Fatty acid modified diphtheria toxin antigen and application thereof

Abstract

The invention particularly relates to a diphtheria toxin antigen modified by fatty acid and application thereof, and belongs to the field of biological medicine, in the diphtheria toxin antigen modified by fatty acid, the fatty acid modification comprises N-terminal cysteine (Cys) modified by fatty acid, the N-terminal Cys modified by fatty acid is characterized in that sulfydryl containing Cys is modified by diacylglycerol and / or alpha amino is modified by fatty acyl. Compared with the existing diphtheria toxin protein antigen, the fatty acid modified diphtheria toxin antigen not only has faster induction and higher immune protection effect than the conventional intramuscular injection induction of the existing diphtheria toxin antigen, but also can induce respiratory tract mucosa immunity, and provides a basis for blocking diphtheria infection and diffusion propagation and providing a vaccine for immunoprophylaxis.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a fatty acid-modified diphtheria toxin antigen and an application thereof. Background Art

[0002] Vaccines are one of the most important inventions in the treatment of infectious diseases, which have greatly reduced the morbidity and mortality of infectious diseases. In many countries and regions, vaccines have reduced the mortality of infectious diseases by 99%, such as smallpox, polio, diphtheria and measles. The vaccines currently on the market mainly include live attenuated vaccines, inactivated vaccines, subunit vaccines, etc. Since attenuated vaccines and inactivated vaccines based on whole pathogens have many safety risks and ineffective immune responses, with the in-depth study of the life cycle, genetic characteristics, pathogenic mechanism, etc. of pathogens, the selection and design of vaccine antigens have transitioned from whole pathogen antigens to one or several clear protective components, entering the era of subunit vaccines. Subunit vaccines have excellent safety, but weak immunogenicity and antigens are easily cleared. Therefore, the selection of appropriate antigens and antigen design schemes, immune stimulants and antigen delivery systems are key factors for the success of subunit vaccines.

[0003] Bacterial lipoproteins are a class of fatty acid-modified proteins that are bound to the membrane through fatty acid chains. The in vivo synthesis of bacterial lipoproteins is completed under the guidance of signal peptides. The bacterial lipidation signal peptide is composed of three regions: the N-terminal positively charged region, the middle hydrophobic region, and the C-terminal lipidation conserved sequence [LVI][ASTVI][GAS][C]. Among them, the C-terminal cysteine ​​is the lipidation modification site. Under the guidance of the signal peptide, the target protein is secreted to the outside of the cell membrane through the Sec or TAT secretion pathway. The Lgt enzyme uses phosphatidylglycerol (PG) as a substrate to connect diacylglycerol to the sulfhydryl group of cysteine ​​(Cys). The LspA enzyme removes the part of the signal peptide except the C-terminal Cys. The Lnt enzyme can use phosphatidylethanolamine (PE) as a substrate to transfer another fatty acid chain to the amino group of Cys.

[0004] Diphtheria toxin is an exotoxin produced by Corynebacterium diphtheriae. It has high biological activity and toxicity. The toxin binds to the ribosomes in the host cell, inhibits the function of the ribosomes, and then interferes with the protein synthesis process. The inhibition has an important impact on the growth, division and repair of cells, and is an important pathogenic factor causing diphtheria disease. CRM197 is a non-toxic variant of diphtheria toxin isolated from the culture of Corynebacterium diphtheriae C7 (β197). The difference between CRM197 and wild-type diphtheria toxin is that the 52nd amino acid mutates from glycine to glutamic acid, thereby eliminating the activity and toxicity of the enzyme, and there are more lysine side chains available for polysaccharide binding. CRM197 is a single-chain polypeptide composed of 535 residues with a relative molecular mass of 58,000 Da. The protein crystal structure can be divided into three independent domains, namely the catalytic domain (C domain), the transmembrane domain (T domain) and the receptor binding domain (R domain). Among them, the C region has adenosine diphosphate (ADP) ribosyltransferase activity and is an important catalyst for protein synthesis in eukaryotic cells; the T region includes two hydrophilic A helices and one hydrophobic A helices. The connecting regions of each helical layer are Asp and Glu. Under neutral conditions, the T region is hydrophilic, and under acidic conditions, some polar groups in the T region are protonated, so that the non-polar hydrophobic layer is inserted into the membrane and further assists the C region to translocate through the membrane to the cytoplasm; the R region is used to bind to cell receptors. These three domains do not cross in the primary structure and are also independent of each other in the tertiary structure. Summary of the invention

