Pharmaceutical compositions containing the tuberculin allergen eh and uses thereof

Abstract

The present disclosure provides a use of a fusion protein EH containing a tuberculous allergen or a pharmaceutical composition containing the fusion protein EH in the preparation of a reagent or kit for immunological diagnosis of tuberculosis, auxiliary diagnosis of tuberculosis or screening of latent tuberculosis infection of Mycobacterium tuberculosis, the fusion protein EH containing ESAT6 and HspX. The recombinant allergen EH provided by the present disclosure has good specificity and high sensitivity, can be suitable for large-scale screening, and is a new generation of LTBI screening and diagnosis product technology with application and development potential. The recombinant allergen EH provided by the present disclosure has lower toxicity compared with EC, and is safer.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of medical detection, in particular to a pharmaceutical composition containing EH containing tuberculin allergen and uses thereof. BACKGROUND

[0002] According to the World Health Organization (WHO) estimates, about 1.7 billion people in the world are infected with Mycobacterium tuberculosis, accounting for about 23% of the total global population. Among the patients with latent tuberculosis infection (LTBI) infected with Mycobacterium tuberculosis, about 10%-15% of people will develop tuberculosis at some time in their life. Screening and prevention of latent tuberculosis infection population is an important means of tuberculosis prevention and control, and is also an important part of the World Health Organization's "End Tuberculosis Strategy".

[0003] Currently, there are two methods for screening of latent tuberculosis infection (LTBI), namely γ-interferon release test (IGRA) or tuberculin skin test (TST). Since IGRA is complex to operate and expensive, it is not suitable for large-scale population screening and use in poor areas, while the traditional TST has low specificity and cannot distinguish between BCG vaccination and Mycobacterium tuberculosis infection. Therefore, it is necessary to introduce a new screening method with high specificity and simple operation to identify Mycobacterium tuberculosis infected people in the population.

[0004] According to the World Health Organization's "2020 Global Tuberculosis Report", the current global application of recombinant allergen skin test reagents mainly includes DST (Diaskintest, Generium, Russia), C-Tb (Danish Serum Institute), and EC (Anhui ZhiFei LongKeMa Biological Pharmaceutical Company). These three detection methods are based on the early secretory protein (ESAT6) and culture filtrate protein (CFP10) of Mycobacterium tuberculosis as antigens to develop skin test methods. Since ESAT6 and CFP10 are absent in BCG and most environmental Mycobacterium, these three methods have better specificity than the traditional PPD (protein purified derivative) method.

[0005] Table 1 Comparison of domestic and imported detection methods of LTBI allergen

[0006] Features EC C-Tb DST Antigen 1 ESAT6 ESAT6 CFP10 Antigen 2 CFP10 CFP10 ESAT6 Antigen preparation Fusion protein Antigen 1 : 1 mix Fusion protein Comparison to PPD protein amount 10 fold Equal amount 2 fold Sensitivity 83-86％ 70％ 84％ Specificity >95％ >95％ >95％

[0007] Comparing the allergens of the three reagents in Table 1, it can be seen that the antigen proteins selected by the three methods are the same, ESAT6 and CFP10, and the detection sensitivities of these products are similar (70-86%). According to the literature reports, the sensitivity of the EC product is the highest among the three products, reaching 86%, and the protein content is higher, equivalent to 10 times the content of the traditional PPD product. Generally, the higher the content of the allergen, the easier it is to trigger the body's hypersensitivity, thereby easily causing hidden dangers in safety, especially CFP10, which has been reported to cause guinea pig tuberculin shock when used in large doses in guinea pig experiments, and reducing the protein content of the product will significantly reduce its sensitivity (among the three products, the protein content of C-Tb is the lowest, and its sensitivity is also the lowest, only reaching 70%). Therefore, it can be seen that CFP10 is not the best choice as an allergen.

[0008] In 2005, Reece et al. conducted guinea pig experiments with purified CFP-10 and found that high purity CFP10 (which can rule out that it is caused by impurities) can cause tuberculin shock in guinea pigs (Reece ST, et al. Skin test performed with highly purified Mycobacterium tuberculosis recombinant protein triggers tuberculin shock in infected guinea pigs. Infection and Immunity. 2005 Jun; 73(6): 3301-3306.). In the article, the authors conducted DTH experiments on guinea pigs with highly purified CFP-10 (10 micrograms) and used Mtb39 as a control. The results showed that within 6-36 hours, 50% of the guinea pigs died and were accompanied by symptoms of tuberculin shock, but the control protein did not. Although the amount of CFP10 tested is higher than the amount of CFP10 in the EC product, it is easy to cause an allergic reaction in low doses for subjects who have been infected with tuberculosis or sensitive testers. Combined with other researchers' studies on CFP10 stimulating the production of TNF-alpha (Trajkovic V, et al. Effect of Mycobacterium tuberculosis-specific 10-kilodalton antigen on macrophage release of tumor necrosis factor alpha and nitric oxide. Infect Immun. 2002 Dec; 70(12): 6558-66), it also suggests that there is a hidden danger of safety for CFP10 as an antigen protein.

