Mannan binding lectin modified protein ppg-rep-MBL, DNA molecule, expression vector, host cell, and use thereof

Abstract

The present invention relates to a mannan-binding lectin modified protein ppg-rep-MBL, a DNA molecule, an expression vector, a host cell, and uses, and belongs to the field of biomedical technology. The present invention provides a mannan-binding lectin modified protein ppg-rep-MBL, whose amino acid sequence is shown in SEQ ID NO:3 and whose nucleotide sequence is shown in SEQ ID NO:4. The present invention also provides the use of the mannan-binding lectin modified protein ppg-rep-MBL in the preparation of a drug for preventing or / and treating coronavirus infection. The modified protein of the present invention does not bind to the MASP2 protein, thereby significantly weakening the activation effect on the complement lectin pathway, while retaining its binding properties with mannose, and can effectively bind to and block the infection of the new coronavirus pseudovirus and its Delta mutant strain to HEK293T-ACE2 cells, providing a candidate possibility for the protein to be designed as an inhibitor or drug for the development of a broad-spectrum anti-coronavirus infection.

Description

Technical Field

[0001] The present invention relates to a mannan binding lectin modified protein ppg-rep-MBL, a DNA molecule, an expression vector, a host cell, and use of the same in preparing a drug for preventing and / or treating coronavirus infection, belonging to the technical field of biomedicine. Background Art

[0002] As an important component of innate immunity and an inherent component of the complement activation process (complement lectin pathway), mannan-binding lectin (MBL) has attracted widespread attention for its recognition and regulatory functions in viral infection. Human MBL has obvious antibacterial and antiviral activity and good drugability. From the perspective of the rationality of developing new drugs, applying MBL as a drug to the human body itself will have advantages such as low immunogenicity compared to exogenous proteins. However, if MBL is injected into the body in large quantities in the form of a medicinal protein, it will inevitably lead to problems such as overactivation of the complement lectin pathway (complement system) and over-promotion of phagocytosis of phagocytes, causing various adverse reactions, causing inflammatory damage to the body and even death. Therefore, in the process of severe inflammatory diseases, balancing or controlling the activation of the complement system and inhibiting adverse reactions without destroying its protective function has become the focus of current research.

[0003] At present, the research reports on MBL mainly focus on the function of specific sequences. For example, Ying L et al. reported that three point mutations CGT 52 TGT, GGC 54 GAC and GGA 57 GAA in the collagen-like region (CLR) of the human MBL gene would cause the ability of the MBL mutant protein to bind mannan to be much lower than that of the wild-type MBL protein, and the MBL mutant could not effectively activate the complement lectin pathway. (Ying L, Feng-Ling L, Zhi-Jun B, et al. Defective activities, but not secretions, resulting from gene point mutations of human mannan-binding lectin. [J]. Molecular medicine reports, 2012, 5 (4): 1121-7.) Russell W et al. made two mutations (G25D and G28E) in rat MBL. It was found that the efficiency of G28E mutant in activating complement was more than 10 times lower than that of G25D mutant, and the activity of G25D mutant was 7 times lower than that of wild-type MBL. It was further found that the reduction of complement activation caused by the two mutant MBL was due to the failure of MASP-2 to be effectively activated when bound to the mannan-coated surface. Kurata H et al. purified and identified two forms of human MBL, liver MBL (L-MBL) and serum MBL (S-MBL) from liver and serum, respectively, and found that newly synthesized MBL in human liver was processed into the above two forms after translation, and the two forms differed by 9 amino acids. Recombinant human MBL was synthesized in COS-1 cells and found that it lacked a sequence containing 9 amino acid residues (Ser-Ser-Pro-Gly-Ile-Asn-Gly-Phe-Pro) at the front end of the collagen-like domain. At the same time, the protein had no ability to activate complement, indicating that this sequence plays an important role in the process of complement activation by human MBL.(Kurata H, Sannoh T, Kozutsumi Y, Yokota Y, Kawasaki T.Structure and function of mannan-binding proteins isolated from human liver and serum. J Biochem. 1994 Jun;115(6):1148-54.) Girija, U. V et al. found that the lysine residue at position X of the Gly-XY collagen repeat sequence, i.e. Lys(56) in ficolin-A, is essential for MASP-2 binding. This residue is present in all ficolins and MBL known to activate complement. A recombinant protein with K56P and M57O double mutations was prepared. The data showed that Lys56 and / or Met57 are essential for binding to MASP-2 and for effective MASP activation. In rat MBL, equivalent mutations (K46P and V47O) in MBL protein also abolished MASP-2 binding and complement activation. (Girija, UV, Dodds, AW, Roscher, S., Reid, KB,&Wallis, R. (2007). Localization and characterization of the mannose-binding lectin (MBL)-associated-serine protease-2 binding site in ratficolin-A: equivalent binding sites within the collagenous domains of MBLsand ficolins. Journal of immunology (Baltimore, Md. : 1950), 179(1), 455–462.) Daming Zuo et al. designed and synthesized a series of peptides based on the CLR sequence in human MBL, which are believed to block the MBL-MASP interaction, to locate the serine protease binding motif on human MBL. The results showed that MASP binds to the C-terminus of the hinge region of the collagen-like domain (formed by the interruption of the Gly-XY repeat sequence). In addition, it was found that the R32C, G35D and G37E mutant proteins had similar serine protease binding properties to wild-type MBL, but the binding of the mutant MBL proteins to MASPs was much weaker than that of the wild-type MBL protein to MASPs.(Zuo, D., Cai, X., Zhao, N., Zhang, L., & Chen, Z. (2010). Location of MBL-associated serine proteases binding motifs on human mannan-binding lectin (MBL). Protein and peptide letters, 17(1), 131–136) Application number CN201910560238.2, invention name: A recombinant protein containing human IgG1 Fc and the C-terminus of mannan-binding lectin, providing a recombinant protein containing human IgG1 Fc and the C-terminus of mannan-binding lectin and a polynucleotide encoding the protein with an optimized sequence. The invention also provides a preparation method for expressing the recombinant protein. The recombinant protein expressed by the plasmid transfected cells containing the polynucleotide with the optimized sequence has a purified concentration of 1.50 mg / ml, which is 3 times the concentration of the protein expressed by the coding sequence without codon optimization. The recombinant protein has good binding activity with 5 standard strains and clinical strains of Candida, which account for more than 90% of blood fungal infections, proving that the recombinant protein can bind to all 5 Candida species, and this binding is mediated by the CRD region of the recombinant protein MBL. Chen Ade et al. studied the effect of mannan-binding lectin (MBL) on the function of human neutrophils (PMN). They proved that MBL enhances PMN phagocytosis in a complement lectin pathway-dependent manner and promotes the secretion of inflammatory cytokines (Effects of mannan-binding lectin on human neutrophil function, J South Med Univ, 2013, 33(6): 842-846).

