, it was found that small intestinal secretory IgA1 was still strongly positive (see Fig.29 ), indicating that the recombinant protease for injection of the invention has no clearing effect on extravascular mucosal IgA1. It can be inferred that this drug will not affect the intestinal mucosal defense barrier mediated by secretory IgA1.

[0124] Experimental Example 3. Detection of neutralizing antibodies of recombinant fusion protein and its effect on activity

[0125] In order to examine the antigenic response of the recombinant fusion protease Fc-AK183 after multiple injections, we implemented an intermittent injection schedule to verify: 1. whether the mice produced anti-AK183 antibodies, and 2. whether the antibodies inhibited protease activity.

[0126] The serum of BALB / c mice was collected on the 30th day after intravenous injection of the recombinant fusion protease of Example 2 at a dose of 5 mg / kg. The ELISA method was used to use 2.5 μg / ml AK183 and the recombinant fusion protease as the plate antigens, and the lower wells of the plate were set with equal concentrations of BSA. After blocking with 1% BSA, the mouse serum was diluted in a gradient (1:10, 1:100, 1:500, 1:1000, 1:10000) and incubated. HRP-labeled anti-mouse IgG was used as the detection antibody to detect antibodies against AK183 and the recombinant fusion protease. The results are shown in FIG. Fig.30 As shown, in the mouse serum on the 30th day after the treatment with the recombinant fusion protease Fc-AK183 of Example 2, there were high titer antibodies that recognized the complete recombinant fusion protease Fc-AK83, but no significant antibodies that recognized AK183, indicating that the antigenicity of the Fc fragment was stronger than that of AK183. Four wild BALB / c mice that did not receive AK183 injection served as the negative control group. The antibodies against AK183 and human IgG1Fc were detected by ELISA method again together with the mice treated with AK183. The results were as follows: Fig.31 As shown, compared with the non-intervention group, the recombinant fusion protease Fc-AK83 treatment group of Example 2 was verified from different angles to have very low titer of antibodies recognizing AK183 and high titer of antibodies recognizing human Fc.

[0127] Furthermore, we tested in vitro whether antibodies against the recombinant protease would affect the activity of the protease. First, 5 μg of the recombinant fusion protease of Example 2 was added to different volumes of the above-mentioned 30th day antiserum (20ul, 10ul, 2ul) in vitro, reacted at 37°C for 1 hour, and then 10 μg of human IgA1 was added for enzymatic cleavage at 37°C overnight. The immunoblotting method was used to detect whether the recombinant fusion protease still had the activity of enzymatic cleavage of human IgA1 in vitro after reacting with serum antibodies that can recognize Fc. The results are as follows: Fig.32 As shown, human IgA1 was well cleaved into IgA-Fc fragments, suggesting that neutralizing antibodies against the fusion protease IgG1Fc did not affect the enzymatic activity of the recombinant fusion protease under whole serum conditions in vitro.

[0128] The experimental design of whether neutralizing antibodies will affect the in vivo activity of the recombinant fusion protease is as follows: 3 mice were intravenously injected with 5 mg / kg of the recombinant fusion protease of Example 2, and 5 mg / kg of the recombinant fusion protease of Example 2 was intravenously injected again on the 23rd day after administration. One hour later, 20 mg / kg of human IgA1 was injected into the tail vein. The plasma was collected at 0.05h, 0.25h, 0.75h, 1.25h, 1.75h and 2.25h after the injection of IgA1, and the concentration of human IgA1 in the plasma was detected by the ELISA method in Experimental Example 1. The group that was initially injected with the recombinant fusion protease of Example 2 and the PBS group in Experimental Example 1 were used as the control group. The results are shown in Figure 1. Fig.33 As shown, 23 days later, the recombinant fusion protease of Example 2 was injected into mice again, and it still had strong IgA1 cleavage activity after reacting with neutralizing antibodies in vivo, indicating that neutralizing antibodies do not affect the enzymatic activity of the recombinant fusion protease in vivo.

[0129] At the same time, the three humanized IgA1-α1KI mice that received intravenous injection of the recombinant fusion protease Fc-AK83 in Example 2 in Experimental Example 2 were + / WT On the 30th day after injection, the mice were again treated intravenously with the same dose of 5 mg / kg of the recombinant fusion protease Fc-AK183 of Example 2, and plasma was collected before administration and 3 minutes, 4 hours, 24 hours, 72 hours, and 120 hours after administration. The levels of plasma human IgA and mouse IgA at different time points after administration were detected by the immunoblotting method in Experimental Example 2. The results are shown in Fig.34 As shown, after re-administration of IgA1-α1 KI+ / WT The mouse level decreased rapidly within 4 hours and maintained the lower limit of 24-hour immunoblot detection. On the third day, the blood human IgA increased, but was still significantly lower than the baseline level before administration (about 50% of the baseline level), and maintained this level until the seventh day, while the mouse IgA maintained a stable level before and after administration. KI+ / WTAntibodies produced by mice after the first injection of the recombinant protease had little effect on the in vivo activity of the second administration.