[0005] According to the technical problems existing in the prior art, the present invention provides a fatty acid-modified diphtheria toxin antigen and its application, in particular, its application in the preparation of diphtheria toxin vaccine, especially respiratory tract immunity vaccine.

[0006] According to the first aspect of the technical solution of the present invention, a fatty acid-modified diphtheria toxin antigen is provided.

[0007] Furthermore, the fatty acid modification comprises a fatty acid-modified N-terminal cysteine ​​(Cys).

[0008] Furthermore, the fatty acid modification is modification of the N-terminal cysteine ​​Cys thiol group by diacylglycerol containing fatty acid chain residues and / or acylation of the fatty acid chain.

[0009] Preferably, the fatty acid chain is a long chain fatty acid comprising C16-C18 carbon atoms.

[0010] Preferably, the fatty acid-modified diphtheria toxin antigen is the non-toxic mutant CRM197.

[0011] More preferably, the fatty acid modified diphtheria toxin antigen includes a variant that retains the same function as diphtheria toxin. The fatty acid modified diphtheria toxin antigen includes the amino acid sequence 33-566 as shown in SEQ ID NO.1.

[0012] Furthermore, the fatty acid-modified diphtheria toxin antigen is a polymer.

[0013] According to the second aspect of the technical solution of the present invention, a method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen is provided, the method comprising the steps of expressing the diphtheria toxin protein gene containing a fatty acid-modified signal peptide in Escherichia coli, and purifying and preparing the fatty acid-modified diphtheria toxin protein. Wherein, the signal peptide contains three regions: an N-terminal positive charge region, a middle hydrophobic region, and a C-terminal lipid conserved sequence [LVI][ASTVI][GAS][C]. Preferably, the signal peptide is the amino acid sequence of SEQ ID NO.3. Furthermore, the C-terminal cysteine ​​is a fatty acid modification site.

[0014] According to the third aspect of the technical solution of the present invention, there is provided a use of the fatty acid modified diphtheria toxin antigen as described above in the preparation of a product that can induce an animal to produce antigen-specific antibodies, wherein the immune composition can be administered by intramuscular injection or subcutaneous injection, or by nasal drops, or by aerosol immunization. Preferably, the product is a vaccine.

[0015] According to a fourth aspect of the technical solution of the present invention, a pharmaceutical composition is provided, comprising the above-mentioned fatty acid-modified diphtheria toxin antigen.

[0016] According to the fifth aspect of the technical solution of the present invention, a pharmaceutical composition is provided, which comprises a nucleic acid encoding the above-mentioned fatty acid-modified diphtheria toxin antigen.

[0017] According to the sixth aspect of the technical solution of the present invention, a pharmaceutical composition is provided, comprising a vector encoding the above-mentioned fatty acid-modified diphtheria toxin antigen.

[0018] According to a seventh aspect of the technical solution of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following: C1) a nucleic acid molecule encoding the fatty acid-modified diphtheria toxin antigen according to any one of claims 1 to 8; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

[0019] According to an eighth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned fatty acid-modified diphtheria toxin antigen is provided.

[0020] According to the ninth aspect of the technical solution of the present invention, a method for preparing a vaccine using the above-mentioned biological material is provided.

[0021] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The present invention provides a fatty acid-modified diphtheria toxin antigen, which can be polymerized into nanoparticles, significantly improving its immunogenicity. It is used as the main component of an intramuscular injection vaccine, significantly improving the mucosal immune effect, and inducing specific IgG antibodies, which has a very high practical application prospect.