[0004] MBL is a multimeric mannose / mannan sugar-binding protein. Its single chain is divided into four parts: N-terminus, collagen-like region (CLR), neck region and sugar recognition region (CRD). The N-terminus is the key to the polymerization of the three monomers of MBL protein, while the neck and CRD regions are the key to whether MBL can recognize and bind to pathogens. Its amino acid sequence is SEQ ID NO.1 and its nucleotide sequence is SEQ ID NO.2. During the activation of the complement lectin pathway, the prerequisite for complement activation is the recognition and binding of the OGKXGP short motif in MBL-CLR by mannan-binding lectin-associated serine protease 2 (MASP2) (Ichise, Steven H. Sacks, Development of a collagen-like peptide polymer via end-to-end disulfide cross-linking and its application as a biomaterial[J]. Acta Biomaterialia, 2019, 9(4): 361-371). If this sequence is replaced or deleted, CLR will be unable to form a collagen-like subunit structure, thereby affecting other biological functions of MBL protein.

[0005] Currently, there are no literature reports on weakening / eliminating the effect of MBL on activating the complement lectin pathway, and there are no literature reports on weakening / eliminating the activation effect of MBL on the complement lectin pathway while retaining its mannan / mannose binding properties.

[0006] Infection caused by the novel coronavirus (SARS-CoV-2) may induce over-activation of the immune system and the release of high concentrations of pro-inflammatory cytokines, leading to a "cytokine storm". The overproduction of such inflammatory cytokines may lead to disease progression, organ failure, acute respiratory distress syndrome, and death. In this context, the human immune system, especially the innate immune response and its complement system, how to respond to viral infections, and how to develop drugs that utilize this line of defense and its related proteins have also become the focus of research.

[0007] At present, there are patents reporting the application of MBL gene in SARS-CoV detection reagents (patent number: CN200510082997.0) and recombinant MBL in new crown detection kits (patent application number: CN202311287315.4), recombinant MBL for blocking new coronavirus infection (patent number CN202110490146.9) and the use of MBL polypeptides, or their functional fragments, derivatives, and mutants to inhibit new coronavirus infection (patent number: US20240123029A1).

[0008] There are currently no literature reports on the use of low-immunogenicity MBL modified proteins that reduce / prevent complement system activation for the prevention and / or treatment of coronavirus infection. Summary of the invention

[0009] The present invention aims at the problems existing in the above-mentioned prior art and provides a modified protein ppg-rep-MBL with low immunogenicity that can inhibit coronavirus infection. The inventor successfully expressed the modified protein MBL by modifying the gene of MBL, which reduces the activation of the complement system while ensuring its sugar binding ability. The present invention also provides a preparation method and use of ppg-rep-MBL.

[0010] The present invention provides a mannan binding lectin modified protein ppg-rep-MBL, the amino acid sequence of which is shown in SEQ ID NO:3, and the nucleotide sequence of which is shown in SEQ ID NO:4.

[0011] The modified protein replaces the OGKXGP short motif in the collagen-like region (CLR region) of MBL that recognizes and binds to MASP2 to activate the complement pathway, referring to the biological functional sequence CCCPP[GPP]4GPR[GPP]4GCCC of natural collagen and retains the motif sequence GEKGEP that mediates binding to C1qR to enhance the phagocytic effect of phagocytes in the body. The present invention introduces the above collagen sequence to replace the MBL-CLR region to ensure collagen-like formation, but in order to make the length of the replaced MBL protein amino acid sequence closer to the natural MBL protein amino acid sequence, the present invention repeats the PP[GPP]4GPR[GPP]4G sequence when introducing the collagen sequence, that is, introduces: CCCPP[GPP]4GPR[GPP]4GPP[GPP]4GPR[GPP]4G sequence, only retains the three cysteines at the N terminal, and removes the three cysteines at the C terminal, so that the N-terminal Cys can cross-link the triple helix through a disulfide bond, while not changing the bonding angle of the C-terminal CRD region of MBL that specifically recognizes the sugar residue. In order to ensure the integrity of the function of the modified MBL protein, the motif sequence GEKGEP in the original position is retained at the C-terminal of the sequence. Finally, CCCPP[GPP]4GPR[GPP]4GPP[GPP]4GPR[GPP]4GGEKGEP (ppg-rep peptide) was used to replace it.

[0012] The present invention also provides use of the mannan-binding lectin modified protein ppg-rep-MBL in preparing a drug for preventing and / or treating coronavirus infection.

[0013] Among them, the drug is a drug that blocks SRAS-CoV-2 infection.

[0014] Among them, the SARS-CoV-2 includes the original strain and the mutant strain.

[0015] The present invention also provides a method for preparing the mannan-binding lectin modified protein ppg-rep-MBL, which comprises the following steps:

[0016] a. Using the wild-type mannan binding lectin gene sequence SEQ ID NO: 1 as a template and the nucleic acid sequence SEQ ID NO: 2, a recombinant mannan binding lectin ppg-rep-MBL amino acid sequence was constructed; the MBL-CLR region of the amino acid sequence of the wild-type mannan binding lectin was replaced with CCCPP[GPP]4GPR[GPP]4GPP[GPP]4GPR[GPP]4GGEKGEP; the ppg-rep-MBL gene was designed and synthesized, the amino acid sequence was SEQ ID NO. 3, and the nucleotide sequence was SEQ ID NO. 4;

[0017] b. Construction of modified mannan-binding lectin recombinant plasmid: Insert the ppg-rep-MBL gene between the HindIII and XbaI restriction enzyme sites of the vector pCMV3 to construct the plasmid pCMV3-ppg.

[0018] c. Preparation of mannan-binding lectin recombinant protein ppg-rep-MBL.

[0019] According to the instructions of the transfection reagent, calculate the ratio of cells, plasmid and transfection reagent, mix DMEM and plasmid in one sterile EP tube, mix DMEM and PEI transfection reagent in another sterile EP tube, and place at room temperature for 5 minutes. Mix the liquids in the two EP tubes and turn them over to mix them, let them stand at room temperature for 20 minutes, add an appropriate amount of the mixed liquid to the 293T cells to be transfected, and place the cells in a 5% CO2, 37℃ cell culture incubator for 6-8 hours. After 6 hours, add 1% ITS and 60µM / L VC. After continuing to culture for 72 hours, collect the cell supernatant containing ppg-rep-MBL protein, and add PMSF at one thousandth of the supernatant volume. Put the supernatant in a dialysis bag (MD44) with a pore size of 8~12kDa, sprinkle PEG20000 evenly for concentration, and concentrate it to about 1 / 10 of the original volume and take it out. Take 500µl of Mannan-Agarose protein purification column and place it in a 10ml column filter. Then use 15-20 column volumes of ultrapure water, 15 column volumes of TBS-Ca 2+After the buffer is drained, rinse the column with 2 column volumes of TBS-Ca 2+ Resuspend the column material with buffer, mix the ppg-rep-MBL protein sample with the Mannan-Agarose agarose purification column at a ratio of 100 µl column material per 1 ml sample, place the mixture on a vertical mixer, and hang the column at 4°C overnight. The next day, place it in a 10 ml column filter, drain the excess liquid, and then filter with TBS-Ca 2+ The column was washed with buffer to remove impurities. 2+ The ppg-rep-MBL protein was eluted with the eluent and collected in a centrifuge tube. Then, ultrafiltration was performed with an ultrafiltration tube with a pore size of 30 kDa, and centrifuged at 4°C, 4000 g for 10 min, and the liquid inside the ultrafiltration tube was retained. The target protein was obtained by overnight dialyzing with a dialysate of 20 mmol Tris-HCl and 150 mmol NaCl (pH = 7.4), and the dialysate was replaced every 6-8 hours.