[0009] Mycobacterium tuberculosis has a region of deletion in BCG genome, which is called RD region. Other genes in non-RD region, although cross with BCG, can also achieve the purpose of identifying BCG immunization and tuberculosis infection by controlling the dosage according to relevant research. The protein HspX (16KD protein) encoded by Rv2031c is a widely researched and applied tuberculosis detection antigen. BCG only expresses a small amount of Rv2031c (HspX) under hypoxic conditions. Rv2031c (HspX) is not expressed in NTM (non-tuberculosis mycobacterium) strains. In 2014, Silva reported the use of HspX antigen to detect potential LTBI patients in rheumatoid arthritis patients. This is very important for controlling rheumatoid arthritis patients who are prone to active tuberculosis patients after being infected with Mycobacterium tuberculosis. In 2017, Castro-Garza reported that the antibody of HspX in LTBI patients is much higher than that in healthy people and active tuberculosis patients, so the HspX protein is also defined as a specific protein of latent infection. SUMMARY

[0010] In view of the deficiencies of the prior art, the present application aims to provide a tuberculosis allergen EH with small toxicity and high safety or a pharmaceutical composition containing the same, thereby opening up a new way for tuberculosis immunological diagnosis, auxiliary diagnosis of tuberculosis, screening of latent tuberculosis infection, and differential diagnosis of tuberculosis infection and BCG immunization.

[0011] In one aspect, the present disclosure provides a use of a fusion protein EH containing a tuberculosis allergen or a pharmaceutical composition containing the fusion protein EH in the preparation of a reagent or kit for tuberculosis immunological diagnosis, auxiliary diagnosis of tuberculosis, or screening of latent tuberculosis infection.

[0012] In another aspect, the present application also provides a use of a fusion protein EH containing a tuberculosis allergen or a pharmaceutical composition containing the fusion protein EH in the preparation of a reagent or kit for differential diagnosis of tuberculosis infection and BCG immunization.

[0013] The present disclosure also provides a preparation method of the above-mentioned fusion protein or pharmaceutical composition, and the specific steps are as follows:

[0014] (1) Construct a recombinant expression vector for expressing a fusion protein, wherein the fusion protein comprises ESAT6 and HspX;

[0015] (2) Culture the host cell by transforming the recombinant expression vector, to obtain the fusion protein.

[0016] The present disclosure applies the recombinant allergen EH screened autonomously, selects the fusion protein of ESAT6 which is deleted in BCG and most environmental mycobacteria and HspX which is deleted in environmental mycobacteria and only expressed in small amount under anaerobic conditions in BCG as a new allergen. The recombinant allergen EH has good specificity and high sensitivity, and takes into account the specificity of IGRA detection and the sensitivity of traditional TST detection, and can be adapted to large-scale screening, and is a new generation of LTBI screening and diagnosis product technology with application and development potential. Moreover, the recombinant allergen EH provided by the present disclosure has lower toxicity compared with EC, and is safer. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is the SDS-PAGE electrophoresis gel map of SUMO-EH and purified EH obtained in Example 1 of the present disclosure.

[0018] Figure 2 It is a schematic diagram of the guinea pig skin test experimental detection results in Example 2 of the present disclosure. Among them, EH, ECH and EC are all negative (the value is 0) in BCG. DETAILED DESCRIPTION

[0019] The present disclosure will be further described below. It should be noted that the present embodiment is based on the technical solution, and gives detailed implementation and specific operation process, but the protection scope of the present disclosure is not limited to the present embodiment.

[0020] I. Definitions

[0021] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding field. At the same time, in order to better understand the present disclosure, the definitions and explanations of related terms are provided as follows.

[0022] As used herein and unless otherwise indicated, the terms "comprise", "comprising", "have", "having", "include", "including", and grammatical equivalents thereof, are generally understood to be open-ended and non-limiting, for example, not excluding additional unrecited elements or steps.

[0023] As used herein, the term "fusion protein" refers to a polypeptide having two moieties that are covalently bound to one another, each moiety having a distinct property. The property can be, for example, a biological property such as an in vitro or in vivo activity. In addition, the property can even be a single chemical or physical property, such as binding to an antigen of interest, catalysis of a reaction, etc. The two moieties can be bound directly by a single peptide bond, or via a peptide linker comprising one or more amino acid residues. Typically, the two moieties and the linker are present in the same reading frame. Preferably, the two moieties of the polypeptide are obtained from heterologous or different polypeptides.

[0024] As used herein, "expression" refers to the process by which a polynucleotide is transcribed and translated to produce a polypeptide. The level of expression of a polypeptide can be evaluated using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods can include, but are not limited to, quantifying a polypeptide in a cell lysate by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0025] As used herein, a "host cell" is a cell that is used to accept, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. When a host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Suitable host cells include, but are not limited to, eukaryotic cells, prokaryotic cells, and insect cells.

[0026] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. With respect to vectors include those into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction enzyme digestion and ligation. With respect to vectors also include those comprising a nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically episomal, but can be designed to integrate a gene or portion thereof into a chromosome of the genome. Vectors of artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles is well known to those skilled in the art.