[0022] 2. The fatty acid-modified diphtheria toxin antigen disclosed in the present invention can produce high-titer IgG binding antibodies in animals after two intramuscular injections of immunized animals; and can produce diphtheria toxin-specific IgG binding antibodies in animals after three respiratory tract immunizations.

[0023] 3. Compared with the existing diphtheria toxin protein antigen, this antigen not only has the effect of inducing faster and higher immune protection by conventional intramuscular injection, but also can induce respiratory mucosal immunity, providing a basis for blocking the infection and spread of diphtheria toxin at the infection site and providing comprehensive immune protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results are shown in the supernatant and precipitate protein expression results of the fatty acid-modified CRM197 positive clone after ultrasonic lysis.

[0025] Figure 2 The SDS-PAGE and Western Blot images are of the purified samples of CRM197 modified by fatty acid according to the present invention.

[0026] Figure 3A , Figure 3B and Figure 3C This is the antibody detection after mouse immunization according to the present invention. Figure 3A Two weeks after the first Figure 3B Two weeks after the second vaccination, Figure 3CThis is the antibody titer against CRM197 protein in mouse serum two weeks after the third immunization.

[0027] Figure 4A and Figure 4B Figure 2 is a diagram showing the particle size of the fatty acid-modified CRM197 protein according to the present invention.

[0028] Figure 5 This is a dynamic light scattering diagram of the fatty acid-modified CRM197 protein according to the present invention.

[0029] Figure 6 The figure is a transmission electron micrograph of the fatty acid-modified CRM197 protein according to the present invention.

[0030] In each quantitative result graph, **** indicates P < 0.0001. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0032] The present invention specifically relates to a fatty acid modified diphtheria toxin antigen and its application, which belongs to the field of biomedicine. The fatty acid modified diphtheria toxin antigen disclosed in the present invention can produce high titer IgG binding antibodies by intramuscular injection of animals for immunization twice; and can produce detectable diphtheria toxin specific IgG binding antibodies in respiratory tract immunization animals after three immunizations. Compared with the existing diphtheria toxin protein antigen, the fatty acid modified diphtheria toxin antigen of the present invention not only has the effect of inducing faster and higher immune protection by conventional intramuscular injection, but also can induce respiratory mucosal immunity, providing a basis for blocking the infection and spread of diphtheria toxin at the infection site, and providing all-round immune protection.

[0033] The present invention provides a diphtheria toxin antigen containing fatty acid modification, and its innovation lies in fatty acid modification of diphtheria toxin antigen, and the diphtheria toxin antigen is a non-toxic mutant CRM197. Further, the diphtheria toxin antigen includes the 33-566 amino acid sequence shown in SEQ ID NO.1, and a variant that retains the same function as the diphtheria toxin, preferably a variant with a homology greater than 90%. In one embodiment, the fatty acid-modified diphtheria toxin antigen includes an N-terminal cysteine ​​(Cys) modified by fatty acid, and the N-terminal Cys modified by fatty acid is a thiol group containing Cys modified by diacylglycerol and / or an alpha amino group modified by a fatty acyl group. Alternatively, the fatty acid modification is that the N-terminal cysteine ​​Cys thiol group is modified by diacylglycerol containing a fatty acid chain residue and / or the fatty acid chain is acylated. The fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms. The diphtheria toxin described in the present invention is an exotoxin produced by Corynebacterium diphtheriae, which can bind to the ribosomes in the host cell, inhibit the function of the ribosomes, and then interfere with the protein synthesis process. The inhibitory effect has an important influence on the growth, division and repair of cells, and is an important pathogenic factor causing diphtheria disease. CRM197 is a non-toxic variant of diphtheria toxin isolated from the culture of Corynebacterium diphtheriae C7 (β197). The difference between CRM197 and wild-type diphtheria toxin is that the 52nd amino acid mutates from glycine to glutamic acid; CRM197 is a single-chain polypeptide composed of 535 residues with a relative molecular mass of 58,000 Da. The protein crystal structure can be divided into three independent domains, namely the catalytic domain (C region), the transmembrane domain (T region) and the receptor binding domain (R region).