[0130] Reference

[0131] 1. Lai KN, Tang SC, Schena FP, et al. IgA nephropathy. Nat Rev Dis Primers, 2:16001, 2016.

[0132] 2. Wyatt RJ, Julian BA IgA nephropathy. N Engl J Med, 368: 2402-2414, 2013.

[0133] 3. Trimarchi H, Barratt J, Monteiro RC, et al. IgA nephropathy: "State of the art": a report from the 15th International Symposium on IgA Nephropathy celebrating the 50th anniversary of its first description. Kidney Int, 95:750-756, 2019.

[0134] 4.https: / / kdigo.org / wp-content / uploads / 2017 / 02 / KDIGO-GN-GL-Public-Review-Draft_1-June-2020.pdf,

[0135] 5. Suzuki H, Kiryluk K, Novak J, et al. The pathophysiology of IgAnephropathy. J Am Soc Nephrol, 22: 1795-1803, 2011.

[0136] 6.Knoppova B,Reily C,Maillard N,et al.The Origin and Activities ofIgA1-Containing Immune Complexes in IgA Nephropathy.Front Immunol,7:117,2016.

[0137] 7. Eitner F, Floege J Bacterial protease for the treatment of IgAnephropathy. Nephrol Dial Transplant, 23: 2173-2175, 2008.

[0138] 8. Oruc Z, Oblet C, Boumediene A, et al. IgA Structure VariationsAssociate with Immune Stimulations and IgA Mesangial Deposition. J Am SocNephrol, 27: 2748-2761, 2016.

[0139] 9. Lamm ME, Emancipator SN, Robinson JK, et al. Microbial IgA proteaseremoves IgA immune complexes from mouse glomeruli in vivo: potential therapy for IgA nephropathy. Am J Pathol, 172: 31-36, 2008.

