RU2350654C2 - Method of mass production of multimeasured mannan-binding lectine - Google Patents

Abstract

FIELD: pharmacology; biotechnology.

SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.

EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.

6 cl, 11 dwg, 9 ex

Description

Technical field

The present invention relates to the development of a method by which mass production of a multimeric recombinant mannose-binding human lectin (rhMBL) is possible.

BACKGROUND OF THE INVENTION

Mannose-binding lectin (hereinafter referred to as “MBL”) is a serum protein that is an element of innate immunity (genetically determined immunity). The molecular weight of MBL is 32 kDa. MBL consists of a C-terminal carbohydrate recognition domain (CRD), a collagen domain, and a cysteine-rich region at the N-terminus. Three MBL molecules form a complex through the formation of a triple helix using collagen domains, and then up to 6 complexes can be combined together via an intermolecular disulfide bond (-ss-) between cysteine residues at the N-terminus, leading to the formation of a multimeric MBL molecule consisting of up to 18 MBL molecules.

The oligomeric form of MBL can form a complex with other proteins, such as MBL-associated serine proteases (MBSP-1, MASP-2 or MASP -3) or an MBL-associated protein (Map19). Although the overall molecular structure and function of the MBL involved in complement activation is similar to C1q, which is the first component of the complement system, unlike C1q, the MBL activates the complement system, splitting C4 and C2. This complement activation process, called the lectin-dependent pathway, is carried out using the MBL-associated serine protease, which is activated by binding MBL to the microorganism. Activation of the complement system via MBL is one of the functions of MBL in the primary defense against microbial infection before an adaptive immune response appears. The binding of MBL to the microorganism via carbohydrate recognition domains (CRD), which recognize unique glycosylation patterns on the surface protein of the microorganism, is a necessary condition for complement activation. The binding of MBL to the microorganism activates the MBL-associated precursors of serine proteases, MASP-1 and MASP-2, leading to the cleavage of the C4 and C2 complement system, leading to the formation of C4b2a and C3b. Some of the proteins produced by complement activation directly attach to the surface of the microorganism as opsonin, and MBL can also function as opsonin to more efficiently carry out the phagocytosis of MBL-associated microorganisms by phagocytes. Some activated complement proteins directly lyse penetrating microorganisms to form a membrane-attacking complex (MAC), and other protein fragments of complement obtained during the activation process contribute to the release of the cytokine, which causes inflammation.

Previous reports have described the expression of the MBL gene in various cells, such as CHO cells, HLF hepatoma cells, or HEK293-EBNA cells. In particular, the expression level in CHO cells was very high, but the MBL produced was mainly monomers or dimers (Katsuki Ohtani et al., J. Immunol. Methods, 222: 135-144, 1999). In the HEK293-EBNA cells, the oligomeric form of MBL was produced, but the total productivity was less than 1 μg / ml (T. Vorup-Jensen et al, International Immunopharmacology, 1: 677-687, 2001). The production of the oligomeric form of MBL is extremely important, as monomers and dimers consisting of 3 subunits and 6 subunits, respectively, have small, if any, activities. Another important issue in the development of a pharmaceutical product from MBL is to obtain a super-producer cell line, since it is needed in the amount of several milligrams.

MBL can also play an important role in adaptive immunity. Cells involved in phagocytosis, such as neutrophils and macrophages, play an important role in the removal of penetrating foreign microorganisms through phagocytosis. In addition, the macrophage is an antigen presenting cell (APC) that activates T cells, leading to the induction of an acquired immune response. Therefore, multimeric MBL, involved in the activation of the complement system as opsonin, plays an important role not only in primary defense as a component of the innate immune system, but also in the induction of acquired immune responses.

The amount of MBL in human serum varies depending on the individuals, ranging from 50 ng / ml to several μg / ml due to the genetic variation of the MBL gene. For example, if a point mutation occurs in codon 52, 54 or 57 of exon 1 of the MBL gene, the MBL molecules do not form oligomeric forms, leading to the appearance of non-functional smaller complexes consisting of no more than 3 MBL molecules. If the mutation occurs in the promoter regions of the MBL gene, the level of expression of MBL is reduced, leading to a significant change in the level of MBL in the blood among affected people. Basically, those infants or patients with neutropenia who have an MBL level below a certain level have weak immunity, becoming highly susceptible to microbial infections (Sumiya, M. et al., Lancet, 337: 1569-1570, 1991; Summerfield, JA et al., Lancet, 345: 886, 1995; Garred, P. et al., Lancet, 346: 941, 1995; Summerfield, J. et al., Br. Med. J., 314: 1229, 1997; Mullighan, CG et al., Scand. J. Immunol, 51: 111-122, 2000; Nefh, O. et al., Lancet, 358: 614-618, 2001; Peterslund, NA et al., Lancet, 358: 637-638, 2001; Mullighan CG et al., Blood, 99: 3524-3529, 2002).

According to a study of patients with chronic hepatitis B virus, the level of MBL in the blood is an important factor in predicting the development of the disease (Hakozaki Y. et al., Liver 22, 29-34, 2002). In particular, among patients who developed transient liver failure during chronic hepatitis B virus infection, the mortality rate in patients with blood MBL levels above 3 μg / ml was 0%, while in patients with blood MBL levels was 1.5 μg / ml and below 0.5 μg / ml, mortality was 58% and more than 80%, respectively. This study shows a significant relationship between blood MBL and mortality in patients with an infectious disease. This study also suggests that adding MBL to patients with low or no MBL may be beneficial.