[0020] The present invention also provides a DNA molecule, wherein the DNA molecule encodes the mannan-binding lectin modified protein ppg-rep-MBL.

[0021] The present invention also provides an expression vector, which contains the DNA molecule.

[0022] The present invention also provides a host cell, wherein the host cell contains the expression vector.

[0023] The recombinant mannan-binding lectin ppg-rep-MBL of the present invention has low immunogenicity and can inhibit the new coronavirus.

[0024] Studies have reported that (Pro-Hyp-Gly)10 is a self-associating triple-helical peptide with collagen molecular characteristics, and many of its assembly characteristics are similar to those of collagen fiber formation. (Wuding Zhou, Self-association of Collagen Triple Helic Peptides into Higher Order Structures*[J]. Journal of Biological Chemistry, 2006, 281(44): 33-40) CPP(GPP)4GPR(GPP)4GC, CCPP(GPP)5GPR(GPP)5GCC, CCCPP(GPP)5GPR(GPP)5GCCC, CPP(GPP)4GPR(GPP)4GC, YCCPP(GPP)5GPR(GPP)5GCCY, CCPP(GPP)4GPR(GPP)4GCC, CCCPP(GPP)4GPR(GPP)4GCCC are a series of biological functional sequences derived from natural collagen, composed of collagen-like triple helical peptides, with good gelation and rheological properties. Among them, CPP(GPP)4GPR(GPP)4GC, CCPP(GPP)4GPR(GPP)4GCC, and CCCPP(GPP)4GPR(GPP)4GCCC have low inflammatory properties, and CCCPP(GPP)4GPR(GPP)4GCCC causes the lowest inflammatory response, which can be ignored. (Ichise, SF (2019). Development of a collagen-like peptide polymervia end-to-end disulfide cross-linking and its application as a biomaterial. Acta biomaterialia, 94, 361–371. ). The motif sequence GEKGEP in MBL has been shown to enhance the phagocytic function of macrophages in the body by mediating binding to C1qR (Al-Refaei MA, Makki RM, AliH M. Structure prediction of transferrin receptor protein 1 (TfR1) by homology modelling, docking, and molecular dynamics simulation studies[J]. Heliyon, 2020, 6(1): 523-528.).

[0025] Therefore, a protein sequence that does not contain the OGKXGP short motif and can normally form a collagen-like structure is used for replacement. Under the premise of ensuring that the replaced collagen-like structure is similar to the original structure, the binding with the MASP2 protein is interrupted, and the MBL-MASP2 complex cannot be formed to activate the downstream complement components, and ultimately the complement lectin pathway cannot be activated, so that the modified MBL can be used as a drug without overactivating the complement system, causing inflammatory damage or even death. The low immunogenicity MBL protein modified by this strategy has become a breakthrough in its safe medicinal research and development, and this strategy is also a new strategy proposed by the inventor for the first time.

[0026] Compared with the recombinant mannan binding lectin with CRD region modification disclosed in patent application number CN201910560238.2, the mannan binding lectin recombinant protein ppg-rep-MBL of the present invention selects to modify its collagen-like region, so that ppg-rep-MBL does not bind to MSAP2 protein, thereby significantly weakening the activation effect on the complement lectin pathway. Compared with the paper (Effects of mannan binding lectin on human neutrophil function, J South Med Univ, 2013, 33(6): 842-846), the modified protein ppg-rep-MBL of the present invention has a significantly stronger promoting effect on the secretion of TNF-α and IL-6 by PMN stimulated by Candida albicans than MBL, and both can be inhibited by Mannan. Compared with the recombinant rhMBL in the paper (KaurS, Gupta VK, Thiel S, Sarma PU, Madan T. Protective role of mannan-bindinglectin in a murine model of invasive pulmonary aspergillosis. [J]. Clinical and experimental immunology, 2007, 148 (2): 382-9), ppg-rep-MBL cannot bind to MASP2, so the amount of C4b deposited by complement activation is significantly less than that of rhMBL. The present invention tested the binding activity of ppg-rep-MBL to MASP2 protein, S protein and mannan, and found that it did not bind to MASP2 protein, and its activation effect on the complement lectin pathway was weakened. At the same time, it retained its binding ability to mannan and could specifically bind to the S protein sugar chain. The results of the pseudovirus neutralization experiment showed that the modified protein could effectively block the infection of the new coronavirus to HEK293T-ACE2 cells and effectively neutralize the new coronavirus.

[0027] The beneficial effects of the present invention are:

[0028] 1. The modified protein described in the present invention does not bind to the MASP2 protein, thereby significantly weakening the activation effect on the complement lectin pathway, while retaining its binding properties with mannose, and can effectively bind to and block the infection of the new coronavirus pseudovirus and its Delta mutant strain to HEK293T-ACE2 cells.

[0029] 2. The new MBL protein with greatly reduced complement activation ability but unchanged other biological activities has a great role in promoting the treatment of antibacterial, antiviral and immune system diseases. The present invention provides theoretical and technical support for the creation of a new generation of innovative drugs with antiviral and low immunogenicity, and provides basic research support for the direction of reducing the inflammatory response caused by the new coronavirus infection through the complement system. It lays a foundation for the application research of new medicinal mannan-binding lectins with high efficiency and low toxicity and side effects and their possible future MBL deficiency diseases, and provides a candidate for the design of the protein as a broad-spectrum inhibitor or drug against coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The models of the MBL-CLR and ppg-rep peptide segments were constructed using the MODELLER4.0 software in Example 1 (wherein A. homology modeling structure of the trimer of the MBL-CLR region; B. homology modeling structure of the trimer of the ppg-rep region);

[0031] Figure 2 This is a hydrogen bond analysis diagram of the binding surface between MBL-CLR and MASP2 in Example 1;

[0032] Figure 3 This is a hydrogen bond analysis diagram of the binding surface between ppg-rep and MASP2 in Example 1;

[0033] Figure 4 This is a graph showing the concentration of ppg-rep-MBL protein determined by BCA in Example 3;

[0034] Figure 5The SDS-PAGE electrophoresis test result diagram of ppg-rep-MBL in Example 3 (wherein, A. Coomassie Brilliant Blue staining: M1: Hi protein maker; M2: pre-stained maker; lane 1: non-reduced natural MBL; lane 2: reduced natural MBL protein; lane 3: non-reduced ppg-rep-MBL protein; lane 4: reduced ppg-rep-MBL protein; B. Western blot: Maker: pre-stained maker; lane 1: non-reduced ppg-rep-MBL protein; lane 2: non-reduced natural MBL protein; lane 3: reduced ppg-rep-MBL protein; lane 4: reduced natural MBL protein);

[0035] Figure 6 This is a graph showing the binding ability of ppg-rep-MBL protein to different pathogens detected by ELISA in Experimental Example 2;

[0036] Figure 7 The sugar inhibition experiment diagram of ppg-rep-MBL protein in Experimental Example 3 (wherein, A. native MBL protein; B. ppg-rep-MBL protein (***p<0.001, **p<0.01, *p<0.05 VS TBS-Ca2+));