[0140] 10.Wang L, Li Sequence Listing <110> Peking University First Hospital Northwestern University <120> A recombinant fusion protease capable of clearing immunoglobulin A in vivo, a preparation method thereof, and application thereof in treating IgA nephropathy <130> LP0484 <140> 2021103854437 <141> 2021-04-10 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 230 <212> PRT <213> human <400> 1 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro 225 230 <210> 2 <211> 1174 <212> PRT <213> Clostridium <400> 2 Gly Ser Ser Lys Pro Asp Ile Lys Val Gly Asp Tyr Val Lys Met Gly 1 5 10 15 Val Tyr Asn Asn Ala Ser Ile Leu Trp Arg Cys Val Ser Ile Asp Asn 20 25 30 Asn Gly Pro Leu Met Leu Ala Asp Lys Ile Val Asp Thr Leu Ala Tyr 35 40 45 Asp Ala Lys Thr Asn Asp Asn Ser Asn Ser Lys Ser His Ser Arg Ser 50 55 60 Tyr Lys Arg Asp Asp Tyr Gly Ser Asn Tyr Trp Lys Asp Ser Asn Met 65 70 75 80 Arg Ser Trp Leu Asn Ser Thr Ala Ala Glu Gly Lys Val Asp Trp Leu 85 90 95 Cys Gly Asn Pro Pro Lys Asp Gly Tyr Val Ser Gly Val Gly Ala Tyr 100 105 110 Asn Glu Lys Ala Gly Phe Leu Asn Ala Phe Ser Lys Ser Glu Ile Ala 115 120 125 Ala Met Lys Thr Val Thr Gln Arg Ser Leu Val Ser His Pro Glu Tyr 130 135 140 Asn Lys Gly Ile Val Asp Gly Asp Ala Asn Ser Asp Leu Leu Tyr Tyr 145 150 155 160 Thr Asp Ile Ser Glu Ala Val Ala Asn Tyr Asp Ser Ser Tyr Phe Glu 165 170 175 Thr Thr Thr Glu Lys Val Phe Leu Leu Asp Val Lys Gln Ala Asn Ala 180 185 190 Val Trp Lys Asn Leu Lys Gly Tyr Tyr Val Ala Tyr Asn Asn Asp Gly 195 200 205 Met Ala Trp Pro Tyr Trp Leu Arg Thr Pro Val Thr Asp Cys Asn His 210 215 220 Asp Met Arg Tyr Ile Ser Ser Ser Gly Gln Val Gly Arg Tyr Ala Pro 225 230 235 240 Trp Tyr Ser Asp Leu Gly Val Arg Pro Ala Phe Tyr Leu Asp Ser Glu 245 250 255 Tyr Phe Val Thr Thr Ser Gly Ser Gly Ser Gln Ser Ser Pro Tyr Ile 260 265 270 Gly Ser Ala Pro Asn Lys Gln Glu Asp Asp Tyr Thr Ile Ser Glu Pro 275 280 285 Ala Glu Asp Ala Asn Pro Asp Trp Asn Val Ser Thr Glu Gln Ser Ile 290 295 300 Gln Leu Thr Leu Gly Pro Trp Tyr Ser Asn Asp Gly Lys Tyr Ser Asn 305 310 315 320 Pro Thr Ile Pro Val Tyr Thr Ile Gln Lys Thr Arg Ser Asp Thr Glu 325 330 335 Asn Met Val Val Val Val Cys Gly Glu Gly Tyr Thr Lys Ser Gln Gln 340 345 350 Gly Lys Phe Ile Asn Asp Val Lys Arg Leu Trp Gln Asp Ala Met Lys 355 360 365 Tyr Glu Pro Tyr Arg Ser Tyr Ala Asp Arg Phe Asn Val Tyr Ala Leu 370 375 380 Cys Thr Ala Ser Glu Ser Thr Phe Asp Asn Gly Gly Ser Thr Phe Phe 385 390 395 400 Asp Val Ile Val Asp Lys Tyr Asn Ser Pro Val Ile Ser Asn Asn Leu 405 410 415 His Gly Ser Gln Trp Lys Asn His Ile Phe Glu Arg Cys Ile Gly Pro 420 425 430 Glu Phe Ile Glu Lys Ile His Asp Ala His Ile Lys Lys Lys Cys Asp 435 440 445 Pro Asn Thr Ile Pro Ser Gly Ser Glu Tyr Glu Pro Tyr Tyr Tyr Val 450 455 460 His Asp Tyr Ile Ala Gln Phe Ala Met Val Val Asn Thr Lys Ser Asp 465 470 475 480 Phe Gly Gly Ala Tyr Asn Asn Arg Glu Tyr Gly Phe His Tyr Phe Ile 485 490 495 Ser Pro Ser Asp Ser Tyr Arg Ala Ser Lys Thr Phe Ala His Glu Phe 500 505 510 Gly His Gly Leu Leu Gly Leu Gly Asp Glu Tyr Ser Asn Gly Tyr Leu 515 520 525 Leu Asp Asp Lys Glu Leu Lys Ser Leu Asn Leu Ser Ser Val Glu Asp 530 535 540 Pro Glu Lys Ile Lys Trp Arg Gln Leu Leu Gly Phe Arg Asn Thr Tyr 545 550 555 560 Thr Cys Arg Asn Ala Tyr Gly Ser Lys Met Leu Val Ser Ser Tyr Glu 565 570 575 Cys Ile Met Arg Asp Thr Asn Tyr Gln Phe Cys Glu Val Cys Arg Leu 580 585 590 Gln Gly Phe Lys Arg