[0034] In one embodiment, the present invention provides a diphtheria toxin antigen containing fatty acid modification, wherein the fatty acid modification comprises a fatty acid modified N-terminal cysteine ​​(Cys); the fatty acid modification is that the N-terminal cysteine ​​Cys thiol group is modified by diacylglycerol containing a fatty acid chain residue and / or the fatty acid chain is acylated. The fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms. Preferably, the fatty acid modified diphtheria toxin antigen is a non-toxic mutant CRM197. The fatty acid modified diphtheria toxin antigen is a variant comprising the 33-566 amino acid sequence shown in SEQ ID NO.1, and a variant that retains the same function as the diphtheria toxin. More preferably, the fatty acid modified diphtheria toxin antigen is a polymer.

[0035] Furthermore, a method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen is provided, the method comprising the steps of expressing the diphtheria toxin protein gene containing a fatty acid-modified signal peptide in Escherichia coli, and purifying and preparing the fatty acid-modified diphtheria toxin protein. In the method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen, the signal peptide contains three regions, namely, an N-terminal positive charge region, a middle hydrophobic region, and a C-terminal lipid conserved sequence [LVI][ASTVI][GAS][C], and the C-terminal cysteine ​​is a lipid modification site; the signal peptide is the amino acid sequence of SEQ ID NO.3.

[0036] Furthermore, the invention provides a use of the fatty acid modified diphtheria toxin in the preparation of a product that can induce an animal to produce antigen-specific antibodies, wherein the immune composition can be administered by intramuscular injection, subcutaneous injection, nasal immunization, or aerosol immunization. A method for preparing a vaccine using the fatty acid modified diphtheria toxin is also provided.

[0037] In another embodiment, the fatty acid modified diphtheria toxin antigen is a polymer, preferably a particle greater than 10 nm. Compared with the non-fatty acid modified antigen, the fatty acid modified diphtheria toxin antigen of the present invention has the characteristics of increased molecular weight, expression on the cell membrane, and can be dissolved by a solution containing a detergent; further, the fatty acid modification is that the N-terminal cysteine ​​(Cys) thiol group is modified by diacylglycerol containing a fatty acid chain residue, and / or the further amino group is also modified by acylation of the fatty acid chain. The fatty acid chain is a long-chain fatty acid containing C16-C18 carbon atoms. The fatty acid modified diphtheria toxin antigen can form a polymer, preferably a particle greater than 10 nm.

[0038] In one embodiment, a method for preparing the above-mentioned fatty acid-modified diphtheria toxin antigen is provided, which comprises expressing the diphtheria toxin protein gene containing a fatty acid-modified signal peptide in Escherichia coli (the amino acid sequence encoded by the gene is shown in SEQ ID NO.1), purifying and preparing the fatty acid-modified diphtheria toxin protein. Wherein, the signal peptide contains three regions, namely, the N-terminal positive charge region, the middle hydrophobic region and the C-terminal lipid conservative sequence [LVI][ASTVI][GAS][C], wherein the C-terminal cysteine ​​is the lipid modification site. Preferably, the signal peptide is the amino acid sequence of SEQ ID NO.3. Further, the protein may be a protein containing the above-mentioned various diphtheria toxins and / or diphtheria toxoid antigens and fragments thereof. The bacterium is preferably Escherichia coli (the diphtheria toxin protein gene containing a fatty acid-modified signal peptide is expressed in Escherichia coli, and the amino acid sequence encoded by the gene is SEQ ID NO.1). The bacterial fatty acid modified signal peptide (the diphtheria toxin protein gene containing the fatty acid modified signal peptide is SEQ ID NO.2) is composed of three regions: the N-terminal positive charge region, the middle hydrophobic region and the C-terminal lipid conservative sequence [LVI][ASTVI][GAS][C], wherein the C-terminal cysteine ​​is the lipid modification site, and the amino acid sequence 1-21 of the sequence SEQ ID NO.3 is the signal peptide sequence. Purification and preparation of fatty acid modified proteins can include various bacterial separation, fragmentation, dissolution, purification and other technologies.