[0027] As used herein, a vector also includes a "viral vector" or a "vector of a virus." A vector of a virus is an engineered virus that is operably linked to a foreign gene to transfer (as a vehicle or shuttle) the foreign gene into a cell.

[0028] As used herein, "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences such as a promoter region that direct transcription of such DNA fragments. Such additional fragments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells and expression vectors that remain episomal or integrate into the host cell genome.

[0029] As used herein, the term "percent identity" is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, depending on the context, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences, "Identity" can be readily calculated by known methods, including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between sequences tested. Methods to determine identity are codified in publicly available computer programs. Sequence alignments and percent identity calculations can be performed using sequence analysis software, such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisconsin), the GCG Wisconsin Programs Package (Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wisconsin 53715 USA).

[0030] As used herein, the term "pharmaceutical composition" refers to a preparation which is effective for the biological activity of the active ingredient contained therein, and which does not contain additional ingredients which are unacceptable for the subject to whom the preparation is administered. It will be appreciated that the fusion proteins provided herein can be administered with a suitable pharmaceutically acceptable carrier, excipient, and other agents that are incorporated to provide improved transfer, delivery, tolerance, etc. Numerous appropriate formulations are known to all pharmaceutical chemists; see, e.g., Remington's Pharmaceutical Sciences (15th Ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 by Blaug, Seymour, incorporated herein by reference. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (e.g., Lipofectin TM ), DNA conjugates, anhydrous abs, oil-in-water and water-in-oil emulsions, emulsions in polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol.

[0031] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific feature, structure, material or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. The illustrative expressions of the above terms in the present specification do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0032] II. Tuberculin allergen EH

[0033] The present disclosure provides a pharmaceutical composition containing tuberculin allergen EH, the combination allergen EH is a fusion protein recombined from ESAT6 and HspX.

[0034] The present disclosure utilizes HspX (RV2031c) to replace CFP10 in the existing allergen, and forms ESAT6-HspX recombinant protein. The fusion protein (EH) of ESAT6 and HspX is used as an allergen reagent, and CFP10 with potential risks is removed. In the case of the same amount of EC, EH is positive for guinea pigs infected with Mycobacterium tuberculosis, and negative for all tested BCG immune guinea pigs. Therefore, EH improves the safety and convenience of application while maintaining high specificity and high sensitivity.

[0035] The present disclosure recombines ESAT6 and HspX into a fusion protein for use as an allergen reagent, and removes CFP10 with potential risks. EH is positive for guinea pigs infected with Mycobacterium tuberculosis, and negative for all tested BCG immune guinea pigs. Therefore, EH improves the safety and convenience of application while maintaining the same high specificity and high sensitivity as EC (Anhui Zhi Fei Long Ke Ma Biological Pharmaceutical Co., Ltd.). The recombinant allergen EH provided by the present disclosure has lower toxicity compared to EC, and is therefore safer.

[0036] The present disclosure replaces CFP10 with HspX while retaining safe and effective ESAT6, and forms ESAT6-HspX recombinant protein, thereby reducing the safety risks caused by the use of CFP10. At the same time, by comparing the DTH of guinea pig experiments of the fusion protein (EH) of ESAT6 and HspX and the fusion protein (ECH) of ESAT6-CFP10-HspX with CFP10 added, the latter does not further improve immunogenicity, and therefore it can be confirmed that CFP10 is not essential.

[0037] The present disclosure also provides a method for efficiently producing recombinant allergen EH. Compared with traditional methods, the present disclosure uses SUMO as a tag to greatly improve the soluble expression of recombinant EH protein. At the same time, the SUMO tag can be removed using SUMO protease, and there is no residual amino acid left after enzyme digestion, which can well guarantee the integrity of the structure of the EH recombinant protein.

[0038] In one aspect, the present disclosure provides the use of a fusion protein EH containing a tuberculosis allergen or a pharmaceutical composition comprising the fusion protein EH in the preparation of a reagent or kit for immunodiagnosis of tuberculosis, auxiliary diagnosis of tuberculosis, or screening for latent Mycobacterium tuberculosis infection.

[0039] In some embodiments, the foregoing immunodiagnosis of tuberculosis includes skin diagnosis, cytokine release test, or serological diagnosis.

[0040] In some embodiments, the foregoing screening for latent Mycobacterium tuberculosis infection includes a skin test.

[0041] In some embodiments, the aforementioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0042] In another aspect, the present disclosure provides use of the fusion protein EH containing tuberculin allergens or the pharmaceutical composition comprising the aforementioned fusion protein EH in the preparation of a reagent or kit for differential diagnosis of tuberculosis infection and BCG vaccination.

[0043] In some embodiments, the aforementioned pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0044] In some embodiments, the fusion protein EH in the aforementioned use comprises ESAT6 and HspX.

[0045] In some embodiments, the aforementioned fusion protein EH comprises ESAT6 and HspX in sequence from N-terminus to C-terminus.

[0046] In some embodiments, the aforementioned fusion protein EH comprises HspX and ESAT6 in sequence from N-terminus to C-terminus.

[0047] In some embodiments, the aforementioned ESAT6 and HspX comprise a linker therebetween.