Met Ser Gln Leu Val Lys Asp Val Asp Leu Tyr 595 600 605 Val Ala Thr Pro Glu Val Lys Glu Tyr Thr Gly Ala Tyr Ser Lys Pro 610 615 620 Ser Asp Phe Thr Asp Leu Glu Thr Ser Ser Tyr Tyr Asn Tyr Thr Tyr 625 630 635 640 Asn Arg Asn Asp Arg Leu Leu Ser Gly Asn Ser Lys Ser Arg Phe Asn 645 650 655 Thr Asn Met Asn Gly Lys Lys Ile Glu Leu Arg Thr Val Ile Gln Asn 660 665 670 Ile Ser Asp Lys Asn Ala Arg Gln Leu Lys Phe Lys Met Trp Ile Lys 675 680 685 His Ser Asp Gly Ser Val Ala Thr Asp Ser Ser Gly Asn Pro Leu Gln 690 695 700 Thr Val Gln Thr Phe Asp Ile Pro Val Trp Asn Asp Lys Ala Asn Phe 705 710 715 720 Trp Pro Leu Gly Ala Leu Asp His Ile Lys Ser Asp Phe Asn Ser Gly 725 730 735 Leu Lys Ser Cys Ser Leu Ile Tyr Gln Ile Pro Ser Asp Ala Gln Leu 740 745 750 Lys Ser Gly Asp Thr Val Ala Phe Gln Val Leu Asp Glu Asn Gly Asn 755 760 765 Val Leu Ala Asp Asp Asn Thr Glu Thr Gln Arg Tyr Thr Thr Val Ser 770 775 780 Ile Gln Tyr Lys Phe Glu Asp Gly Ser Glu Ile Pro Asn Thr Ala Gly 785 790 795 800 Gly Thr Phe Thr Val Pro Tyr Gly Thr Lys Leu Asp Leu Thr Pro Ala 805 810 815 Lys Thr Leu Tyr Asp Tyr Glu Phe Ile Lys Val Asp Gly Leu Asn Lys 820 825 830 Pro Ile Val Ser Asp Gly Thr Val Val Thr Tyr Tyr Tyr Lys Asn Lys 835 840 845 Asn Glu Glu His Thr His Asn Leu Thr Leu Val Ala Ala Lys Ala Ala 850 855 860 Thr Cys Thr Thr Ala Gly Asn Ser Ala Tyr Tyr Thr Cys Asp Gly Cys 865 870 875 880 Asp Lys Trp Phe Ala Asp Ala Thr Gly Ser Val Glu Ile Thr Asp Lys 885 890 895 Thr Ser Val Lys Ile Pro Ala Pro Gly His Thr Ala Gly Thr Glu Trp 900 905 910 Lys Ser Asp Asp Thr Asn His Trp His Glu Cys Thr Val Ala Gly Cys 915 920 925 Gly Val Ile Ile Glu Ser Thr Lys Ser Ala His Thr Ala Gly Glu Trp 930 935 940 Ile Val Asp Thr Pro Ala Thr Ala Thr Thr Ala Gly Thr Lys His Lys 945 950 955 960 Glu Cys Thr Val Cys His Arg Val Leu Glu Thr Gln Pro Ile Pro Ser 965 970 975 Thr Gly Thr Glu Leu Lys Ile Ile Ala Gly Asp Asn Gln Ile Tyr Asn 980 985 990 Lys Ala Ser Gly Ser Asp Val Thr Ile Thr Cys Asn Gly Asp Phe Ala 995 1000 1005 Lys Phe Thr Gly Ile Lys Val Asp Gly Ser Val Val Asp Ser Ser Asn 1010 1015 1020 Tyr Thr Ala Val Ser Gly Ser Thr Val Leu Thr Leu Lys Ala Ser Tyr 1025 1030 1035 1040 Leu Gly Thr Leu Thr Asp Gly Ser His Thr Ile Thr Phe Val Tyr Thr 1045 1050 1055 Asp Gly Glu Ala Asn Ala Asn Leu Thr Val Arg Thr Ala Gly Ser Gly 1060 1065 1070 His Ile His Asp Tyr Gly Thr Glu Trp Lys Ser Asn Ala Asp Asn His 1075 1080 1085 Trp His Glu Cys Asn Cys Gly Asp Lys Lys Asp Glu Ala Ala His Ser 1090 1095 1100 Phe Lys Trp Val Val Asp Lys Glu Ala Thr Ala Thr Lys Lys Gly Ser 1105 1110 1115 1120 Lys His Glu Glu Cys Lys Ile Cys Gly Tyr Lys Arg Ser Ala Val Glu 1125 1130 1135 Ile Pro Ala Thr Gly Thr Ser Thr Ala Pro Thr Asp Thr Thr Lys Pro 1140 1145 1150 Asn Asp Thr Thr Lys Pro Gly Asn Thr Asn Gly Ser Glu Lys Ser Pro 1155 1160 1165 Gln Thr Gly Asp Asn Ser 1170 <210> 3 <211> 720 <212> DNA <213> Artificial Sequence <400> 3 atccatggga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60 aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120 tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240 aggagcagta caacagcacg taccgggtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg 360 agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480 atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540 ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccacta cacgcagaag agcctctccc tgtctccggg taaaggtggc