In order for the recombinant MBL to be used as a therapeutic agent, the recombinant MBL protein must be produced in large quantities at a reasonable price in the form of active oligomeric molecules. Unlike cytokines, it is needed in the amount of several milligrams for the patient at each dose. For example, human blood volume averages about 5 liters, which requires 25 mg to produce 5 μg / ml MBL. Considering the volume of other body fluids, a much larger amount may be required at each dose. Therefore, it was an extremely important problem in developing a commercial MBL product that not only a recombinant superproducer recombinant cell line is created, but also a cell line that produces active forms of polymer MBL.

Thus, the authors of the present invention made every effort to create a recombinant cell line that could produce MBL on an industrial scale and, at the same time, that could produce a functionally active oligomeric form of MBL that was able to activate the complement system by specifically binding with MBL binding glycoproteins in the presence of MASP. As a result of these efforts, the inventors of the present invention prepared the invention, stating that active rhMBL suitable for therapeutic use can be produced on an industrial scale in the form of a multimeric polymer in a CHO cell line transfected with a cloning vector containing a polynucleotide encoding the rhMBL sequence.

Description of drawings

Figure 1 is a schematic diagram showing the genetic map of the plasmid vector pMSG-MBL.

2A and 2B are images of MBL produced by the recombinant CHO cell line, CHO MBL / D1-3, which is transformed with pMSG-MBL. Fig. 2A is a view of an SDS-PAGE assay, and Fig. 2B shows a Western blot assay using an MBL-specific monoclonal antibody.

Figure 3 is an electron microscope photograph showing oligomeric forms of MBL. The oligomeric MBL molecules consisting of 2, 3, 4, 5, and 6 trimeric subunits of MBL are clearly visible.

Figure 4 is an electron microscope photograph showing a complex of recombinant MBL protein and a gold nanoparticle coated with pre-S in the presence of a calcium ion.

FIGS. 5A and 5B show that rhMBL specifically binds to pre-S, glycoprotein, or mannan with activity quantitatively similar to natural MBL.

Figure 6 shows the relative binding capacity of large polymeric forms of MBL and smaller forms, consisting mainly of monomers and dimers of three-dimensional subunits. Smaller forms have a small, if any, binding ability.

7 shows a comparison of the ability to activate C4 large forms and smaller forms of rhMBL.

On Fig shows that rhMBL specifically binds to various microorganisms.

Figure 9 shows that rhMBL inhibits the infection of fetal monkey kidney cells (FRhK-4) SARS-CoV.

10 is a series of phase contrast microscope images showing FRhk-4 cells infected with SARS-CoV. Without rhMBL treatment, cells look unhealthy, while healthy cells are present with MBL.

11 is a schematic view showing a diagnostic kit obtained using recombinant human MBL.

Description

The purpose of the invention

An object of the present invention is to construct a CHO cell line that is transfected with a vector containing the human MBL polynucleotide coding sequence to produce a large quantity of the functionally active oligomeric form of MBL, which will be used commercially.

Additionally, the purpose of the present invention is to provide a method for purification of rhMBL from the culture medium of the recombinant CHO cell line.

SUMMARY OF THE INVENTION

The present invention provides an expression vector pMSG-MBL containing the sequence of the coding region of rhMBL presented on the map of the plasmid in figure 1.

The present invention also provides a host cell line transfected with an expression vector.

The present invention further provides a method for mass production of multimeric recombinant MBL in accordance with the following steps:

(1) obtaining host cells transfected with an expression vector containing a sequence encoding a human MBL region;

(2) production of recombinant human MBL by a cell line transformed with an expression vector with a sequence encoding a region of human MBL in a culture system; and

(3) purification of recombinant human MBL obtained in stage (2).

The present invention also provides a kit for determining recombinant human MBL, comprising i) a solid phase support with a test line and a control line, where murine anti-mannos-binding human lectin antibodies are fixed in the test line and mouse anti-mouse IgG antibodies or pre-S antibodies are fixed in the control line ; ii) a dye substrate with an adsorbed dye conjugate and connected to the bottom of the solid phase substrate, where if the anti-mouse IgG antibodies are fixed in the control line, the dye conjugate is a conjugate of mouse antibodies against mannos-binding human and gold lectins and if fixed in the control line anti-pre-S antibodies; the dye conjugate is a pre-S and gold conjugate; and iii) a sample substrate connected to the bottom of the dye substrate onto which the sample is loaded.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an expression vector pMSG-MBL containing a polynucleotide encoding rhMBL shown on the plasmid map of FIG. 1, which is sufficient for expression of a human oligomeric recombinant MBL that can activate the complement system by specifically binding to MBL binding glycoprotein in the presence of MBL serine proteases.

In a preferred embodiment of the present invention, human MBL cDNAs (Gene Bank NM_000242) were inserted into the pMSG vector (Access No: KCCM (Korea Center for Microorganisms) 10202) containing the MAR element (nuclear matrix binding site element) of the beta-globin gene, poly-A SV40 virus and gastrin gene transcription terminator complex, which led to the creation of a unique vector designation designated pMSG - MBL.

The present invention also provides host cells transfected with an expression vector containing a polynucleotide encoding rhMBL, which is sufficient for expression of a recombinant human MBL that is capable of activating the complement system by specifically binding to a glycoprotein on the surface of microorganisms in the presence of MBL-associated serine proteases.

The host cells used in the present invention are mainly animal cells and are preferably selected from the group consisting of CHO cells (Chinese hamster ovary), hepatocytes, HEK cells (human fetal kidney), etc.