[0039] In another embodiment, the present invention also provides the use of the above-mentioned fatty acid-modified diphtheria toxin in the preparation of a product that can induce animals to produce antigen-specific antibodies, and the immune composition can be immunized by nasal drops or atomization. The present invention also provides a method for preparing a vaccine using the above-mentioned fatty acid-modified diphtheria toxin. The present invention also includes expression vectors, strains and fatty acid-modified proteins prepared according to the above-mentioned invention. The present invention also includes a method for preparing a disease caused by diphtheria toxin using the above-mentioned fatty acid-modified diphtheria toxin antigen, including preparing various single-dose or multi-dose vaccines, and mixing with aluminum adjuvants, MF59, CPG, etc. to prepare various vaccines. The vaccine prepared by the present invention can be injected, including intramuscular injection, subcutaneous injection, etc.; it can also be immunized for respiratory mucosa by inhalation and other methods.

[0040] The present invention is further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0041] In the following examples, the diphtheria toxin antigen is specifically a fatty acid-modified diphtheria toxin, i.e., fatty acid-modified CRM197, which is recorded in the non-patent document "NCBI: toxin CRM197 [synthetic construct]". The public may obtain the biological material from the applicant in accordance with the relevant provisions of national biosafety. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0042] In the following examples, the carrier PET30(a) is a product of Invitrogen.

[0043] In the following examples, nickel ion affinity chromatography (Chelating Fast Flow column) and gel exclusion chromatography (Superdex-G200) are both products of GE Healthcare.

[0044] In the following examples, goat anti-rabbit IgG secondary antibody (SAB3700885) was a product of Sigma.

[0045] In the following examples, kana is a product of Invitrogen, catalog number ant-zn-1.

[0046] In the following examples, unless otherwise specified, whole gene synthesis, nucleotide synthesis, primer synthesis, sequencing, etc. were performed by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. and Beijing Qingke Biotechnology Co., Ltd.

[0047] In the following examples, BALB / C mice are products of Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0048] In the following examples, unless otherwise specified, the first position of each nucleotide sequence is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.

[0049] SEQ ID NO.1 (Lipo-CRM197 amino acid sequence) MKATKLVLGAVILGSTLLAGCGSGGSGGSGGSGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKGSGGSGGSGGSLEHHHHHH*; SEQ ID NO.2 (Nucleotide sequence of Lipo-CRM197) SEQ ID NO.3 (ML signal peptide amino acid sequence) MKATKLVLGAVILGSTLLAGC.

[0050] The present invention will be further described below through specific embodiments.

[0051] Example 1. Construction of fatty acid modified CRM197 recombinant Escherichia coli strain (1.1) Acquisition of fatty acid-modified CRM197 gene and construction of Escherichia coli expression vector According to the published sequence of CRM197 (NCBI: KU521393.1), the whole genome amino acid sequence of CRM197 was selected, and the inventors artificially designed a codon DNA sequence, added a signal peptide and a CGS connecting peptide at the N-terminus, and added a GGS connecting peptide and 6 his tags at the C-terminus, which is a DNA fragment with a sequence as shown in SEQ ID No. 2. Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the relevant DNA fragment (from the 5' end to the 3' end, there are NdeI restriction site, fragment codon DNA sequence shown in SEQ ID No. 2, and NotI restriction site), and the synthesized fragment was inserted between the NdeI and NotI restriction sites of the PET30(a) vector to obtain the recombinant expression vector PET30(a)-Lipo-CRM197, i.e., the Lipo-CRM197 expression vector.