[0048] In some embodiments, the aforementioned amino acid sequence of ESAT6 comprises an amino acid sequence having 80% or above identity with the amino acid sequence shown in SEQ ID NO. 1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or above identity, more preferably an amino acid sequence having 98% or 99% or above identity; more preferably, the aforementioned amino acid sequence of ESAT6 is shown in SEQ ID NO. 1.

[0049] In some embodiments, the aforementioned amino acid sequence of HspX comprises an amino acid sequence having 80% or above identity with the amino acid sequence shown in SEQ ID NO. 3, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or above identity, more preferably an amino acid sequence having 98% or 99% or above identity; more preferably, the aforementioned amino acid sequence of HspX is shown in SEQ ID NO. 3.

[0050] In some embodiments, the aforementioned linker is a flexible polypeptide; preferably, the aforementioned flexible polypeptide consists of flexible amino acids, preferably, the aforementioned flexible polypeptide consists of 2-20 flexible amino acids, the aforementioned flexible amino acids being selected from at least one of Gly, Ser, Ala and Thr; preferably, the amino acid sequence of the aforementioned linker is shown in SEQ ID NO. 5.

[0051] In some embodiments, the amino acid sequence of the aforementioned fusion protein EH comprises an amino acid sequence that is 80% or above identical to the amino acid sequence set forth in SEQ ID NO. 7, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or above identical, more preferably an amino acid sequence that is 98% or 99% or above identical; more preferably, the amino acid sequence of the aforementioned fusion protein is set forth in SEQ ID NO. 7.

[0052] In some embodiments, the nucleic acid encoding the aforementioned ESAT6 comprises a nucleotide sequence that is 80% or above identical to the nucleotide sequence set forth in SEQ ID NO. 2, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or above identical, more preferably a nucleotide sequence that is 98% or 99% or above identical; more preferably, the nucleic acid encoding the aforementioned ESAT6 is set forth in SEQ ID NO. 2.

[0053] In some embodiments, the nucleic acid encoding the aforementioned HspX comprises a nucleotide sequence that is 80% or above identical to the nucleotide sequence set forth in SEQ ID NO. 4, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or above identical, more preferably a nucleotide sequence that is 98% or 99% or above identical; more preferably, the nucleic acid encoding the aforementioned HspX is set forth in SEQ ID NO. 4.

[0054] In some embodiments, the nucleic acid sequence of the aforementioned linker is set forth in SEQ ID NO. 6.

[0055] In some embodiments, the nucleic acid encoding the aforementioned fusion protein EH comprises a nucleotide sequence that is 80% or above identical to the nucleotide sequence set forth in SEQ ID NO. 8, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or above identical, more preferably a nucleotide sequence that is 98% or 99% or above identical; more preferably, the nucleic acid encoding the aforementioned fusion protein is set forth in SEQ ID NO. 8.

[0056] In another aspect, the present disclosure also provides a method for preparing the aforementioned fusion protein, comprising the following steps:

[0057] (1) constructing a recombinant expression vector for expressing a fusion protein, wherein the fusion protein comprises ESAT6 and HspX;

[0058] (2) transforming the recombinant expression vector into a host cell culture to obtain the fusion protein.

[0059] In some implementations, the aforementioned method further includes step (3) extracting the aforementioned fusion protein.

[0060] In some implementations, the aforementioned method further includes step (4) where the fusion protein is a fusion protein containing a protein tag, the protein tag of the fusion protein is enzymatically cleaved, and the fusion protein is collected.

[0061] In some implementations, the aforementioned protein tag is selected from His tag and / or SUMO tag.

[0062] In some implementations, the recombinant expression vector in step (1) is a prokaryotic expression vector or a eukaryotic expression vector.

[0063] In some implementations, the aforementioned prokaryotic expression vector can be based on any form such as the pET system, pGEX system, or pMAL system; preferably, it is selected from pET-28a-SUMO, pET-28a, pET-30a, pET-31b, pET-34b, pET-35b, pET22b, or pET-43.1; more preferably, the aforementioned recombinant expression vector is pET-28a-SUMO.

[0064] In some implementations, the aforementioned eukaryotic expression vector is selected from pPICZαA, pPICZαB, pPICZαC, pPIC9, pHIL-S1, and pPIC9K.

[0065] In some implementations, the host cell in step (2) is a prokaryotic cell, a eukaryotic cell, or an insect cell.

[0066] In some embodiments, the aforementioned prokaryotic cells are selected from Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, Streptomyces, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Staphylococcus, Neisseria, Pseudomonas, Salmonella, and Ureaplasma; more preferably, the aforementioned prokaryotic cells are Escherichia coli; even more preferably, the aforementioned Escherichia coli is BL21(De3).

[0067] In some embodiments, the aforementioned eukaryotic cells are selected from yeast cells, such as cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Saccharomyces*, or *Yersinia*, such as *Kluyveromyces lactis*, *Kalvatia*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Douglas*, *Kluyveromyces rufiformis*, *Nordicia*, *Ovoyces*, or *Yersinia lipolytica*.

[0068] In some implementations, the aforementioned insect cells are selected from sf9 cells, sf21 cells, and Hi5 cells.