[0037] Figure 8 This is a graph showing the binding ability of ppg-rep-MBL to MASP2 detected by ELISA in Experimental Example 4;

[0038] Fig. 9 This is a graph showing the complement activation ability of ppg-rep-MBL protein detected by ELISA in Experimental Example 5;

[0039] Fig.10 This is a diagram showing the regulatory effect of ppg-rep-MBL protein on PMN phagocytic function in Experimental Example 6;

[0040] Fig.11 This is a diagram showing the regulatory effect of ppg-rep-MBL on cytokine secretion by human peripheral blood neutrophils (PMN) as determined by ELISA in Experimental Example 7 (wherein, A. the effect of ppg-rep-MBL protein on TNF-α secretion by PMN stimulated by Candida albicans; B. the effect of ppg-rep-MBL protein on IL-6 secretion by PMN stimulated by Candida albicans);

[0041] Fig.12The binding diagram of ppg-rep-MBL and the trimeric S protein of SARS-CoV-2 determined by BLI in Experimental Example 8 (wherein, A. Binding analysis of rMBL, ppg-rep-MBL and trimeric S protein (BLI); B. Binding of rMBL, ppg-rep-MBL and trimeric S protein (with / without PNGase F treatment));

[0042] Fig.13 The binding diagram of ppg-rep-MBL and the trimeric S protein of the SARS-CoV-2Delta mutant strain measured by BLI in Experimental Example 8 (wherein, A. Binding analysis (BLI) of rMBL, ppg-rep-MBL and the trimeric S protein of the Delta mutant strain; B. Binding of rMBL, ppg-rep-MBL and the trimeric S protein of the Delta mutant strain (with / without PNGase F treatment));

[0043] Fig.14 This is a graph for evaluating the anti-SARS-CoV-2 pseudovirus activity of rMBL and ppg-rep-MBL in Experimental Example 9 (wherein A. wild type; B. Delta mutant strain). DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0045] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0046] Unless otherwise specified, the materials and reagents used in the following examples were obtained from commercial sources.

[0047] Example 1: Determination of collagen replacement sequence and construction of trimer structure model and evaluation

[0048] (1) First, the sequence of MBL protein was downloaded from NCBI. Based on the artificial collagen sequences reported in the literature, CPP(GPP)4GPR(GPP)4GC, CCPP(GPP)5GPR(GPP)5GCC, CCCPP(GPP)5GPR(GPP)5GCCC, CPP(GPP)4GPR(GPP)4GC, YCCPP(GPP)5GPR(GPP)5GCCY, CCPP(GPP)4GPR(GPP)4GCC, and CCCPP(GPP)4GPR(GPP)4GCCC, MODELLER4.0 and Swiss-Model were used to construct the MBL protein model after replacing the MBL-CLR region sequence with the above sequences, and their structural stability and credibility were evaluated. DOPE is an atomic potential energy statistical model evaluation program that uses the credibility of different conformations of the same complex as the standard. Here, the optimal conformation and score of each structure were selected for comparison. The lower the MolPDFEnergy and DOPE score values, the more credible the results and the more stable the structure. The higher the GMQE and Identity scores, the higher the structural credibility and the closer the modeled structure is to the crystal analysis structure. The results are shown in Table 1. The MolPDFEnergy, DOPE score, GMQE, and Identity of the MBL protein structure after the sequence CCCPP (GPP) 4GPR (GPP) 4GCCC replaced MBL-CLR were the best, that is, the protein structure after the sequence was replaced was the most stable and the credibility was the highest. And the corresponding inflammatory response caused by this sequence has been confirmed by research to be the lowest among the above sequences, so this sequence was selected for subsequent experiments.

[0049] Table 1. Evaluation of protein structure credibility after different collagen-like sequences replaced MBL-CLR

[0050] sequence name Sequence Mol PDF Energy (kcal / mol) Discrete Optimized Protein Energy (DOPEScore) Global Model Quality Evaluation (GMQE) Identity SEQ ID NO. 5 CPP(GPP)4GPR(GPP)4GC 721.61 -6263.27 0.55 52.15 SEQ ID NO.6 CCPP(GPP)5GPR(GPP)5GCC 717.42 -6109.53 0.56 54.32 SEQ ID NO. 7 CCCPP(GPP)5GPR(GPP)5GCCC 749.42 -6461.18 0.57 56.18 SEQ ID NO. 8 CPP(GPP)4GPR(GPP)4GC 805.27 -6719.85 0.53 47.61 SEQ ID NO. 9 YCCPP(GPP)5GPR(GPP)5GCCY 816.67 -6864.20 0.51 46.15 SEQ ID NO. 10 CCPP(GPP)4GPR(GPP)4GCC 690.56 -6018.10 0.63 60.25 SEQ ID NO. 11 CCCPP(GPP)4GPR(GPP)4GCCC 680.24 -5969.61 0.66 72.73

[0051] (2) In order to make the length of the replaced MBL protein amino acid sequence closer to the natural MBL protein amino acid sequence, the PP[GPP]4GPR[GPP]4G sequence was repeated when introducing the above selected collagen sequence, that is, the CCCPP[GPP]4GPR[GPP]4GPP[GPP]4GPR[GPP]4G sequence was introduced, only the three cysteines at the N-terminus were retained, and the three cysteines at the C-terminus were removed. This allowed the N-terminal Cys to cross-link the triple helix through a disulfide bond, while not changing the bonding angle of the C-terminal CRD region of MBL that specifically recognizes sugar residues. In order to ensure the functional integrity of the modified MBL protein, the motif sequence GEKGEP was retained at the C-terminus of the sequence. That is, the MBL-CLR region was replaced with CCCPP[GPP]4GPR[GPP]4GPP[GPP]4GPR[GPP]4GGEKGEP, and the replaced MBL was named ppg-rep-MBL. ppg-rep-MBL includes three parts: the N-terminus, neck and CRD of MBL, and its amino acid sequence is shown in SEQ ID NO:3.

[0052] (3) Further, the protein structures before and after the replacement were modeled and evaluated. Models of MBL-CLR and ppg-rep peptides were constructed using MODELLER4.0, and homology modeling of natural MBL-CLR and ppg-rep peptides was performed using the BLAST program. The corresponding stable trimer structures of MBL-CLR and ppg-rep were constructed using MODELLER4.0, as shown in the following figure. Figure 1 As shown. The ppg-rep protein before and after replacement is similar to most of the MBL-CLR region, both of which have a relatively regular trimeric collagen-like structure. The difference is that the N-terminus of the ppg-rep protein has a section of intertwined structures, some of which are embedded in the protein, and some are exposed on the surface of the ppg-rep protein.

[0053] (4) Hydrogen bond analysis

[0054] In protein complexes, the presence of hydrogen bonds plays a key role in the stability of the complexes. The increase in hydrogen bonds enhances the affinity of the active centers between proteins. The more hydrogen bonds there are, the more stable the complex is. In order to evaluate the stability of the simulation system, the number of hydrogen bonds between MASP2 and MBL-CLR and ppg-rep at the binding interface was analyzed respectively.