[0052] The structure of the recombinant expression vector PET30(a)-Lipo-CRM197 is described as follows: the DNA fragment between the NdeI and NotI recognition sequences of the PET30(a) vector is replaced with a recombinant plasmid of the DNA fragment shown in SEQ ID No. 2. SEQ ID No. 2 is a coding gene sequence obtained after artificial codon optimization based on Lipo-CRM197, encoding Lipo-CRM197 with 6 His tags and a GGS connecting peptide as shown in SEQ ID No. 1. Positions 1-20 of SEQ ID No. 1 are lipidation signal peptides, and positions 33-566 are CRM197 proteins.

[0053] (1.2) Transformation of C43(DE3) competent cells with the recombinant expression vector PET30(a)-Lipo-CRM197 E. coli strain C43 (DE3) was streaked on LB plates to recover and isolate single clones. The recovered single clone was picked and inoculated into LB liquid medium. After incubation in a test tube to its logarithmic phase, 1 mL was transferred to a 100 mL LB shake flask and cultured at 37℃ 200rpm shaking incubator until OD600 reached 0.4-0.8, centrifuged at 5000 rpm 4℃ for 5 min, discarded the supernatant, resuspended with 20 mL pre-cooled distilled water, centrifuged at 5000 rpm 4℃ for 5 min, discarded the supernatant, and repeated this step 3 times; resuspended with 20 mL pre-cooled 10% glycerol, centrifuged at 5000 rpm 4℃ for 5 min, discarded the supernatant, and repeated this step 3 times. After the above bacterial cell precipitation was added with distilled water three times and 10% glycerol three times, 1 mL of 10% glycerol was added to suspend the cell, and 100 μL of each cell was dispensed into sterile centrifuge tubes and stored at -80°C to obtain Escherichia coli C43 (DE3) electroporation transformation competent cells.

[0054] Add 2 μL of the expression plasmid PET30(a)-Lipo-CRM197 constructed in step 1.1 to 100 μL of E. coli C43 (DE3) electroporation competent cells, mix gently, transfer to a pre-cooled 0.2 cm electroporation cup, and place on ice for 5 min. According to the E. coli electroporation manual, quickly add 900 μL of pre-cooled LB medium without antibiotics after 2 kV voltage electroporation, transfer to a clean test tube, and culture at 37°C, 200 rpm shaking for 1 hour. Take 100 μL of the bacterial solution obtained by the above shaking culture and spread it on the LB plate for screening resistance to kana, and culture it in an incubator at 37°C for 8-16 hours.

[0055] (1.3) Screening of recombinant expression strains After a single clone grows out on the plate, pick one single clone and inoculate it into 3 mL of LB liquid culture medium containing 50 μg / mL Kana, and culture it at 37°C with a shaker at 200 rpm. After the bacterial liquid grows and becomes concentrated, transfer it to 3 mL of 1# culture medium according to the inoculation amount of 1% (volume percentage), and culture it at 37°C with a shaker at 200 rpm until OD600 reaches 0.4-0.8. Add 0.5 mM IPTG, and induce it at 15°C with a shaker at 200 rpm for 18 h. Collect the precipitate at 12000 rpm for 5 min, resuspend the precipitate with an equal volume of water, and ultrasonicate at 80 V for 5 min (working 3 s, stopping 3 s), separate the supernatant and precipitate at 12000 rpm for 5 min, and resuspend the precipitate with an equal volume of water.

[0056] The supernatant and precipitate separated by ultrasonic sterilization were screened by Western Blot (WB). The Western Blot steps are as follows: (1) Separate the samples on 10% SDS-PAGE gel; (2) Transfer the samples on the SDS-PAGE gel to a PVDF membrane; (3) Block the PVDF membrane with the target protein transferred with 5% milk blocking solution for 1 h at room temperature; (4) Transfer to an anti-His tag antibody (Anti-his, SIGMA, A7058-1VL) diluted with 5% milk at a dilution of 1:2000 and incubate for 1 h; (5) Wash with PBST for 5 min, wash 5 times; (6) Use Pro-light HRP Chemiluminescent colorimetric solution (Tiangen Biochemical, PA112-02) for color development.