ggtggcggcg 720 <210> 4 <211> 3534 <212> DNA <213> Artificial Sequence <400> 4 ggatccagca aaccggacat caaagtgggc gactacgtga aaatgggtgt gtataataac 60 gcaagcatcc tgtggcgctg tgtgagcatc gacaacaatg gcccgctgat gctggccgat 120 aaaattgttg acacgctggc gtatgatgct aaaaccaacg acaattcgaa cagcaaatct 180 catagtcgtt cctacaaacg cgatgactac ggcagcaact attggaaaga tagtaatatg 240 cgctcctggc tgaactcaac cgcggccgag ggtaaagtgg attggctgtg cggcaatccg 300 ccgaaagacg gttacgtcag cggcgtgggt gcatataatg aaaaagctgg ttttctgaac 360 gcgttctcaa aatcggaaat tgcagctatg aaaacggtga cccagcgtag cctggtttct 420 catccggaat ataataaagg cattgttgat ggtgacgcga actcggatct gctgtattac 480 accgacatca gcgaagcagt ggctaactac gatagctctt attttgaaac cacgaccgaa 540 aaagttttcc tgctggatgt caaacaggcg aacgccgtct ggaaaaatct gaaaggctat 600 tacgtggctt acaacaatga tggtatggca tggccgtatt ggctgcgtac cccggtgacg 660 gattgtaatc atgacatgcg ctatattagt tcctcaggcc aggttggtcg ttacgctccg 720 tggtattctg atctgggcgt ccgtccggcg ttttacctgg acagtgaata tttcgtgacg 780 accagcggct ctggtagtca gtcgagcccg tacattggtt ccgcgccgaa caaacaagaa 840 gatgactata ccatctcaga accggcggaa gatgccaacc cggactggaa tgtttcgacg 900 gaacagagca ttcaactgac cctgggcccg tggtactcga atgatggtaa atatagcaac 960 ccgaccattc cggtgtatac catccagaaa acgcgctcgg ataccgaaaa catggtggtt 1020 gtcgtgtgcg gcgaaggtta taccaaatca cagcaaggca aatttatcaa tgatgttaaa 1080 cgtctgtggc aggacgctat gaaatatgaa ccgtaccgta gctatgcgga tcgctttaat 1140 gtgtatgcac tgtgtacggc ttccgaatca accttcgata acggcggttc tacctttttc 1200 gatgtgatcg ttgacaaata caactctccg gttatcagta acaatctgca tggcagtcag 1260 tggaaaaatc acattttga acgctgcatc ggtccggaat tcattgaaaa aatccatgat 1320 gcccacatta agaaaaaatg tgacccgaac accatcccgt cgggtagcga atacgaaccg 1380 tattactatg tgcatgatta tattgcacag tttgctatg ttgtcaatac caaatccgac 1440 ttcggcggtg catataacaa tcgcgaatac ggctttcact atttcatctc tccgagtgat 1500 tcctaccgtg cctctaaaac ctttgcacat gaattcggcc acggtctgct gggcctgggt 1560 gatgaatact cgaatggtta tctgctggat gacaaagaac tgaaaagcct gaacctgtct 1620 agtgtggaag atccggaaaa aattaaatgg cgtcagctgc tgggctttcg caatacgtac 1680 acctgccgta acgcgtatgg ttctaaaatg ctggtttcct catacgaatg tatcatgcgc 1740 gataccaact atcaattttg cgaagtctgt cgcctgcagg gcttcaaacg tatgagccaa 1800 ctggttaaag atgtcgacct gtatgtggcc acgccggaag ttaaagaata caccggtgca 1860 tatagtaaac cgtccgattt tacggacctg gaaacctcga gctactacaa ctacacctac 1920 aaccgtaacg atcgcctgct gagtggcaac tcaaaatcgc gtttcaatac gaacatgaat 1980 ggcaagaaaa ttgaactgcg caccgttatt cagaacatca gcgataaaaa cgcccgtcaa 2040 ctgaaattca aaatgtggat caaacattca gatggctcgg tggcaaccga ctctagtggt 2100 aacccgctgc agaccgtcca aacgtttgat attccggtgt ggaacgacaa agccaatttc 2160 tggccgctgg gcgcactgga tcacatcaaa tccgacttta attcaggtct gaaaagctgc 2220 tctctgattt atcagatccc gtctgatgct caactgaaaa gtggcgacac cgtggcgttc 2280 caggttctgg atgaaaacgg taatgtgctg gcggatgaca acacggaaac ccagcgctac 2340 acgaccgttt ctatccaata caaattcgaa gatggcagtg aaatcccgaa tacggcgggc 2400 ggtaccttca ccgttccgta tggtaccaaa ctggatctga cgccggccaa aaccctgtac 2460 gattacgaat tcatcaaagt tgacggcctg aataaaccga tcgtcagcga tggtaccgtg 2520 gttacgtact actacaaaaa caaaaacgaa gaacatacgc acaacctgac cctggtggcg 2580 gccaaagcag ctacctgtac gaccgcgggc aatagcgcct attacacctg cgatggttgt 2640 gacaaatggt ttgcagatgc taccggctcc gtggaaatta ccgacaaaac gtcagttaaa 2700 atcccggctc cgggtcatac cgccggtacg gaatggaaaa gcgatgacac gaaccattgg 2760 cacgaatgca ccgtcgcagg ctgtggtgtg