[0055] The protein amino acid residues and bond lengths involved in hydrogen bond formation at the junction of MASP2 protein and MBL-CLR and ppg-rep peptides are listed in Table 1. It is generally believed that a bond length of 2.4 to 2.7 Å is a strong hydrogen bond, 2.7 to 3.2 Å is a medium-strong hydrogen bond, and a bond length greater than 3.2 Å is considered a weak hydrogen bond. Figure 2 It can be seen that there are 2 strong hydrogen bonds, 3 medium-strong hydrogen bonds, and 2 weak hydrogen bonds between the amino acid residues at the binding interface between MASP2 protein and MBL-CLR complex. Figure 3 At the binding interface between MASP2 and ppg-rep, there are only three weak hydrogen bonds larger than 3.2Å, and no strong hydrogen bonds or medium-strong hydrogen bonds. From this result, it can be determined that the stability of the ppg-rep-MASP2 complex is weaker than that of the MBL-CLR-MASP2 protein complex.

[0056] Example 2: Construction and expression of purified ppg-rep-MBL

[0057] Step 1. Transform the ppg-rep-MBL plasmid constructed by the company (synthesized by Beijing Qingke Biotechnology Co., Ltd.) into the competent E. coli DH5α, pick a single clone and use sequencing technology to identify the correctness of the recombinant plasmid. The sequencing work was completed by Beijing Qingke Biotechnology Co., Ltd. Select the verified clone to amplify, culture and extract the plasmid for use.

[0058] Step 2. Use 293T cells to secrete and express ppg-rep-MBL protein. Insulin-transferrin-Selenium (ITS) at a concentration of 1% and ascorbic acid (vitamin C) at a concentration of 60µM / L were added to the DMEM medium to make the expressed protein polymer structure more stable. According to the instructions of the transfection reagent, the ratio between cells, plasmid and transfection reagent was calculated, and the plasmid in step 1 was transfected into 293T cells. After that, the cells were placed in a 5% CO2, 37°C incubator for 72h. The cell supernatant containing ppg-rep-MBL protein was collected, and PMSF was added at 1 / 1000 of the supernatant volume, and it was temporarily stored at 4°C.

[0059] Step 3. The supernatant collected in step 2 was placed in a dialysis bag (MD44) with a pore size of 8-12 kDa, and PEG20000 was evenly sprinkled on it for concentration. The concentrate was concentrated to about 1 / 10 of the original volume and then taken out. The concentrate was separated and purified using a Mannan-Agarose agarose protein purification column (10 ml), washed with 15-20 times ultrapure water and 15 times column volume of TBS-Ca 2+After washing the column with buffer, use 2 column volumes of TBS-Ca 2+ Resuspend the column material with buffer and mix the ppg-rep-MBL protein sample with the Mannan-Agarose agarose purification column at a ratio of 100µl column material per 1ml sample. Place the protein and column material mixture on a vertical mixer and hang it on the column at 4℃ overnight. The next day, take out the mixture and add it to a 10ml column filter. Allow the excess liquid to flow out of the filter column by gravity and then use TBS-Ca 2+ The column was washed with buffer to remove impurities. 2+ The ppg-rep-MBL protein was eluted with the eluent and collected in a centrifuge tube. The eluent was then ultrafiltered with an ultrafiltration tube with a pore size of 30 kDa, centrifuged at 4°C, 4000g for 10 min, and the liquid inside the ultrafiltration tube was retained. The ultrafiltration liquid was dialyzed overnight in a dialysate of 20 mmol Tris-HCl, 150 mmol NaCl (pH = 7.4), and the dialysate was replaced every 6-8 hours to finally obtain the target protein.

[0060] Example 3: Detection and concentration determination of purified ppg-rep-MBL

[0061] The ppg-rep-MBL protein concentration was determined using the Biotech BCA kit, and the protein integrity was tested using SDS-PAGE and Western Blot.

[0062] The results are as follows Figure 4 As shown, according to the standard curve formula, the concentrations of ppg-rep-MBL and native MBL proteins were measured to be 2.27 mg / ml and 1.39 mg / ml, respectively. Figure 5As shown in the figure, according to the results of Coomassie Brilliant Blue staining, under non-reducing conditions, the relative molecular mass (Mr) of the purified ppg-rep-MBL and natural MBL proteins is greater than 460kD, and they mainly exist in the form of pentamers and above oligomers. The remaining proteins have obvious protein bands near 32kD and 70kD, which exist in the form of ppg-rep-MBL and natural MBL single chains or double chains; under reducing conditions, the two proteins exist in the form of single peptide chains or double peptide chains near 32kD and 70kD; Western blot analysis shows that under non-reducing conditions, the molecular weight of some proteins is greater than 460kD and cannot be successfully transferred to the PVDF membrane. It can be seen that under non-reducing conditions, corresponding protein bands appear near 32kD and 96kD in lanes 1 and 2, which are ppg-rep-MBL and 96kD, respectively. The protein and the natural MBL protein exist in the form of single chain or three chains. Under reducing conditions, the ppg-rep-MBL protein and the natural MBL protein both exist in the form of single chain.

[0063] The beneficial effects of the present invention are demonstrated by the following efficacy tests.

[0064] Test Example 1: Yeast agglutination test to detect the agglutination ability of ppg-rep-MBL protein

[0065] The fresh yeast was centrifuged, washed 3 times with sterile TBS and resuspended to adjust the density of the yeast to 1 × 10 10 cfu / ml, heat inactivated at 65℃ for 1h, cooled and used. 2+ Dilute native MBL protein and ppg-rep-MBL to 20µg / ml, add to the agar plate in multiple dilutions, and mix with yeast. 2+ As negative control, react at room temperature for 1 hour, observe under a microscope and record the results. The judgment criteria are as follows:

[0066] (1) Yeast has no aggregation, which is the same as the negative control and is recorded as (-);

[0067] (2) 2 to 3 yeasts aggregated, recorded as (+ / -);

[0068] (3) 3 to 5 yeasts agglutinated, recorded as weak positive (+);

[0069] (4) 5 to 10 yeasts agglutinated, which was recorded as positive (++);

[0070] (5) There are 10 to 20 dense yeast agglomerates, which is recorded as (+++);

[0071] (6) Large agglomerations of more than 20 densely packed yeast cells are marked as (++++).

[0072] Table 2. Yeast agglutination assay to analyze the binding activity of ppg-rep-MBL with native MBL protein

[0073]

[0074] The agglutination effect of purified ppg-rep-MBL and natural MBL proteins was compared and analyzed. The results are shown in Table 2. The agglutination titer of purified ppg-rep-MBL protein was 1:512, and the agglutination titer of natural MBL protein was 1:512. At this time, the concentrations of both proteins were 0.039µg / ml. The experimental results show that the minimum concentration of purified ppg-rep-MBL protein causing agglutination is 0.039µg / ml. That is, when the concentration of ppg-rep-MBL protein is less than 0.039µg / ml, the protein no longer agglutinates yeast. Compared with natural MBL protein, the minimum agglutination concentration of modified ppg-rep-MBL protein is the same as that of natural MBL protein, which roughly reflects that the activity of ppg-rep-MBL protein is not affected before and after modification.