[0057] The results are as follows Figure 1 As shown, the positive clones were able to express the target protein Lipo-CRM197 at a high level. The positive clones were selected as the next experimental cloning strains and named PET30(a)-Lipo-CRM197.

[0058] Example 2. Expression and purification of recombinant Lipo-CRM197 protein (2.1) Cultivation of recombinant strain PET30(a)-Lipo-CRM197 The positive clone identified in Example 1 (i.e., the recombinant strain PET30(a)-Lipo-CRM197) was picked and inoculated into LB / Kana liquid medium (containing 50 μg / mL Kana), and cultured at 37°C, 200 rpm until OD600 reached 1.5-2.0, and then transferred to 1# medium at an inoculum volume of 1% (V / V), and cultured at 37°C, 200 rpm until OD600 reached 0.4-0.8, and 0.5 mM IPTG was added, and the culture was carried out at 15°C, 200 rpm for 18 h to induce the expression of Lipo-CRM197, and the bacteria were collected by centrifugation.

[0059] (2.2) Purification of Lipo-CRM197 1. Nickel ion affinity chromatography Resuspend the cells induced for 18 h in water at a ratio of 1:20, homogenize (550-700 Bar), add 1% Triton X-100, stir at room temperature for 2 h, add 20 mM Tris Hcl pH 7.5, 0.5 M NaCl + 20 mM imidazole, adjust the pH to 7.5, centrifuge and collect the supernatant, add the pre-equilibrated Ni column filler, stir and adsorb for 5 h; let stand at 4°C overnight, discard the supernatant on the next day, add solution A to resuspend, let stand and then load the column; Purify with nickel ion chromatography medium, the mobile phase composition is: Solution A: 20 mM Tris Hcl 7.5 + 0.5 M NaCl + 20 mM imidazole + 1% Triton X-100; Solution B: 20 ​​mM Tris Hcl 7.5 + 0.5 M NaCl + 1 M imidazole + 1% Triton X-100; After the column is loaded, use solution A to balance it, then use solution B to elute at 15%-25%-50%-100% respectively, and finally wash with NaOH.

[0060] 2. Gel Exclusion Chromatography The sample purified by nickel column was purified by Supdex G200, and the mobile phase composition was: Solution C: 1×PBST (0.2% TWEEN-20) + 150 mM NaCl; After loading, balance with mobile phase and collect samples in segments.

[0061] Purify samples such as Figure 2 As shown in the figure, it can be seen that the Lipo-CRM197 protein is expressed correctly, which is consistent with the design.

[0062] Example 3: Mouse Immunization Experiment The immunization method has been disclosed in many documents, such as "Replication of Animal Models of Human Diseases, edited by Li Cai, published by People's Medical Publishing House". The details are as follows: 45 female BALB / C mice aged 6-8 weeks were randomly divided into the following 9 groups: Immunization group 1: i.e. control group: 100 μL PBST (0.2% TWEEN-20) and 150 mM NaCl were injected intramuscularly on days 0, 14, and 28, respectively.

[0063] Immunization group 2: 100 μL of vaccine was injected intramuscularly on days 0, 14, and 28. The vaccine used was 1 μg Lipo-CRM197. Among them, Lipo-CRM197 was the Lipo-CRM197 protein expressed by C43 (DE3) prepared in the previous article, and the vaccine was prepared with 1× PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 1 μg Lipo-CRM197 in 100 μL volume.

[0064] Immunization group 3: 100 μL of vaccine was injected intramuscularly on days 0, 14, and 28. The vaccine used was 5 μg Lipo-CRM197. Among them, Lipo-CRM197 was the Lipo-CRM197 protein expressed by C43 (DE3) prepared in the previous article, and the vaccine was prepared with 1× PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 5 μg Lipo-CRM197 in 100 μL volume.