attatcgaaa gcacgaaatc tgcgcacacc 2820 gccggcgaat ggattgtgga taccccggca acggcaacga ccgccggtac gaaacataaa 2880 gaatgcaccg tctgtcaccg tgtgctggaa acccagccga tccccgagcac gggtaccgaa 2940 ctgaaaatta tcgccggtga taaccaaatc tacaacaaag caagtggctc cgatgtcacg 3000 atcacctgca acggtgactt tgccaaattc accggcatta aagtggatgg tagcgtcgtg 3060 gactcctcaa attacaccgc cgtttcaggc tcgaccgtcc tgacgctgaa agcatcctat 3120 ctgggcacgc tgaccgatgg ttcacatacg attaccttcg tttacaccga cggtgaagca 3180 aacgctaatc tgaccgtccg cacggctggc tctggtcata tccacgatta tggcaccgaa 3240 tggaaaagta acgcggacaa tcattggcac gaatgcaatt gtggtgataa aaaagacgaa 3300 gcggcccatt cctttaaatg ggttgtcgat aaagaagcga cggccaccaa aaaaggctca 3360 aaacacgaag aatgcaaaat ctgtggttac aaacgttcgg ccgtggaaat cccggcaacg 3420 ggtaccagca cggcaccgac cgatacgacc aaaccgaacg acacgacgaa accgggtaat 3480 acgaatggct ccgaaaaatc tccgcaaacg ggcgacaata gttaatgaaa gctt 3534 <210> 5 <211> 1412 <212> PRT <213> Artificial Sequence <400> 5 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Ser 225 230 235 240 Ser Lys Pro Asp Ile Lys Val Gly Asp Tyr Val Lys Met Gly Val Tyr 245 250 255 Asn Asn Ala Ser Ile Leu Trp Arg Cys Val Ser Ile Asp Asn Asn Gly 260 265 270 Pro Leu Met Leu Ala Asp Lys Ile Val Asp Thr Leu Ala Tyr Asp Ala 275 280 285 Lys Thr Asn Asp Asn Ser Asn Ser Lys Ser His Ser Arg Ser Tyr Lys 290 295 300 Arg Asp Asp Tyr Gly Ser Asn Tyr Trp Lys Asp Ser Asn Met Arg Ser 305 310 315 320 Trp Leu Asn Ser Thr Ala Ala Glu Gly Lys Val Asp Trp Leu Cys Gly 325 330 335 Asn Pro Pro Lys Asp Gly Tyr Val Ser Gly Val Gly Ala Tyr Asn Glu 340 345 350 Lys Ala Gly Phe Leu Asn Ala Phe Ser Lys Ser Glu Ile Ala Ala Met 355 360 365 Lys Thr Val Thr Gln Arg Ser Leu Val Ser His Pro Glu Tyr Asn Lys 370 375 380 Gly Ile Val Asp Gly Asp Ala Asn Ser Asp Leu Leu Tyr Tyr Thr Asp 385 390 395 400 Ile Ser Glu Ala Val Ala Asn Tyr Asp Ser Ser Tyr Phe Glu Thr Thr 405 410 415 Thr Glu Lys Val Phe Leu Leu Asp Val Lys Gln Ala Asn Ala Val Trp 420 425 430 Lys Asn Leu Lys Gly Tyr Tyr Val Ala Tyr Asn Asn Asp Gly Met Ala 435 440 445 Trp Pro Tyr Trp Leu Arg Thr Pro Val Thr Asp Cys Asn His Asp Met 450 455 460 Arg Tyr Ile Ser Ser Ser Gly Gln Val Gly Arg Tyr Ala Pro Trp Tyr 465 470 475 480 Ser Asp Leu Gly Val Arg Pro Ala Phe Tyr Leu Asp Ser Glu Tyr Phe 485 490 495 Val Thr Thr Ser Gly Ser Gly Ser Gln Ser Ser Pro Tyr Ile Gly Ser 500 505 510 Ala Pro Asn Lys Gln Glu Asp Asp Tyr Thr Ile Ser Glu Pro Ala Glu 515 520 525 Asp Ala Asn Pro Asp Trp Asn Val Ser Thr Glu Gln Ser Ile Gln Leu 530 535 540 Thr Leu Gly Pro Trp Tyr Ser Asn Asp Gly Lys Tyr Ser Asn Pro Thr 545 550 555 560 Ile Pro Val Tyr Thr Ile Gln Lys Thr Arg Ser Asp Thr Glu Asn Met 565 570 575 Val Val Val Val Cys Gly Glu Gly Tyr Thr Lys Ser Gln Gln Gly Lys 580 585 590 Phe Ile Asn Asp Val Lys Arg Leu Trp Gln Asp Ala Met Lys Tyr Glu 595 600 605 Pro Tyr Arg Ser Tyr Ala Asp Arg Phe Asn Val Tyr Ala Leu Cys Thr 610 615 620 Ala Ser Glu Ser Thr Phe Asp Asn Gly Gly Ser Thr Phe Phe Asp Val 625 630 635 640 Ile Val Asp Lys Tyr Asn Ser Pro Val Ile Ser Asn Asn Leu His Gly 645 650 655 Ser Gln Trp Lys Asn His Ile Phe Glu Arg Cys Ile Gly Pro Glu Phe 660 665 670 Ile Glu Lys Ile His Asp Ala His Ile Lys Lys Lys Cys Asp Pro Asn 675 680 685 Thr Ile Pro Ser Gly Ser Glu Tyr Glu Pro Tyr Tyr Tyr Val His Asp 690 695 700 Tyr Ile Ala Gln Phe Ala Met Val Val Asn Thr Lys Ser Asp Phe Gly 705 710 715 720 Gly Ala Tyr Asn Asn Arg Glu Tyr Gly Phe