[0075] Experimental Example 2 ELISA to detect the binding ability of ppg-rep-MBL protein to different pathogens

[0076] (1) Take the logarithmic growth of Candida albicans, yeast, and Escherichia coli and wash them three times with PBS to adjust the pH to 10. 8 cfu / ml, serially diluted and added to the ELISA plate, 100 µl / well, coated at 4°C for 12 h;

[0077] (2) Aspirate the liquid in each well and wash with TBS-T-Ca 2+ Wash, 300 µl / well, repeated 3 times;

[0078] (3) Drain the detergent and absorb the remaining liquid on clean absorbent paper;

[0079] (4) Allow the plate to air dry at room temperature, then add 100 µl / well of 4% paraformaldehyde and fix at room temperature for 30 min.

[0080] (5) Repeat steps 2-3, add blocking solution, block for 1 h at room temperature, 300 µl / well;

[0081] (6) Repeat steps 2-3 and use TBS-Ca 2+ Dilute ppg-rep-MBL protein to 20 µg / ml per well and incubate at room temperature for 2 h;

[0082] (7) Repeat steps 2-3, then add 100 µl / well of 1:2000 rabbit anti-human MBL monoclonal antibody (diluted in TBS buffer) and use the dilution as a blank control. Cover with tape and incubate at room temperature for 2 h.

[0083] (8) Repeat steps 2-3, then add 100 µl / well of 1:5000 diluted HRP-goat anti-rabbit (diluted in TBS buffer) and incubate at room temperature for 20 min. Avoid placing the plate in direct light.

[0084] (9) Repeat steps 2-3; add a 1:1 mixture of A and B in the ELISA universal kit, 100 µl / well, and react at room temperature in the dark for 20 min;

[0085] (10) Add 50 µl of reaction stop solution to each well and tap the plate gently to ensure thorough mixing;

[0086] (11) Measure the A450nm value on an ELISA reader within 15 min.

[0087] The results are as follows Figure 6 , ppg-rep-MBL protein can effectively bind to pathogens. However, the binding ability with the three bacteria is different. Among them, ppg-rep-MBL protein binds Candida albicans and yeast more strongly than Escherichia coli. This is because Candida albicans and yeast cell walls contain a large amount of mannan components, while Escherichia coli only has flagella that adsorb a small amount of mannan in the culture medium, so the binding ability between Escherichia coli and ppg-rep-MBL protein is weaker than that of the other two bacteria.

[0088] Experimental Example 3: Sugar inhibition experiment of ppg-rep-MBL protein

[0089] (1) Dilute Mannan to 20 µg / ml in coating solution, add to ELISA plate, 100 µl / well, and coat at 4°C for 12 h.

[0090] (2) Aspirate the liquid in each well and wash with TBS-T-Ca 2+ Wash, 300 µl / well, repeat 3 times;

[0091] (3) Drain the detergent and absorb the remaining liquid on clean absorbent paper;

[0092] (4) Add blocking solution and block at room temperature for 1 h, 300 µl / well;

[0093] (5) Repeat steps 2-3, adding 100 µl of 20 µg / ml purified ppg-rep-MBL and native MBL protein respectively. The experimental group diluent contains only TBS-Ca 2+ , the control group dilution solution contained 20mmol / LEDTA, mannose, mannotriose, and mannopentaose at a concentration of 20μg / ml, respectively, and incubated at room temperature for 1.5h;

[0094] (6) Repeat steps 2-3, then add 100 µl / well of 1:2000 rabbit anti-human MBL monoclonal antibody (diluted in TBS buffer) and use the dilution as a blank control. Cover with tape and incubate at room temperature for 2 h.

[0095] (7) Repeat steps 2-3, then add 100 µl / well of 1:5000 diluted HRP-goat anti-rabbit (diluted in TBS buffer) and incubate at room temperature for 20 min. Avoid placing the plate in direct light.

[0096] (8) Repeat steps 2-3; add 100 µl / well of the 1:1 mixture of A and B in the ELISA universal kit and react at room temperature in the dark for 20 min;

[0097] (9) Add 50 µl of reaction stop solution to each well and tap the plate gently to ensure thorough mixing;

[0098] (10) Measure the A450nm value on an ELISA reader within 15 min.

[0099] The results are as follows Figure 7 ,exist Figure 7 In A, native MBL protein can effectively bind to mannan (TBS-Ca 2+ In the other experimental groups, due to the presence of sugars such as manno-3-ose and manno-5-ose in the respective reaction systems, they can compete with mannan to bind to the sugar recognition region (CRD) of the natural MBL protein, thereby inhibiting the binding of mannan to the natural MBL protein to varying degrees. Among them, the EDTA group was set up because the natural MBL protein is a Ca2+-dependent protein. EDTA was added to the diluent to chelate Ca2+. 2+ , making the Ca-dependent 2+ The CRD site activity of α-MLB was lost, thus inhibiting the binding of native MBL protein to mannan. Figure 7In B, the binding of the modified ppg-rep-MBL protein to mannan was also inhibited to varying degrees, and the inhibition trend was similar to that of the natural MBL protein, which fully demonstrated that the binding activity of the modified ppg-rep-MBL protein to different sugars was close to that of the natural MBL protein, and the modification of the MBL collagen-like region did not significantly affect its sugar binding activity.

[0100] Experimental Example 4: ELISA to detect the binding ability of ppg-rep-MBL to MASP2

[0101] (1) Dilute the purified ppg-rep-MBL protein and native MBL protein from 20 µg / ml to 0.625 µg / ml (TBS-Ca2+ dilution), add to the ELISA plate, 100 µl / well, and coat at 4°C for 12 h. Set the uncoated well as a blank control.

[0102] (2) Aspirate the liquid in each well and wash with TBS-T-Ca2+, 300 µl / well, repeat washing 3 times;

[0103] (3) Drain the detergent and absorb the remaining liquid on clean absorbent paper;

[0104] (4) Add blocking solution and block at room temperature for 1 h, 300 µl / well;

[0105] (5) Repeat steps 2-3, dilute MASP2 protein to 20 µg / ml with TBS-Ca2+, 100 µl / well, and incubate at room temperature for 2 h;

[0106] (6) Repeat steps 2-3, add 100 µl / well of 1:2000 rabbit anti-human MASP2 monoclonal antibody (diluted in TBS buffer), and incubate at room temperature for 2 h;

[0107] (7) Repeat steps 2-3, then add 100 µl / well of 1:5000 diluted HRP-goat anti-rabbit (diluted in TBS buffer) and incubate at room temperature for 20 min. Avoid placing the plate in direct light.

[0108] (8) Repeat steps 2-3; add 100 µl / well of the 1:1 mixture of A+B in the ELISA universal kit, and react at room temperature in the dark for 20 min. Add 50 µl of the reaction stop solution to each well, tap the plate gently to ensure thorough mixing, and measure the A450nm value on a microplate reader within 15 min.

[0109] The graph was drawn using GraphPad Prism 6. The results are as follows: Figure 8 As shown, ppg-rep-MBL no longer has the ability to bind to MASP2 compared with rMBL.

[0110] Experimental Example 5: ELISA to detect the complement activation ability of ppg-rep-MBL protein

[0111] (1) Add 20 µg / ml Mannan to the ELISA plate, 100 µl / well, and coat at 4°C for 12 h. Set the uncoated well as a blank control.