In a preferred embodiment of the present invention, a transformant was prepared by inserting a pMSG-MBL cloning vector containing a polynucleotide encoding rhMBL, which is sufficient for expression of a recombinant human MBL capable of activating the complement system by specifically binding to a glycoprotein on the surface of microorganisms in the presence of MBL-associated serine proteases in a host cell line, such as CHO cells, and then adapting the transformants to conditions with MTX (methotrexate) leading to selection of transformers manta, which is able to express in large quantities the rhMBL protein as a multimeric polymer. The selected transformant was designated MBL D1-3 (CHO cell line) and deposited with the Korean Type Culture Collection, Daejeon, Korea May 16, 2003 (Access No: KCTC 10472BP).

The recombinant human MBL produced by the transformant described in the present invention is mainly in the form of a multimeric form, which is similar to natural MBL from human blood. The cell line obtained in the present invention produced mainly a functionally active multimeric form of MBL and showed a high level of expression, making it possible to use the transformed CHO cell line to obtain large quantities of functionally active rhMBL protein.

The present invention further provides a method for producing recombinant human MBL, comprising the following steps:

(1) A method for producing host cells transfected with a DNA construct comprising a cloning vector containing a polynucleotide encoding rhMBL that is sufficient for expression of a recombinant human MBL capable of activating the complement system by specifically binding to a glycoprotein on the surface of microorganisms in the presence of MBL-associated serine proteases .

(2) A method for producing recombinant human MBL by expressing a polynucleotide encoding a human MBL in and from an animal cell using a massive cell culture system.

(3) A purification method for the recombinant MBL obtained in step (2).

In particular, the use of MBL-glycoprotein binding patterns in step (1) and MBL-binding glycoprotein, examples of which are a glycosylated viral envelope protein containing pre-S HBV, a glycosylated bacterial protein, a glycosylated fungal protein, and a synthetic glycoprotein.

In a preferred embodiment of the present invention, the recombinant MBL production step of step (2) is characterized by the steps of suspension culturing cells in a serum-free / protein-free medium and inoculating such cells well adapted to serum-free / protein-free medium in order to gradually increase production, resulting in a large the amount of rhMBL protein in the form of a multimeric polymer.

In step (3), the recombinant MBL protein was purified from the culture medium of the MBL gene transformants using anion exchange chromatography and MBL binding capacity for pre-S of hepatitis B. The MBL purification method consists of the following steps: (a) separating samples containing multimeric recombinant human MBL using anion exchange chromatography; (b) preparing the column by filling it with a substrate on which pre-S of hepatitis B virus is immobilized; (c) specific capture of recombinant MBLs on immobilized pre-S hepatitis B virus when passing through a column of a sample containing recombinant MBL proteins in the presence of calcium ions, after equilibrating the column with a buffer containing calcium ions; and (d) eluting the recombinant MBL proteins by adding a buffer solution supplemented with EDTA or EGTA to the column on which the recombinant MBL was captured in step (c).

In step (a), a substance was used that is commonly used for anion exchange chromatography, and Q-Sepharose was one of such column materials used. A sample containing MBL proteins as a monomer or dimer was separated in the presence of 150-200 mM NaCl, and another portion containing a high molecular weight MBL protein as a copolymer was eluted in the presence of 350-400 mM NaCl.

In step (b), a substrate was used, which is usually used for affinity chromatography, and Sepharose was one of the commonly used substrates.

In step (c), column balancing was performed by adding a buffer solution or the same solution as used for the affinity binding of recombinant MBL and pre-S hepatitis B virus. The binding of recombinant MBL with pre-S hepatitis B virus was preferably carried out in the presence of calcium ions and then the concentration calcium was preferably 2 to 20 mm. The sample containing the recombinant protein may be a supernatant obtained from a culture medium of a transformant cell producing MBL or an MBL solution eluted from the MBL fractionation column mentioned above.

In step (d), elution of the rhMBL protein was performed using EDTA or EGTA buffer solution in the absence of calcium ions. The buffer solution may be a solution containing 2 to 10 mM EDTA or EGTA, for example, distilled water or Tris-Cl buffer, pH 7.4.

The eluent obtained in step (d) was lyophilized or dialyzed before lyophilization, resulting in a purified recombinant MBL protein.

The rhMBL protein purification method of the present invention is also applicable for purification of MBL obtained from natural biological samples, in addition to the recombinant MBL protein obtained from transformant. A biological sample may be exemplified by blood, plasma, or serum.

The present invention also provides a kit for determining recombinant human MBL, comprising: i) a solid phase support comprising a test line and a control line, where murine anti-mannos-binding human lectin antibodies are fixed in the test line and mouse anti-mouse IgG antibodies or pre-S antibodies are fixed in control line; ii) a dye substrate with an adsorbed dye conjugate and connected to the bottom of the solid phase substrate, where if the anti-mouse IgG antibodies are fixed in the control line, the dye conjugate is a conjugate of mouse antibodies against human and gold mannose-binding lectin and if fixed in the control line anti-pre-S antibodies; the dye conjugate is a pre-S and gold conjugate; and iii) a sample substrate connected to the bottom of the dye substrate into which the sample is loaded.

In the present invention, a nitrocellulose membrane, polyvinylidene difluoride (PVDF) and a nylon membrane can be used as a solid phase support. As a substrate with a dye, you can use, but not limited to, polyester, fiberglass, etc.