[0069] For the purpose of clarity and concise description, features are described herein as part of some identical or separate implementations; however, it will be understood that the scope of this disclosure may include some implementations having combinations of all or some of the features described.

[0070] Example

[0071] Example 1: Construction of recombinant vector and protein expression

[0072] This embodiment constructed an expression vector for the fusion protein EH and expressed the protein using the expression vector. The fusion protein EH comprises the following fragments from its N-terminus to its C-terminus: ESAT6, a linker, and HspX. The amino acid sequence of the linker is shown in SEQ ID NO. 5, and its nucleotide sequence is shown in SEQ ID NO. 6. The amino acid sequence of the fusion protein EH is shown in SEQ ID NO. 7, and its nucleotide sequence is shown in SEQ ID NO. 8.

[0073] The specific steps for vector construction and protein expression are as follows:

[0074] (1) Plasmid construction: Completed by Suzhou Genewiz Co., Ltd. First, the codon sequence was optimized (using the optimized E. coli sequence) and the gene ESAT6 was synthesized (its nucleotide sequence is shown in SEQ ID NO.2, and its encoded amino acid sequence is shown in SEQ ID NO.1). 5' (BamHI) and 3' (XhoI) restriction endonucleases were added, and the gene was cloned into the vector pET-28A-SUMO (kanamycin resistant) through the restriction sites 5'BamHI and 3'XhoI (using the recombination method) to construct the plasmid pET-28A-SUMO-ESAT6. Then, the codon sequence was optimized (using the optimized E. coli sequence) and the gene HspX (its nucleotide sequence is shown in SEQ ID NO.4, and its encoded amino acid sequence is shown in SEQ ID NO.3) was synthesized. A 3' (XhoI) restriction endonuclease was added, and the gene was cloned into the vector pET-28A-SUMO-ESAT6 (kanamycin resistant) via the 3'XhoI restriction site (using a recombination method) to construct the plasmid pET-28A-SUMO-ESAT6-HspX. The SUMO tag contains a His tag. The amino acid sequence of the SUMO tag containing the His tag is shown in SEQ ID NO.9.

[0075] MGSSHHHHHHSSGLVPRGSHMASMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKI

[0076] KKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG(SEQ ID NO9)

[0077] (2) pET-28A-SUMO-ESAT6-HspX was transformed into Escherichia coli strain BL21(DE3) by chemical transformation to obtain an engineered strain abbreviated as BL21_SUMO-EH.

[0078] (3) Inoculate BL21_SUMO-EH engineered bacteria into 5 mL of LB medium containing 50 μg / mL kanamycin sulfate, and incubate overnight in a 37°C constant temperature shaker. Then, inoculate 1% of the culture into 400 mL of LB medium containing 50 μg / mL kanamycin sulfate, and incubate overnight in a 37°C constant temperature shaker until OD (outcome limit) is reached. 600 Once the concentration reaches 0.4-0.5, add IPTG to a final concentration of 0.25 mmol / L and continue induction at 37 degrees Celsius for 3 hours.

[0079] (4) Collect bacterial cells, add lysis buffer (1×PBS + 0.3M NaCl + 30mM imidazole + 30mg / ml lysozyme + 2mM PMSF), sonicate to disrupt, centrifuge at high speed (25,000g, 30 minutes), collect the supernatant, and add it to a His60 nickel column for purification. After washing away impurities with washing buffer (1×PBS + 0.3M NaCl + 30mM imidazole), elute SUMO-EH (the amino acid sequence of SUMO-EH is shown in SEQ ID NO.10) with elution buffer (containing 25mM Tris (pH 8.0) + 0.5M NaCl + 10% glycerol + 0.25M imidazole). SUMO-EH, after one-step purification, was digested with SUMO protease (at a protein mass ratio of 1:100 to the target protein) overnight at 4°C. The digested protein was then reintroduced into a His60 nickel column. Since the cleaved SUMO tag, the incompletely cleaved SUMO-EH, and the SUMO protease all contain the His tag, they were adsorbed onto the His60 nickel column. However, the EH protein itself does not contain any tag and is therefore retained in the flow-through. The flow-through contained the purified fusion protein EH without the SUMO tag.

[0080] This method can be used to construct and express EH proteins with high expression yield (SUMO can help the protein be expressed in a soluble manner), the purification route is simple, and the final product has high purity (>95%) and high yield (>100 mg / L).

[0081] Figure 1The image shown is an SDS-PAGE gel image, from left to right: purified EH, SUMO-EH, and molecular weight marker.

[0082] Example 2: EH fusion protein specifically induces DTH

[0083] This embodiment describes an animal experiment on the fusion protein EH obtained according to Example 1.