[0112] (2) Aspirate the liquid in each well and wash with TBS-T-Ca 2+ Wash, 300 µl / well, repeat this process 3 times;

[0113] (3) Drain the detergent and absorb the remaining liquid on clean absorbent paper;

[0114] (4) Add blocking solution and block at room temperature for 1 h, 300 µl / well;

[0115] (5) Repeat steps 2-3 and use TBS-Ca 2+ Purified ppg-rep-MBL and native MBL proteins were diluted serially from 20 µg / ml to 0.625 µg / ml and incubated at room temperature for 2 h;

[0116] (6) Repeat steps 2-3, add 1:5 diluted MBL-deficient serum, and incubate at 37°C for 1 h.

[0117] (7) Repeat steps 2-3, add 100 µl / well of 1:3000 rabbit anti-human C4b monoclonal antibody (diluted in TBS buffer), use the dilution as a blank control, and incubate at room temperature for 2 h.

[0118] (8) Repeat steps 2-3, then add 100 µl / well of 1:5000 diluted HRP-goat anti-rabbit (diluted in TBS buffer), cover the plate and incubate at room temperature for 20 min, avoiding direct light.

[0119] (9) Repeat steps 2-3, add a 1:1 mixture of A and B, 100 µl / well, and react at room temperature in the dark for 20 min.

[0120] (10) Add 50 µl of stop solution to each well and tap the plate gently to ensure thorough mixing.

[0121] (11) Detect the A450nm value using an ELISA reader.

[0122] The results are as follows Fig. 9As shown in the figure, the natural MBL protein can bind to the mannan in the system and then bind to the MASP2 protein in the serum, activating MASPs and causing C4 to be cleaved to form C4b, thereby activating complement. However, ppg-rep-MBL cannot activate MASPs because it cannot bind to the MASP2 protein. Finally, the ability of ppg-rep-MBL protein to activate complement is significantly reduced (p < 0.001).

[0123] Experimental Example 6: Regulatory Effect of ppg-rep-MBL Protein on PMN Phagocytic Function

[0124] Step 1: Preparation of fluorescein-labeled bacteria

[0125] (1) Take logarithmic phase Candida albicans and adjust to 1x10 9 cfu / ml.

[0126] (2) Resuspend and wash three times in TBS, resuspend in FITC solution dissolved in carbonate buffer, wrap in tin foil, and place in a 37℃ incubator for 40 minutes.

[0127] (3) Wash three times with TBS and fix with 4% paraformaldehyde for 30 min.

[0128] (4) Wash twice with TBS, resuspend in TBS to a certain concentration, and store at 4°C for later use.

[0129] Step 2: Isolation of human peripheral blood neutrophils (PMNs)

[0130] During the entire process, samples, reagents and experimental environment must be kept at a temperature of 25±2℃.

[0131] (1) Add 8 ml of PMN isolation solution to a sterile siliconized centrifuge tube.

[0132] (2) Aspirate the blood and gently drip it onto the surface of the separation liquid. Centrifuge at 450g for 30 minutes.

[0133] (3) Two layers of ring-shaped white cells appear in the tube. The upper layer is monocytes, and the lower layer of PMN cells is absorbed.

[0134] (4) Add the aspirated PMNs to 8 ml of washing solution and resuspend the cells.

[0135] (5) Centrifuge at 300g for 15 min, discard the supernatant, resuspend the cells in 6 ml of washing buffer, and centrifuge at 300g for 15 min.

[0136] (6) If there are remaining red blood cells, add red blood cell lysis solution and wait for 3 minutes.

[0137] (7) Centrifuge at 300g for 15 min, discard the supernatant, and repeat steps 7-8 until no red blood cells are visible to the naked eye.

[0138] (8) Wash 1-2 times: Add 2 ml of 1× PBS to resuspend the cells, centrifuge at 350 g for 6 min, and discard the supernatant.

[0139] (9) Resuspend the cells in neutrophil culture medium and count the cells.

[0140] (10) After counting PMNs, place the cells in a 37°C, 5% CO2 cell culture incubator.

[0141] (11) Trypan blue staining was used to verify the activity of PMNs, Wright-Giemsa staining was used to observe the cell morphology, and flow cytometry was used to analyze the purity of PMNs.

[0142] Step 3: Take FITC-labeled Candida albicans, set up a mixed human serum group, a natural MBL protein group, and a ppg-rep-MBL protein group, and mix them with MBL-deficient serum in a certain proportion, incubate at 37°C, 5% CO2 in the dark for 30 minutes, and then add PMN (1×10 6 The cells were then washed with TBS for 3 times, centrifuged at 600 g for 10 min, and fixed with an equal amount of 4% paraformaldehyde. The phagocytic effect was detected by flow cytometry, and the results were analyzed using FlowJo software. Fig.10 As shown, in the group with only mixed human serum added, the phagocytosis rate of neutrophil (PMN) to Candida albicans was only 12.2%, while the phagocytosis rate of the group with natural MBL protein added was 25.6%, and the phagocytosis rate of the group with ppg-rep-MBL protein added was 23.5%. Compared with the mixed human serum group without any protein added, it can be clearly observed that ppg-rep-MBL protein, like natural MBL protein, has a significant promoting effect on PMN phagocytosis of Candida albicans, indicating that the modified ppg-rep-MBL protein still has the function of promoting PMN phagocytosis of pathogens.

[0143] Experimental Example 7: ELISA was used to detect the regulatory effect of ppg-rep-MBL on cytokine secretion by human peripheral blood neutrophils (PMN).

[0144] Take 1×10 5 cfu / ml Candida albicans and 4×10 5PMN cells were mixed in three groups and inoculated in 96-well plates. MBL-deficient serum with a final concentration of 5% was added. One group was added with ppg-rep-MBL protein at concentrations of 0, 2.5, 5, 10, and 20 μg / ml, and the content of cytokine TNF-α was determined; the other two groups were added with ppg-rep-MBL protein at concentrations of 0, 2.5, and 20 μg / ml, and 100 μg / ml Mannan was added to the 20 μg / ml ppg-rep-MBL protein for stimulation. Three replicates were set up, 200 μl / well, and the content of cytokines TNF-α and IL-6 was determined. The cells were placed in a cell culture incubator and cultured for 24 hours.

[0145] ELISA detection of cytokines secreted by PMN: Collect the cell supernatant, centrifuge at 2500g for 18 minutes, and collect the supernatant in a new centrifuge tube. Use ELISA kits for human tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to detect the content of cytokines in the PMN culture supernatant.

[0146] (1) Standards: Set up standard wells and add 50 µl of serially diluted standard to each well;

[0147] (2) Sample addition: Add 10 µl of cell supernatant and 40 µl of sample diluent to the ELISA plate and mix;

[0148] (3) Add enzyme labeling reagent: add 100 µl to each well, and leave blank wells without adding any reagent.

[0149] (4) Incubation: Seal the 96-well plate with a sealing film and incubate at 37°C for 60 min.

[0150] (5) Washing solution: dilute 20× washing solution to 1×;

[0151] (6) Washing: Pour out the liquid, spin dry, add 300 µl of washing solution, pour out after 30 seconds, and repeat 5 times;

[0152] (7) Color development: Add a 1:1 mixture of A and B, 100 µl / well, and react at room temperature in the dark for 20 min.