The present invention also provides a method for preparing a kit for determining recombinant human MBL, comprising the following steps:

(1) preparing conjugates with a dye by binding antibodies against a mannose-binding human lectin or a pre-S protein with gold particles;

(2) sorbing dye conjugates onto a dye substrate;

(3) binding of anti-mannose-binding human lectin antibodies to the solid phase support in the test line and anti-human pre-S antibodies to the solid-phase support in the control line, if the dye conjugates are gold particles with anti-human mannose-binding lectin antibodies, or binding of anti-pre- S with a solid phase support as a control line, if the dye conjugates are gold particles with murine antibodies against human MBL; and

(4) attaching the substrate for the sample and the substrate with the dye to the bottom of the solid phase substrate.

In the present invention, pre-S proteins or antibodies against human MBL can be used, but not limited to, as a dye conjugate.

The present invention also provides a method for determining MBL using the MBL determination kit, which comprises the following steps:

(1) applying a sample containing MBL to a dye substrate connected to the bottom of the solid phase substrate; and

(2) confirming that the antibodies bind to the test line and the control line of the solid phase support.

The recombinant human MBL of the present invention resembles a natural form purified from human blood. It is an oligomeric form, and it is active against binding to a microorganism, thereby activating the complement system. And the high level of product receipt (50 μg / million cells / day) provides commercial production of functionally active MBL for the development of a new product. Thus, the resulting rhMBL can be effectively used to treat an individual infected with a virus, bacteria or fungi. It can also be used to produce a diagnostic kit for determining recombinant human MBL.

Examples

Practical and currently preferred embodiments of the present invention are illustrative, as shown in the following examples.

However, it will be appreciated that those skilled in the art can make changes and improvements within the spirit and scope of the present invention.

Example 1: Obtaining a transformant of the MBL gene and expression of recombinant human MBL.

<1-1> Construction of an expression vector

pEZ-MBL 2-5 was constructed by cloning MBL cDNA obtained by PCR from a human hepatocyte cDNA library into a pEZ vector, and then it was confirmed that the nucleotide sequence of the MBL cDNA was authentic to the known MBL nucleotide sequence (Gene Bank NM_000242). PCR was performed using pEZ-MBL 2-5 as template and using a set of primers (forward primer 1 and reverse primer 2) to amplify MBL cDNA of approximately 750 kbp. Each primer contained a restriction enzyme recognition site and a Kozak sequence, so that the whole coding region was amplified. Then pMSG-MBL was obtained by cloning the MBL cDNA into the pMSG vector (Access No: KCCM 10202), and then the inserted MBL nucleotide sequence was confirmed (FIG. 1).

Direct primer 1 (SEQ. ID. No. 1)

ctagctagcc accatgtccc tgtttccatc actc (34-mer)

Reverse Primer 2 (SEQ. ID. No. 2)

gaagatctca gatagggaac tcacagacg (29-mer)

<l-2> Transformation of host cells pMSG-MBL

<l-2-l> Preparation of pMSG-MBL Expressing Plasmid DNA

E. coli was transfected with pMSG-MBL and the obtained transformant was cultured in 100 ml of LB medium supplemented with ampicillin at a concentration of 100 μg / ml (Sigma, USA). PMSG-MBL DNA was separated using a plasmid purification kit (QUIAPREP Plasmid Midi Kit, Quiagen, USA). The purified DNA was digested with the restriction enzyme Sea I to make it linear, which was separated using a PCR product purification kit (QIAQuick PCR product purification kit, Quiagen, USA).

<l-2-2> Production of host cells

After culturing the host cells CHO DG44 (dhfr- / dhfr-) in α-MEM medium supplemented with 10% cFBS, the number of cells was counted in a hemocytometer. Cells were distributed (2 × 10 ⁵ cells / well) in α-MEM supplemented with 10% cFBS, which were cultured in a CO ₂ incubator for 24 hours.

<l-2-3> Transformation

2 μg of the pMSG-MBL vector was mixed with 5.3 μl of Dosper ™ reagent and 16 ng of the DNA of the pDCHlP vector (plasmid containing the DHFR gene, Venolia, L. et al., 1987, Somat. Cell MoI. Genet. 13, 491-501 ) to induce a reaction at room temperature for 45 minutes. The resulting product was added to the host cells. Cells were cultured at 37 ° C for 6 hours, then the medium was replaced with freshly prepared α-MEM medium supplemented with 10% cFBS (3 μl / well), followed by further cultivation. After 2-3 days, when the transformed cells grew sufficiently, trypsin was added and 2 μl of α-MEM (w / v) containing 10% dFBS (4 × 10 ⁵ cells / well) was added to the cells, and then again followed by further cultivation. Every 2-3 days, the medium was replaced with freshly prepared and the state of the cells and the formation of single colonies were observed under a microscope. After 10 days, adapted early cells were obtained.

<l-3> Selection of MBL expressing cell line and amplification of the MBL gene

After receiving the adapted early cells, the concentration of MTX (methotrexate) in the medium was gradually increased in order to induce amplification of the MBL gene inserted into the transformed cells. Cells were placed at 4 × 10 ⁵ cells / well and then cultured in medium (α-MEM + 10% dFBS) supplemented with 10 nM MTX until confluence, during which time the medium was replaced with freshly prepared every 2-3 days. The cells were again distributed at 4 × 10 ⁵ cells / well and adapted to the medium supplemented with 100 nM MTX, followed by the same procedure as described above, and then adapted again to the conditions with 1 μM MTX. During amplification of the MBL gene, a Western blot analysis was performed at each step in order to observe changes in the level of MBL expression. Here, the expression of MBL increased with increasing MTX content in the medium.