His Tyr Phe Ile Ser Pro 725 730 735 Ser Asp Ser Tyr Arg Ala Ser Lys Thr Phe Ala His Glu Phe Gly His 740 745 750 Gly Leu Leu Gly Leu Gly Asp Glu Tyr Ser Asn Gly Tyr Leu Leu Asp 755 760 765 Asp Lys Glu Leu Lys Ser Leu Asn Leu Ser Ser Val Glu Asp Pro Glu 770 775 780 Lys Ile Lys Trp Arg Gln Leu Leu Gly Phe Arg Asn Thr Tyr Thr Cys 785 790 795 800 Arg Asn Ala Tyr Gly Ser Lys Met Leu Val Ser Ser Tyr Glu Cys Ile 805 810 815 Met Arg Asp Thr Asn Tyr Gln Phe Cys Glu Val Cys Arg Leu Gln Gly 820 825 830 Phe Lys Arg Met Ser Gln Leu Val Lys Asp Val Asp Leu Tyr Val Ala 835 840 845 Thr Pro Glu Val Lys Glu Tyr Thr Gly Ala Tyr Ser Lys Pro Ser Asp 850 855 860 Phe Thr Asp Leu Glu Thr Ser Ser Tyr Tyr Asn Tyr Thr Tyr Asn Arg 865 870 875 880 Asn Asp Arg Leu Leu Ser Gly Asn Ser Lys Ser Arg Phe Asn Thr Asn 885 890 895 Met Asn Gly Lys Lys Ile Glu Leu Arg Thr Val Ile Gln Asn Ile Ser 900 905 910 Asp Lys Asn Ala Arg Gln Leu Lys Phe Lys Met Trp Ile Lys His Ser 915 920 925 Asp Gly Ser Val Ala Thr Asp Ser Ser Gly Asn Pro Leu Gln Thr Val 930 935 940 Gln Thr Phe Asp Ile Pro Val Trp Asn Asp Lys Ala Asn Phe Trp Pro 945 950 955 960 Leu Gly Ala Leu Asp His Ile Lys Ser Asp Phe Asn Ser Gly Leu Lys 965 970 975 Ser Cys Ser Leu Ile Tyr Gln Ile Pro Ser Asp Ala Gln Leu Lys Ser 980 985 990 Gly Asp Thr Val Ala Phe Gln Val Leu Asp Glu Asn Gly Asn Val Leu 995 1000 1005 Ala Asp Asp Asn Thr Glu Thr Gln Arg Tyr Thr Thr Val Ser Ile Gln 1010 1015 1020 Tyr Lys Phe Glu Asp Gly Ser Glu Ile Pro Asn Thr Ala Gly Gly Thr 1025 1030 1035 1040 Phe Thr Val Pro Tyr Gly Thr Lys Leu Asp Leu Thr Pro Ala Lys Thr 1045 1050 1055 Leu Tyr Asp Tyr Glu Phe Ile Lys Val Asp Gly Leu Asn Lys Pro Ile 1060 1065 1070 Val Ser Asp Gly Thr Val Val Thr Tyr Tyr Tyr Lys Asn Lys Asn Glu 1075 1080 1085 Glu His Thr His Asn Leu Thr Leu Val Ala Ala Lys Ala Ala Thr Cys 1090 1095 1100 Thr Thr Ala Gly Asn Ser Ala Tyr Tyr Thr Cys Asp Gly Cys Asp Lys 1105 1110 1115 1120 Trp Phe Ala Asp Ala Thr Gly Ser Val Glu Ile Thr Asp Lys Thr Ser 1125 1130 1135 Val Lys Ile Pro Ala Pro Gly His Thr Ala Gly Thr Glu Trp Lys Ser 1140 1145 1150 Asp Asp Thr Asn His Trp His Glu Cys Thr Val Ala Gly Cys Gly Val 1155 1160 1165 Ile Ile Glu Ser Thr Lys Ser Ala His Thr Ala Gly Glu Trp Ile Val 1170 1175 1180 Asp Thr Pro Ala Thr Ala Thr Thr Ala Gly Thr Lys His Lys Glu Cys 1185 1190 1195 1200 Thr Val Cys His Arg Val Leu Glu Thr Gln Pro Ile Pro Ser Thr Gly 1205 1210 1215 Thr Glu Leu Lys Ile Ile Ala Gly Asp Asn Gln Ile Tyr Asn Lys Ala 1220 1225 1230 Ser Gly Ser Asp Val Thr Ile Thr Cys Asn Gly Asp Phe Ala Lys Phe 1235 1240 1245 Thr Gly Ile Lys Val Asp Gly Ser Val Val Asp Ser Ser Asn Tyr Thr 1250 1255 1260 Ala Val Ser Gly Ser Thr Val Leu Thr Leu Lys Ala Ser Tyr Leu Gly 1265 1270 1275 1280 Thr Leu Thr Asp Gly Ser His Thr Ile Thr Phe Val Tyr Thr Asp Gly 1285 1290 1295 Glu Ala Asn Ala Asn Leu Thr Val Arg Thr Ala Gly Ser Gly His Ile 1300 1305 1310 His Asp Tyr Gly Thr Glu Trp Lys Ser Asn Ala Asp Asn His Trp His 1315 1320 1325 Glu Cys Asn Cys Gly Asp Lys Lys Asp Glu Ala Ala His Ser Phe Lys 1330 1335 1340 Trp Val Val Asp Lys Glu Ala Thr Ala Thr Lys Lys Gly Ser Lys His 1345 1350 1355 1360 Glu Glu Cys Lys Ile Cys Gly Tyr Lys Arg Ser Ala Val Glu Ile Pro 1365 1370 1375 Ala Thr Gly Thr Ser Thr Ala Pro Thr Asp Thr Thr Lys Pro Asn Asp 1380 1385 1390 Thr Thr Lys Pro Gly Asn Thr Asn Gly Ser Glu Lys Ser Pro Gln Thr 1395 1400 1405 Gly Asp Asn Ser 1410