[0153] (8) Stop: Add 50 µl of stop solution, the blue color will turn to yellow immediately;

[0154] (9) Determination: Determine the absorbance at 450 nm within 10 min after adding the stop solution;

[0155] (10) Establish a standard curve and calculate the content of cytokines in the sample.

[0156] The graph was drawn using GraphPad Prism 6. The results are as follows Fig.11 As shown, ppg-rep-MBL plays a dual role in the state of Candida albicans infection. Low concentration of ppg-rep-MBL has a significant promoting effect on the secretion of TNF-α and IL-6 by PMN cells; high concentration of ppg-rep-MBL inhibits the secretion of these two cytokines by PMN, and this inhibitory effect can be blocked by Mannan.

[0157] Experimental Example 8: BLI detection of the binding ability of ppg-rep-MBL to SARS-CoV-2 Spike

[0158] The affinity of ppg-rep-MBL to S protein was detected by biolayer interferometry (BLI). Affinity detection was performed using ForteBio Octet RED 96e and K2 system (Pall ForteBio, USA). Ni-NTA biosensor (Pall ForteBio, USA) was used for quantitative and kinetic characterization of his-tagged biomolecules. The sensor was pre-wetted in dialysis buffer for 15 minutes before use. Trimeric S protein (S_Trimer and S_Trimer_Delta) and sugar-free trimer S protein (S_Trimer ' and S_Trimer_Delta ') formed after co-incubation with PNGase F at 37°C (20μg protein / 1μL PNGase F, PBS 7.4) were loaded and fixed on the surface of the biosensor tip, and the signal value of each group was fixed at 3.0 nm (equilibrium: 100 s; dissociation: 300 s). ppg-rep-MBL at a concentration of 1 μM was dissolved in PBS buffer (0.02% Tween 20). The biosensor loaded with trimeric S protein was applied to the following procedures: equilibration (PBS 7.4 + 0.02% Tween 20, 100 s), binding (1 μM ppg-rep-MBL, PBS 7.4 + 0.02% Tween 20, 180 s), and dissociation (PBS 7.4 + 0.02% Tween 20, 300 s).

[0159] The binding or dissociation of the soluble ppg-rep-MBL-trimeric S protein complex on the biosensor tip surface causes a mode shift in the reflected interference wave, which is represented as a sensorgram. The entire experiment was performed in a black 96-well microplate (Greiner Bio-One, Austria) at 30 °C, 500 rpm, and a working volume of 200 µL per well. The S protein-loaded sensor was added to PBS buffer as a control to correct the baseline drift. The response data were normalized using Octet data analysis software (version 11.1.2.9).

[0160] The results are as follows Fig.12 , Fig.13 As shown in the figure, compared with rMBL, ppg-rep-MBL has a slightly weaker binding ability to the trimeric S protein of SARS-CoV-2 and its Delta mutant, but it can still effectively bind to the S protein. And it cannot bind to the S protein after the sugar chain is removed after PNGase treatment, indicating that ppg-rep-MBL exerts its binding effect by binding to the sugar chain on the surface of the S protein, that is, ppg-rep-MBL can effectively bind to its surface sugar chain.

[0161] Experimental Example 9: Neutralization Activity Detection of ppg-rep-MBL against SARS-CoV-2 Pseudovirus

[0162] (1) 293T cells stably transfected with ACE2 plasmid (293T-ACE2 cells) were digested with trypsin and resuspended in complete culture medium [DMEM medium (Gibco) supplemented with 0.1µg / mL streptomycin (Thermo), 0.06µg / mL penicillin (Thermo), and 10% w / v fetal bovine serum (ExCell Bio)] and counted, and diluted to a concentration of 1x10 5 / mL, with a density of 1×10 4 Cells / well were seeded in 96-well cell culture plates (Corning) and cultured at 37°C.

[0163] (2) Use double-free DMEM to dilute the protein samples (rMBL, ppg-rep-MBL) to a concentration gradient of 0.005-500 µg / ml; dilute ACE2 protein with double-free DMEM to a final protein concentration of 600ug / mL (about 4.6µM), and filter with a 0.22 µm filter for later use. Use double-free DMEM to dilute SARS-CoV-2 pseudovirus of equal titer (including the original strain and Delta mutant strain, both purchased from Genomeditech), add 0.08ul pseudovirus to each well, and add 50ul pseudovirus-DMEM mixture to each well.

[0164] (3) The diluted pseudovirus was mixed with the protein sample to be tested / ACE2 protein at a ratio of 1:1, and the diluted pseudovirus was mixed with double-free DMEM at a ratio of 1:1 (negative control group), and incubated at 37°C for 1 h; then, the protein sample to be tested / pseudovirus mixture was added to HEK293T-ACE2 cells, and fresh complete DMEM medium was replaced after 24 h of infection.

[0165] (4) After incubation at 37 °C for 48 h, the luciferase activity in HEK293T-ACE2 cells was measured using the One-LumiTM II Firefly Luciferase Assay Kit (Beyotime) and the values ​​were read using a fluorescence microplate reader. Finally, the neutralization curve was drawn using GraphPad Prism 6 and the half inhibitory concentration (IC50) value was calculated. The results are shown in Fig.14 , although compared with the inhibitory effect of rMBL on the new coronavirus pseudovirus and Delta mutant pseudovirus (IC 50 7.02µg / ml and 4.398 µg / ml), the inhibitory effect of ppg-rep-MBL was reduced (IC 50 22.91µg / ml and 18.78µg / ml), but it can still effectively neutralize the new coronavirus pseudovirus and Delta mutant pseudovirus.

Claims

1. Use of mannan-binding lectin modified protein ppg-rep-MBL in the preparation of drugs for treating coronavirus infection; The amino acid sequence of the mannan binding lectin modified protein ppg-rep-MBL is shown in SEQ ID NO:3, and the nucleotide sequence is shown in SEQ ID NO:4; The drug described is a drug that blocks SRAS-CoV-2 infection.

2. The use according to claim 1, characterized in that: The SARS-CoV-2 includes the original strain and the Delta mutant strain.

3. The use according to claim 1 or 2, characterized in that: The preparation method of the mannan binding lectin modified protein ppg-rep-MBL comprises the following steps: a. Using the wild-type mannan binding lectin amino acid sequence SEQ ID NO: 1 as a template and the nucleotide sequence SEQ ID NO: 2, a recombinant mannan binding lectin ppg-rep-MBL amino acid sequence was constructed; the MBL-CLR region of the wild-type mannan binding lectin amino acid sequence was replaced with CCCPP[GPP]4GPR[GPP]4GPP[GPP]4GPR[GPP]4GGEKGEP; the ppg-rep-MBL gene was designed and synthesized, the amino acid sequence was SEQ ID NO. 3, and the nucleotide sequence was SEQ ID NO. 4; b. Construction of modified mannan-binding lectin recombinant plasmid: insert the ppg-rep-MBL gene between the HindIII and XbaI restriction enzyme sites of the vector pCMV3 to construct the plasmid pCMV3-ppg; c. Preparation of mannan-binding lectin recombinant protein ppg-rep-MBL.

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