<l-4> Selection of one cell line

In order to separate one cell line showing a high degree of MBL expression, cells cultured in medium (α-MEM + 10% dFBS) containing 1 μM MTX were distributed on a 96-well plate to obtain 0.5 cells / well, then cultured in medium containing 1 μM MTX. After 2 weeks, when the formation of a single colony was confirmed, the cells were transferred to a 24-well plate, which was further cultured until the cells proliferated sufficiently. Some of them were frozen and stored, and some of them were used to compare expression by Western blot analysis. And the D1-3 transformant in the form of a single cell, which was confirmed to produce a high molecular weight form of MBL in large quantities, was selected as the cell line of the present invention. The selected transformant was designated MBL D1-3 (CHO cell line) and deposited with the Korean type culture collection, Daejeon, Korea May 16, 2003 (Access No: KCTC 10472BP). The PAGE analysis of the recombinant MBL protein expressed by the transformant was carried out under non-denaturing conditions and it was found that the protein was a multimeric form of MBL, which is very similar in characteristics to human MBL of natural origin.

<l-5> Quantitative analysis of recombinant MBL expressed by transformant

Based on the standard amount of purified MBL obtained from human serum, the expression level of the recombinant MBL protein from transformed CHO MBL / D1-3 cells was evaluated. Cells were placed in T25 flasks at a concentration of 4 × 10 ⁵ and then cultured in medium (α-MEM (w / v) + 10% dFBS) to 90% fusion. Then, 3 ml of medium (α-MEM (w / v) + 5% dFBS) was added thereto and further cultured for 4 days. The culture medium was diluted 10 times, followed by a Western blot analysis. The result was compared with a standard amount of natural MBL. Moreover, the expression level of the recombinant MBL protein was confirmed to be approximately 50 μg / 10 ⁶ cells / day as cultured in a cell culture flask.

Example 2: Method for mass production of rhMBL protein

<2-l> Obtaining a cell line adapted for suspension culture in serum-free medium

A culture-dependent cell line expressing MBL was cultured in medium (α-MEM (w / v) supplemented with 10% dFBS and 1 μM MTX. After restoration of viable cells, they were inoculated into a 250 ml roller flask containing 100 ml protein-free medium HyQ SFM4 CHO (Hyclone, USA) with the addition of 0.15% sodium bicarbonate at a seed size of 4 × 10 ⁵ cells / ml Cultivation in a roller flask was carried out at 37 ° C in an incubator with 5% CO2, which was stirred all the time at 40 rpm./min When the number of cells reached 1.0-2.0 × 10 ⁵ cells / ml, the cells were again inoculated into HyQ SFM4 CHO medium supplemented with 0.15% sodium bicarbonate at a seed value of 5 × 10 ⁵ cells / ml Cell viability was determined by the exclusion of trypan blue, in particular, viability of over 90% was confirmed at a late stage of adaptation.

Cells adapted to serum-free protein-free medium were cultured until the cell density reached 2.5 × 10 ⁶ cells / μl. Then the cells were restored and resuspended in a freezing medium. Cells were placed in cryovials to obtain a concentration of 2.8 × 10 ⁷ cells / μl, which were then stored in a container with liquid nitrogen.

<2-2> Creating a culture in a bioreactor using a cell line adapted for serum-free suspension culture

Suspension culture cells were inoculated into a 250 ml roller flask containing 100 ml of serum-free / protein-free medium (HyQ SFM4 CHO medium) supplemented with 0.15% sodium bicarbonate at 5 × 10 ⁵ cells / μl seed. When the number of cells increased to 1.0-2.0 × 10 ⁶ cells / μl, the cells were inoculated into a 500 ml roller flask containing 200 ml of HyQ SFM4 CHO medium with a seed size of 5 × 10 ⁵ cells / μl. Then, when the number of cells increased again to 2.0 × 10 ⁶ cells / μl, the cells were inoculated into a 1000 ml roller flask containing 400 ml of HyQ SFM4 CHO medium with a seed of 5 × 10 ⁵ cells / μl. Thus, the cells were cultured with a gradual increase, leading to 1 l for plating a culture with a concentration of 2.5 × 10 ⁶ cells / μl.

The cell culture for plating obtained above was inoculated into a 7.5 L bioreactor (5 L working volume) and cultured at 34 ° C for 5 days (50 rpm, DO 50, pH 7.2-7.4) .

Example 3: Method for purification of multimeric recombinant human MBL protein

MBO-expressing CHO cells were cultured in HyQ SFM4 CHO medium and then the culture medium was centrifuged and passed through a 0.45 μm pore membrane filter to remove cells. Anion exchange chromatography was performed to purify the recombinant MBL proteins. The amount of purified proteins was determined using the MBL ELISA kit.

<3-l> Separation of a sample containing a multimeric polymer using a Q-Sepharose column

After optimizing the pH and supernatant filtration coefficient, smaller forms of MBL, such as monomer and dimer, were removed using a column filled with Q-Sepharose, and only large forms of MBL were collected. A sample containing smaller forms of MBL was separated by passing through 150-200 mM NaCl buffer and a sample containing large forms of MBL was eluted using 350-400 mM NaCl buffer. These results show that more than 80% was expressed as a high molecular weight copolymer form of MBL with the cell line of the invention (Fig. 2A).