[0084] The specific experimental protocol for the guinea pig skin test was based on Section 3.1.5 of the "Pharmacopoeia of the People's Republic of China 2020 Edition, Part III" for in vivo diagnostic tuberculin purified protein derivatives (TB-PPD), specifically the animal method (3.1.5.1). The detailed procedure is as follows:

[0085] Five weeks after sensitization with Mycobacterium tuberculosis H37Ra or BCG (50 mg / ml, 0.2 ml / guinea pig), white female guinea pigs weighing 400-600 g were shaving their fur. After disinfection with alcohol, 0.1 mL of the sample (containing 10 μg / mL of candidate recombinant protein; or 5 IU TB-PPD (1 μg / mL); or 5 U EC reference (10 μg / mL)) was injected intradermally into the corresponding areas on either side of the spine on the back. The longitudinal and transverse diameters of the local induration were observed 24 hours after injection. If a reaction was observed, the longitudinal and transverse diameters of the local induration or erythema were recorded. A negative result was defined as an average induration or erythema diameter (the sum of the longitudinal and transverse diameters divided by 2) less than 5 mm, and a positive result was defined as 5 mm or greater.

[0086] A guinea pig skin test was conducted to detect delayed-type hypersensitivity (DTH) induced by different antigens in guinea pigs infected with Mycobacterium tuberculosis. Four guinea pigs were tested in each group, with replicates. Skin reactions were observed 24 hours after injection, and the DTH responses in guinea pigs sensitized with Mycobacterium tuberculosis H37Ra and BCG were recorded. Results are as follows: Figure 2 As shown.

[0087] from Figure 2 The results show that when the purified novel antigen EH recombinant protein was used to sensitize guinea pigs, the DTH reaction intensity of the EH recombinant fusion protein (1.0 μg / 0.1 ml) in guinea pigs sensitized with Mycobacterium tuberculosis was higher than that of the reference material TB-PPD (mean halo diameters of 14.4 mm and 10.6 mm, respectively), and also slightly higher than that of the EC reference material (5 U / 0.1 ml, 1.0 μg / 0.1 ml) (mean halo diameters of 14.4 mm and 13.9 mm, respectively). Furthermore, it was negative for BCG-immunized guinea pigs, effectively differentiating tuberculosis infection from BCG immunization. Since the recombinant allergen EH does not contain CFP10, which is an antigenic protein and poses safety risks, using EH as an allergen is safer than EC due to its lower toxicity.

[0088] Meanwhile, by comparing the DTH of ESAT6 and HspX fusion protein (EH) with that of ESAT6-CFP10-HspX (ECH) fusion protein with CFP10 added in guinea pig experiments, the latter did not further enhance immunogenicity, thus confirming that CFP10 is not essential.

[0089] This embodiment uses ESAT6 and HspX to form a fusion protein EH. Compared to methods that use a mixture of two proteins as an allergen (requiring separate quality control for each protein), this allows for better quality control of the product. Compared to the existing EC, the tuberculosis allergen EH described in Example 2 not only removes the potentially hazardous CFP10, but also, based on existing guinea pig experiments, maintains the same high specificity as EC, and is expected to have better sensitivity than EC in diagnosing different populations.