Claims

1. Use of a recombinant fusion protein based on AK183 protease in the preparation of a drug for treating diseases mediated by IgA complex deposition, characterized in that: The related diseases mediated by IgA complex deposition are IgA nephropathy, Henoch-Schonlein purpura nephritis or Kawasaki disease; the amino acid sequence of the recombinant fusion protein is shown in SEQ ID No.

5.

2. The use according to claim 1, characterized in that: The recombinant fusion protein based on AK183 protease is prepared into a liquid preparation for administration to mammals suffering from the related diseases mediated by the IgA complex deposition.

3. The use according to claim 2, characterized in that: The liquid preparation is an injection, and the administration is via intravenous injection or infusion.

4. The use according to claim 3, characterized in that: The mammal is a human, and the intravenous injection regimen includes: injecting once every 10-15 days at a dose of 5-10 mg / kg.

5. The use according to claim 4, characterized in that: The intravenous injection regimen includes: taking a dose of 10-15 mg / kg as an initial injection dose, and then changing it to a maintenance dose of 5-10 mg / kg injected once every 10 days.

6. The use according to claim 3, characterized in that: The mammal is a passive human IgA injection mouse model, a human IgA1 transgenic mouse model or a primate model, and the intravenous injection regimen includes: injecting once every 5-10 days at a dose of 5-10 mg / kg.

7. The use according to claim 6, characterized in that: The intravenous injection regimen includes: injecting once every 5 days at a dose of 5 mg / kg.

8. A recombinant fusion protein, the amino acid sequence of which is shown in SEQ ID No.

5.

9. A recombinant fusion protein gene expression vector, which is a plasmid composed of a carrier and a target gene recombined and fused, wherein: The target gene is a recombinant fusion gene encoding a protein having an amino acid sequence as shown in SEQ ID No.

5.

10. The recombinant fusion protein gene expression vector according to claim 9, characterized in that: The vector is suitable for a prokaryotic expression system.

11. The recombinant fusion protein gene expression vector according to claim 9, characterized in that: The carrier is a carrier that matches the Escherichia coli expression system.

12. The recombinant fusion protein gene expression vector according to any one of claims 9 to 11, characterized in that: The carrier is a carrier with a purification tag.

13. The recombinant fusion protein gene expression vector according to any one of claims 9 to 11, characterized in that: The carrier is pET30a plasmid.

14. A method for expressing and purifying a recombinant fusion protein based on AK183 protease, comprising: The recombinant fusion protein gene expression vector of claim 9 is transfected into competent Escherichia coli cells, and protein expression is performed under the induction of an inducer to obtain the recombinant fusion protein; the inducer concentration is 0.1-0.5 mM isopropyl-β-D-thiogalactoside (IPGT); the protein expression temperature is 15-18°C; the protein expression time is 20-30 hours; after the expression is completed, the Escherichia coli cell body is treated according to a conventional method, ultrasonically fragmented, and then centrifuged at high speed and the supernatant is retained, and then affinity chromatography and molecular sieve purification are used to obtain the recombinant fusion protein.

15. The method according to claim 14, characterized in that: The inducing agent is IPGT with a concentration of 0.3 mM; the protein expression temperature is 16-18° C.; and the protein expression time is 24 hours.

16. The method of claim 14, wherein: The affinity chromatography and molecular sieve purification were performed at 4 degrees Celsius, and a buffer containing 0.8 mM EDTA was used to protect the protease activity to the maximum extent. Finally, the purified protein was frozen in a neutral phosphate buffer.

17. A pharmaceutical composition comprising the recombinant fusion protein of claim 8.

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