<3-2> Preparation of a column with pre-S-Sepharose

The recombinant pre-S protein used in the present invention was obtained by expressing the pre-S gene, which is part of the surface protein of hepatitis B virus in Sacharomyces cerevisiae, and then purifying it as highly glycosylated molecules (international patent publication No. WO 02 / 094866).

One gram of CNBr-activated 4B Sepharose powder was dissolved in 1 mM HCl, followed by washing several times. In order to use the recombinant pre-S protein as a ligand, 6.4 mg of the pre-S protein was dissolved in the addition buffer (0.2 M NaHCO ₃ , 0.5 M NaCl and pH 8.3) to obtain a concentration of 0.5 - 10 mg / μl. CNBr activated Sepharose 4B resins were mixed with pre-S solution and reacted at room temperature for 2 hours. The mixture was then transferred to blocking buffer (0.1 M Tris-Cl, pH 8.0), followed by further reaction at room temperature for 2 hours. After washing, the sepharose-immobilized pre-S was confirmed by Western blot analysis and was found to be nearly all recombinant pre-S proteins immobilized on a sepharose column, which was used to purify the recombinant MBL protein. In conclusion, a pre-S-Sepharose column for purification of the recombinant human MBL protein was prepared by filling the column with pre-S-Sepharose 4B resin.

<3-3> Purification of Recombinant Human MBL

A column filled with pre-S-Sepharose was equilibrated with binding buffer (20 mM Tris, 150 mM NaCl and 10 mM CaCl ₂ , pH 7.6). A Q-Sepharose column fraction containing recombinant MBL proteins, or a culture medium containing MBL, was loaded onto an MSL adsorption column. Then the column was washed several times with binding buffer. An elution buffer (20 mM Tris, 150 mM NaCl and 5 mM EDTA, pH 7.6) without Ca ^{2+ was} passed through the column in order to elute recombinant human MBL. The resulting eluent was examined using SDS-PAGE. The result was a purified recombinant human MBL having 99.9% purity (Fig. 2A and Fig. 2B).

Example 4: Verification of multimeric recombinant human MBL by EM and the binding activity of MBL to the surface of a microorganism.

<4-1> Confirmation of the formation of multimeric recombinant human MBL

In order to confirm whether or not the purified recombinant human MBL forms a multimeric polymer in the form of a bunch of flowers, the purified recombinant human MBL is treated with amorphous carbon and then the protein form was checked under a transmission electron microscope (Tecnai 12, FEI, Netherlands) and it was found that the purified recombinant MBL human was multimeric polymers in the form of a bouquet (Fig. 3).

<4-2> Binding of recombinant human MBL to pre-S coated gold particles

The ability of recombinant human MBL to bind to the surface of a microorganism was observed using a pre-S surface protein of the hepatitis B virus. Pre-S proteins were attached to the surface of gold particles that are 20-40 nm in size, which are thus made to appear to be a microorganism. Recombinant human MBL was added to them in the presence of calcium. Then, using a Tecnai 12 transmission electron microscope, it was observed whether recombinant human MBL was attached to pre-S proteins, leading to confirmation of complexation. As shown in FIG. 4, pre-S proteins deposited on gold particles were surrounded by recombinant human MBL in the presence of calcium. This demonstrates how the binding of rhMBL to the surface of a microorganism looks like. In fact, an MBL that binds to a microorganism could mechanically block infectivity specifically for organisms such as viruses to cells, in addition to activating the complement system and acting as opsonin.

Example 5: Confirmation of the binding activity of recombinant human MBL

In order to investigate the biological activity of rhMBL, the binding activity of pre-S and mannan was examined in the presence of calcium ions.

Hepatitis B virus mannan or pre-S protein was dissolved in 50 mM carbonate-bicarbonate buffer solution and placed on a microtiter plate (Nunc Maxisorp Immunoplate) to obtain 1 μg per well, which was then incubated overnight at 4 ° C. A tablet coated with pre-S or mannan was washed four times with wash buffer (20 mM Tris, 150 mM NaCl, 10 mM CaCl ₂ , 0.05% Tween-20, pH 7.6), followed by blocking using 0.2% BSA at room temperature for one hour. After washing with wash buffer three times, a sufficient amount of MBL was added to each well to obtain the amount of MBL in each well, as indicated in the figure. To do this, 1 μg of recombinant human MBL in 1 ml of binding buffer (20 mM Tris, 1 M NaCl, 10 mM CaCl ₂ , 0.1% BSA, 0.05% Tween-20, pH 7.6) was sequentially diluted. to obtain the required amount of MBL in 100 μl, which was then added to each well and incubated at room temperature for 2 hours. The plates were then washed again with wash buffer 6 times. Then, murine monoclonal antibodies against human MBL (MBL8F6, Dobeel Corp., Korea) were diluted 10,000 times and 100 μl was added to each well, followed by incubation at 37 ° C for 1 hour. Anti-mouse IgG-HRP was diluted 10,000 times and 100 μl was added to each well. The plate was incubated at 37 ° C for one hour and then staining was manifested by adding 150 μl of TMB solution. After 20 minutes, the reaction was stopped by adding 50 μl of 2M H ₂ SO ₄ . OD _{450 was} measured using an ELISA plate reader spectrophotometer (Multiskan Ex, Labsystems, USA). In FIG. 5A shows the binding of natural MBL purified from human serum to mannan and a pre-S protein, and FIG. 5B shows the binding of recombinant human MBL to pre-S and mannan. In conclusion, recombinant human MBL and natural human MBL had similar binding activity against pre-S and mannan, and both of them did not bind to bovine serum albumin (Fig. 5A and Fig. 5B).