[0090] For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all such changes and modifications should be included within the scope of protection of the claims disclosed herein. sequence list <110> Anbo Intelligent Link (Beijing) Biotechnology Co., Ltd. <120> Pharmaceutical compositions containing tuberculosis allergen EH and their uses <130> MTI21464 <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 95 <212> PRT <213> Mycobacterium tuberculosis <400> 1 Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser 1 5 10 15 Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly 20 25 30 Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35 40 45 Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu 50 55 60 Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly 65 70 75 80 Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala 85 90 95 <210> 2 <211> 285 <212> DNA <213> Mycobacterium tuberculosis <400> 2 atgaccgaac agcagtggaa ctttgcgggc attgaagcgg ccgcgagcgc gattcaaggc 60 aatgtgacga gcattcatag cctgctggat gaaggcaaac agagcctgac caaactggcg 120 gccgcgtggg gcggcagcgg cagcgaagcg tatcaaggcg tgcagcagaa atgggatgcg 180 accgcgaccg aactgaacaa cgcgctgcag aacctggcgc gcaccattag cgaagcgggc 240 caagcgatgg cgagcaccga aggtaacgtg accggcatgt ttgcg 285 <210> 3 <211> 143 <212> PRT <213> Mycobacterium tuberculosis <400> 3 Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe Pro Glu 1 5 10 15 Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu Arg Pro 20 25 30 Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu Gly 35 40 45 Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys Asp 50 55 60 Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala Glu Arg 65 70 75 80 Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr Gly Ser 85 90 95 Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp Asp Ile 100 105 110 Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala Val Ser 115 120 125 Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr Asn 130 135 140 <210> 4 <211> 432 <212> DNA <213> Mycobacterium tuberculosis <400> 4 gcgacgaccc tgccggtgca gcgccatccg cgcagcctgt ttccggaatt tagcgaactg 60 tttgcggcgt ttccgagctt tgcgggcctg cgcccgacct ttgatacccg cctgatgcgc 120 ctggaagatg aaatgaaaga aggccgctat gaagtgcgcg cggaactgcc gggcgtggat 180 ccggataaag atgtggatat tatggtgcgc gatggtcagc tgaccattaa agcggaacgc 240 accgaacaga aagattttga tggccgcagc gaatttgcgt atggcagctt tgtgcgcacc 300 gtgagcctgc cggtgggcgc ggatgaagat gatattaaag cgacctatga taaaggcatt 360 ctgaccgtga gcgtggcggt gagcgaaggc aaaccgaccg aaaaacatat tcagattcgc 420 agcaccaact aa 432 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <400> 5 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 ggcggtggca gcggcggtgg cggcagcggc 30 <210> 7 <211> 248 <212> PRT <213> Artificial Sequence <400> 7 Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser 1 5 10 15 Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly 20 25 30 Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35 40 45 Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu 50 55 60 Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly 65 70 75 80 Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Gly 85 90 95 Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala Thr Thr Leu Pro Val Gln 100 105 110 Arg His Pro Arg Ser Leu Phe Pro Glu Phe Ser Glu Leu Phe Ala Ala 115 120 125 Phe Pro Ser Phe Ala Gly Leu Arg Pro Thr Phe Asp Thr Arg Leu Met 130 135 140 Arg Leu Glu Asp Glu Met Lys Glu Gly Arg Tyr Glu Val Arg Ala Glu 145 150 155 160 Leu Pro Gly Val Asp Pro Asp Lys Asp Val Asp Ile Met Val Arg Asp 165 170 175 Gly Gln Leu Thr Ile Lys Ala Glu Arg Thr Glu Gln Lys Asp Phe Asp 180 185 190 Gly Arg Ser Glu Phe Ala Tyr Gly Ser Phe Val Arg Thr Val Ser Leu 195 200 205 Pro Val Gly Ala Asp Glu Asp Asp Ile Lys Ala Thr Tyr Asp Lys Gly 210 215 220 Ile Leu Thr Val Ser Val Ala Val Ser Glu Gly Lys Pro Thr Glu Lys 225 230 235 240 His Ile Gln Ile Arg Ser Thr Asn 245 <210> 8 <211> 747 <212> DNA <213> Artificial Sequence <400> 8 atgaccgaac agcagtggaa ctttgcgggc attgaagcgg ccgcgagcgc gattcaaggc 60 aatgtgacga gcattcatag cctgctggat gaaggcaaac agagcctgac caaactggcg 120 gccgcgtggg gcggcagcgg cagcgaagcg tatcaaggcg tgcagcagaa atgggatgcg 180 accgcgaccg aactgaacaa cgcgctgcag aacctggcgc gcaccattag cgaagcgggc 240 caagcgatgg cgagcaccga aggtaacgtg accggcatgt ttgcgggcgg tggcagcggc 300 ggtggcggca gcggcgcgac gaccctgccg gtgcagcgcc atccgcgcag cctgtttccg 360 gaatttagcg aactgtttgc ggcgtttccg agctttgcgg gcctgcgccc gacctttgat 420 acccgcctga tgcgcctgga agatgaaatg aaagaaggcc gctatgaagt gcgcgcggaa 480 ctgccgggcg tggatccgga taaagatgtg gatattatgg tgcgcgatgg tcagctgacc 540 attaaagcgg aacgcaccga acagaaagat tttgatggcc gcagcgaatt tgcgtatggc 600 agctttgtgc gcaccgtgag cctgccggtg ggcgcggatg aagatgatat taaagcgacc 660 tatgataaag gcattctgac cgtgagcgtg gcggtgagcg aaggcaaacc gaccgaaaaa 720 catattcaga ttcgcagcac caactaa 747 <210> 9 <211> 121 <212> PRT <213> Artificial Sequence <400> 9 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu 20 25 30 Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn 35 40 45 Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys 50 55 60 Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly 65 70 75 80 Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln 85 90 95 Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile 100 105 110 Glu Ala His Arg Glu Gln Ile Gly Gly 115 120 <210> 10 <211> 369 <212> PRT <213> Artificial Sequence <400> 10 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu 20 25 30 Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn 35 40 45 Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys 50 55 60 Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly 65 70 75 80 Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln 85 90 95 Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile 100 105 110 Glu Ala His Arg Glu Gln Ile Gly Gly Met Thr Glu Gln Gln Trp Asn 115 120 125 Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn Val Thr 130 135 140 Ser Ile His Ser Leu Leu Asp Glu Gly Lys Gln Ser Leu Thr Lys Leu 145 150 155 160 Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly Val Gln 165 170 175 Gln Lys Trp Asp Ala Thr Ala Thr Glu Leu Asn Asn Ala Leu Gln Asn 180 185 190 Leu Ala Arg Thr Ile Ser Glu Ala Gly Gln Ala Met Ala Ser Thr Glu 195 200 205 Gly Asn Val Thr Gly Met Phe Ala Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220 Ser Gly Ala Thr Thr Leu Pro Val Gln Arg His Pro Arg Ser Leu Phe 225 230 235 240 Pro Glu Phe Ser Glu Leu Phe Ala Ala Phe Pro Ser Phe Ala Gly Leu 245 250 255 Arg Pro Thr Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys 260 265 270 Glu Gly Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp 275 280 285 Lys Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala 290 295 300 Glu Arg Thr Glu Gln Lys Asp Phe Asp Gly Arg Ser Glu Phe Ala Tyr 305 310 315 320 Gly Ser Phe Val Arg Thr Val Ser Leu Pro Val Gly Ala Asp Glu Asp 325 330 335 Asp Ile Lys Ala Thr Tyr Asp Lys Gly Ile Leu Thr Val Ser Val Ala 340 345 350 Val Ser Glu Gly Lys Pro Thr Glu Lys His Ile Gln Ile Arg Ser Thr 355 360 365 Asn