Example 6: Comparison of the biological activity of high molecular weight multimeric recombinant human MBL and low molecular weight oligomeric recombinant human MBL

The biological activity of the larger and smaller forms of recombinant human MBL was investigated in order to show which of the recombinant human MBL proteins, of a larger or smaller form, including a monomer and a dimer, binds more efficiently to pre-S and mannan in the presence of calcium ions.

<6-l> Binding of MBL to Pre-S Glycosylated Protein

Pre-S proteins were coated onto a plate (Nunc Maxisorp Immunoplate) under the same conditions as described in Example 5. Then, a larger or smaller form was dissolved in binding buffer (20 mM Tris, 1 M NaCl, 10 mM CaCl ₂ , 0.1 % BSA, 0.05% Tween-20, pH 7.6) and added to various concentrations of the pre-S coated plate to obtain the required amount in each well. And a further binding analysis was carried out as in Example 5. As described previously, the binding activity of MBL to pre-S proteins was much greater in multimeric recombinant human MBL compared to the smaller form, which included mainly monomers and dimers (FIG. 6).

<6-2> Activation of C4 in the complement system

On a Nunc Maxisorp Immunoplate immunoplate, 500 ng of pre-S proteins were loaded per well, to which various concentrations of recombinant human MBL were added, allowing the MBL to bind to pre-S at room temperature for 2 hours. As a source of MASP protein, 100 μl of MBL-free serum diluted 100-fold was added to each well and incubated for 90 minutes at room temperature. The tablet was then washed with wash buffer 6 times, then 500 ng C4 was added, followed by further reaction at room temperature for 2 hours. Anti-C4 HRP antibodies were diluted 2,000 times and 100 μl was added to each well. The reaction was then allowed to proceed at room temperature for one hour. One hundred and fifty μl OPD solution was added and staining was observed for 20 minutes. The C4b pellet was then measured after adding 50 μl of 3 M HCl and the OD was read at OD ₄₉₂ using a spectrophotometer to read ELISA plates (FIG. 7). For experiments, both the multimeric recombinant human MBL of the larger form proposed in the present invention and the recombinant human MBL of the smaller form, such as monomers or dimers, were used, respectively, to compare their activity. As shown in FIG. 7, C4 was largely activated by larger multimeric recombinant human MBL, but was barely activated by smaller recombinant human MBL proteins. Therefore, it has been confirmed that recombinant human multimeric polymer MBLs of the present invention have superior C4 activation activity.

Example 7: Binding of Recombinant Human MBL to Various Microorganisms

Eleven strains, including both gram-positive bacteria and gram-negative bacteria and fungi, were cultured in a liquid medium suitable for each of them. In order to immobilize cultured bacterial cells, 100 μl of cultured cells were placed on a plate (Maxisorp Immunoplate, Nunc) to obtain 1 × 10 ⁶ cells / well and incubated at 4 ° C overnight. Blocking was performed using 0.2% BSA at 37 ° C for one hour. Five hundred ng of recombinant human MBL was dissolved in 100 μm binding buffer (20 mm Tris, 1 M NaCl, 10 mm CaCl ₂ , 0.1% BSA, 0.05% Tween-20, pH 7.6), was added to each well, followed by interaction at 37 ° C for one hour. The mouse monoclonal anti-human MBL antibody MB1B5 (Dobeel Corp., Korea) was diluted 10,000 times and 100 μl was added to each well, followed by reaction at 37 ° C for one hour. Anti-mouse IgG-HRP was diluted 10,000 times and 100 μl was added to each well, followed by reaction at 37 ° C for one hour. Then, 100 μl of TMB solution was added thereto and staining was observed for 30 minutes. The reaction was stopped by adding 50 μl of a stop solution (H ₂ SO ₄ ) and then OD _{450 was} measured using an ELISA plate spectrophotometer (Multiskan Ex, Labsystems, USA).

The microorganisms used in the present invention exhibited different binding activities to recombinant human MBL and, as shown in FIG. 8, C. albicans, H. influenzae ATCC 51907, S. aureus CCARM 3197 and S. aureus ATCC 29213 showed very high binding activity to recombinant human MBL, but S. pyrogenes ATCC 8668, S. aureus CCARM 3114 and E. faecalis ATCC 29212 showed an average level of binding activity. K. pneumoniae ATCC 10031, S. epidermis ATCC 12228, S. epidermis CCARM 35048, and E. faecium CCARM 5028 had low MBL binding activity. In conclusion, it was proved that recombinant human MBL, like natural MBL, binds to microorganisms, recognizing the pattern of glycosylated surface protein, and the binding activity varies depending on the target microorganism.

Example 8 Inhibition of SARS-CoV Infection with Recombinant Human MBL

FRhk-4 cells, monkey kidney cells, were cultured in MEM. Recombinant human MBL was added to the culture solution, which was then infected with SARS-CoV. Recombinant human MBL was diluted four times with cell culture medium sequentially, starting at a concentration of 2.5 μg / ml, and SARS-CoV, the primary isolate from the patient, was used (Ksiazek TG et al. N. Eng. J. Med 348, 1953-1966 , 2003; Peiris et al., Lancet 361, 1319-1325, 2003).

In order to confirm whether the host cells really were infected with SARS-CoV and propagated, real-time quantitative PCR (GeneAmp PCR system, Applied Biosystems, USA) was performed using a set of primers specific for SARS-CoV. FRhk-4 cells infected with SARS-CoV and not treated with recombinant human MBL were used as a control group.