Claims

1. Use of a fusion protein EH containing tuberculogen or a pharmaceutical composition containing said fusion protein EH in the preparation of a reagent or kit for the immunological diagnosis of tuberculosis or screening for latent tuberculosis infection; wherein the immunological diagnosis of tuberculosis is a skin diagnosis; and the screening for latent tuberculosis infection includes a skin test, wherein the fusion protein EH is composed of ESAT6 and HspX, wherein the structure of the fusion protein EH from the N-terminus to the C-terminus is ESAT6-linker-HspX, wherein the amino acid sequence of ESAT6 is shown in SEQ ID NO. 1, the amino acid sequence of HspX is shown in SEQ ID NO. 3, and the amino acid sequence of the linker is shown in SEQ ID NO.

5.

2. The use as described in claim 1, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

3. Use of a fusion protein EH containing tuberculogen or a pharmaceutical composition containing said fusion protein EH in the preparation of a reagent or kit for the differential diagnosis of tuberculosis infection and BCG immunization; wherein the differential diagnosis is a skin diagnosis, said fusion protein EH is composed of ESAT6 and HspX, said fusion protein EH has the structure from the N-terminus to the C-terminus as ESAT6-linker-HspX, said amino acid sequence of said ESAT6 is shown in SEQ ID NO. 1, said amino acid sequence of said HspX is shown in SEQ ID NO. 3, and said amino acid sequence of said linker is shown in SEQ ID NO.

5.

4. The use as described in claim 3, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

5. The use according to any one of claims 1-4, wherein, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

7.

6. The use according to any one of claims 1-4, wherein, The encoding nucleic acid of ESAT6 is shown in SEQ ID NO.

2.

7. The use according to any one of claims 1-4, wherein, The encoding nucleic acid of HspX is shown in SEQ ID NO.

4.

8. The use according to any one of claims 1-4, wherein, The nucleic acid encoding the adapter is shown in SEQ ID NO.

6.

9. The use according to any one of claims 1-4, wherein, The nucleic acid encoding the fusion protein is shown in SEQ ID NO.

8.

10. The use according to any one of claims 1-4, wherein, The preparation method of the fusion protein includes the following steps: (1) Construct a recombinant expression vector for expressing the fusion protein, wherein the fusion protein is composed of ESAT6 and HspX; (2) The recombinant expression vector is transferred into host cell culture to obtain the fusion protein.

11. The use as described in claim 10, wherein, The method further includes step (3) extracting the fusion protein.

12. The use as described in claim 11, wherein, The method further includes step (4) where the fusion protein is a fusion protein containing a protein tag, the protein tag of the fusion protein is digested with enzymes, and the fusion protein is collected.

13. The use as described in claim 12, wherein, The protein tag is selected from His tag and / or SUMO tag.

14. The use according to claim 10, wherein the recombinant expression vector in step (1) is a prokaryotic expression vector or a eukaryotic expression vector.

15. The use as described in claim 14, wherein, The prokaryotic expression vector is based on the pET system, pGEX system, or pMAL system.

16. The use as described in claim 14 or 15, wherein, The prokaryotic expression vector is selected from pET-28a-SUMO, pET-28a, pET-30a, pET-31b, pET-34b, pET-35b, pET22b or pET-43.

1.

17. The use according to any one of claims 14-15, wherein, The eukaryotic expression vectors are selected from pPICZαA, pPICZαB, pPICZαC, pPIC9, pHIL-S1, and pPIC9K.

18. The use according to claim 10, wherein the host cell in step (2) is a prokaryotic cell or a eukaryotic cell.

19. The use as described in claim 18, wherein, The prokaryotic cells are selected from Bacillus, Clostridium, Enterococcus, Lactobacillus, Lactococcus, Staphylococcus, Streptococcus, Streptococcus, Campylobacter, Escherichia coli, Flavobacterium, Helicobacter, Staphylococcus, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

20. The use as described in claim 18 or 19, wherein, The prokaryotic cells are Escherichia coli.

21. The use as described in claim 20, wherein, The Escherichia coli mentioned is BL21 (De3).

22. The use as described in claim 18, wherein, The eukaryotic cells were selected from yeast cells.

23. The use as described in claim 22, wherein, The eukaryotic cells are selected from Candida, Hansenula, Kluyveromyces, Pichia, Schizosaccharomyces or Yersinia.

24. The use as described in claim 22 or 23, wherein, The eukaryotic cells are selected from Kluyveromyces lactis, Kelvin, Saccharomyces cerevisiae, Saccharomyces sacchariformis, Saccharomyces douglas, Kluyveromyces noviceum, Saccharomyces ovalis, or Yeastra lipolytica cells.

25. The use as described in claim 18, wherein, The eukaryotic cells are insect cells, and the insect cells are selected from sf9 cells, sf21 cells, and Hi5 cells.

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