As shown in FIG. 9, SARS-CoV infection was inhibited by treatment with recombinant human MBL in a dose-dependent manner. For example, when cells were cultured in the presence of recombinant human MBL at a concentration of 2.5 μg / ml, virus proliferation was significantly inhibited (less than 15% proliferation) compared with the control group. These 15% most likely come from a viral inoculum.

FIG. 10 is a photograph from a phase contrast microscope showing that FRhk-4 cells are infected with SARS-CoV. In FIG. 10 (1) healthy cells were not observed in the control group, which was not treated with recombinant human MBL, and in FIG. 10 (2-6) healthy cells were observed in test groups treated with recombinant human MBL. In particular, the number of healthy cells increased with increasing concentrations of recombinant human MBL.

Example 9: Determination of the amount of MBL contained in clinical samples

The inventors of the present invention have developed a method for determining the amount of MBL included in clinical samples such as blood, serum, saliva, etc., which is designed to exploit the ability of the hepatitis B virus pre-S protein to bind to MBL.

<9-l> Immobilization of pre-S on the surface of gold particles

Particles of colloidal gold with a size of 20-80 nm were prepared and pre-S proteins (approximately 100 μg / ml) were added to them under conditions of pH 6.0-8.0, which were stirred at room temperature for conducting. After completion of the reaction, a sufficient amount of 10% BSA was added to obtain a final concentration of 0.3% (w / v) with which it was stirred at room temperature for 15 minutes to further react, resulting in surface blocking. Centrifugation was carried out at 12,000 rpm. Within 15 minutes to separate the supernatant. The precipitate was suspended in a buffer solution with 2% BSA. Since Pre-s proteins uniformly bind to the surface of colloidal gold particles, they created dye conjugates that can be used in the preparation of the MBL test kit. Such dye conjugates exhibit red or purple color due to colloidal gold particles.

<9-2> preparation of the diagnostic strip

Using an inkjet printer or an injection printer, a test line and a control line were applied to a nitrocellulose membrane (S&S, USA) to obtain an MBL test strip. To obtain the test line, a murine monoclonal antibody against human MBL was used, MB1B5 (Dobeel Corp., Korea) and a pre-S specific monoclonal antibody was used for the control line. The dye conjugates prepared above were sorbed on a dye substrate, which is made of polyester or glass fiber fixed to the bottom of the solid-phase substrate of the diagnostic strip.

When a drop of a clinical sample containing human MBL was dropped onto a sample substrate that is attached to a dye substrate of a diagnostic strip, the sample migrated to the nitrocellulose membrane through capillary phenomena and diffusion. When the sample reached the test line, the MBL-pre-S-gold particle complexes were captured by mouse monoclonal antibodies against human MBL on the test line, so that the test line turned red. And when the sample reached the control line, both the MBL-pre-S-gold particle complexes and the pre-S-gold particles were captured by anti-pre-S monoclonal antibodies, so that the control line turned red. Therefore, when the sample contained MBL, both the test line and the control line showed a positive reaction, turning red. On the other hand, when the sample did not contain MBL, only the control line turned red, showing a negative reaction (Fig. 11).

Industrial Applicability

As illustrated above, mass production methods of the multimeric recombinant human mannose-binding lectin (rhMBL) of the present invention represent a significant advance in the development of MBL as an applicable commercial product. In a certain multidimensional MBL, it binds effectively to microorganisms, thereby it could effectively inhibit infection with a virus, bacteria or fungi, so that it could be used to develop drugs to prevent and / or treat infections by microorganisms such as viruses, bacteria or fungi , in particular, SARS-CoV, and the production of a diagnostic kit for determining human MBL.

Those skilled in the art will appreciate that the concepts and specific implementations shown in the foregoing description can easily be used as the basis for modifying or creating other implementations for the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims (6)

1. The Chinese Hamster Ovary (CHO) host cell line transfected with a polynucleotide encoding a human mannosectic lectin (MBL) expressing the pMSG-MBL vector shown on the plasmid map of FIG. 1 is an MBL producer. 2. The host cell line according to claim 1, where the cell line is a cell line CHO MBL D1-3, deposited in the Korean type culture collection under accession number CCC 10472BP. 3. A method for mass production of multimeric recombinant human MBL using the cell line according to claim 1, comprising the following steps:
(1) culturing the cell line according to claim 1 with the accumulation of recombinant human MBL as a result of expression of the coding region of the human MBL region in the cultured cell, and
(2) purification of recombinant human MBL obtained in stage (1). 4. The method according to claim 3, in which stage (1) is characterized by the following stages:
(a) suspension cell culture in serum-free / protein-free medium, and
(b) passaging these cells adapted to serum-free / protein-free medium for their gradual scaling. 5. The method according to claim 3, in which stage (2) is characterized by the following stages:
(a) separating samples containing multimeric recombinant human MBL using anion exchange chromatography;
(b) preparing the column by filling it with a substrate on which the MBL binding protein is immobilized;
(c) specific binding of the recombinant human MBL to the MBL binding protein when a sample containing recombinant human MBL is added to the column in the presence of calcium ions after the column is equilibrated, and
(d) eluting the recombinant human MBL protein from the column by adding buffer solution supplemented with EDTA or EGTA. 6. The method according to claim 5, in which the MBL binding protein is selected from the group consisting of glycosylated pre-S virus envelope protein, glycosylated bacterial protein, glycosylated fungal protein, and synthetic glycoprotein.

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