[0065] Immunization group 4: 50 μL of vaccine was delivered to the lungs on days 0, 14, and 28. The vaccine used was 5 μg of Lipo-CRM197. Among them, Lipo-CRM197 was the Lipo-CRM197 protein expressed by C43 (DE3) prepared in the previous article, and the vaccine was prepared with 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 5 μg of Lipo-CRM197 in a volume of 50 μL.

[0066] Immunization group 5: 20 μL of vaccine was dripped into the nose on days 0, 14, and 28. The vaccine used was 5 μg of Lipo-CRM197. Among them, Lipo-CRM197 was the Lipo-CRM197 protein expressed by C43 (DE3) prepared in the previous article, and the vaccine was prepared with 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution in a volume of 20 μL containing 5 μg of Lipo-CRM197.

[0067] Immunization group 6: 100 μL of vaccine was injected intramuscularly on days 0, 14, and 28. The vaccine used was 1 μg CRM197 (CRM197 protein was purchased from Sinovac Biotech). CRM197 was the CRM197 protein purchased from Sinovac Biotech, and the vaccine was prepared with 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 5 μg CRM197 in 100 μL volume.

[0068] Immunization group 7: 100 μL of vaccine was injected intramuscularly on days 0, 14, and 28. The vaccine used was 5 μg CRM197 (CRM197 protein was purchased from Sinovac Biotech). CRM197 was the CRM197 protein purchased from Sinovac Biotech, and the vaccine was prepared with 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 5 μg CRM197 in 100 μL volume.

[0069] Immunization group 8: 50 μL of vaccine was injected intramuscularly on days 0, 14, and 28. The vaccine used was 5 μg CRM197 (CRM197 protein was purchased from Sinovac Biotech). CRM197 was the CRM197 protein purchased from Sinovac Biotech, and the vaccine was prepared with 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 5 μg CRM197 in a volume of 50 μL.

[0070] Immunization group 9: 50 μL of vaccine was injected intramuscularly on days 0, 14, and 28. The vaccine used was 5 μg CRM197 (CRM197 protein was purchased from Sinovac Biotech). CRM197 was the CRM197 protein purchased from Sinovac Biotech, and the vaccine was prepared with 1×PBST (0.2% TWEEN-20) and 150 mM NaCl solution containing 5 μg CRM197 in a volume of 20 μL.

[0071] Eye blood samples were collected from immunized mice in each group before immunization, two weeks after the first immunization, two weeks after the second immunization, and three weeks after the third immunization.

[0072] The antibody titer of anti-CRM197 in the serum of each group of mice was measured by ELISA. The operation steps are shown in the Concise Molecular Biology Experiment Guide [M]. Science Press, 2008.

[0073] Figure 3A , Figure 3B and Figure 3B This is the antibody detection after mouse immunization according to the present invention. Figure 3A Two weeks after the first Figure 3B VC is the antibody titer of mouse serum against CRM197 protein two weeks after the second immunization, and VC is the antibody titer of mouse serum against CRM197 protein three weeks after the third immunization. Figure 3B and Figure 3C As shown: the antibody titer of the immunized group was much higher than that of the control group. In the intramuscular injection group, the IgG antibody titer of Lipo-CRM197 was significantly different from that of CRM197.

[0074] Example 4: Protein particle size detection experiment (4.1) The protein particle size was detected by gel exclusion chromatography Suppex G200 and compared with the peak position of the standard to determine the protein particle size.

[0075] The sample purified by nickel column was purified by Supdex G200, and the mobile phase composition was: Solution C: 1×PBST (0.2% TWEEN-20) + 150 mM NaCl; After loading, balance with mobile phase and collect samples in segments.

[0076] Figure 4A and Figure 4B Figure 2 is a particle size diagram of the fatty acid modified CRM197 protein according to the present invention. Figure 4A-4B As shown in the figure, the peak positions of Lipo-CRM197 protein and standard protein are the same, indicating that Lipo-CRM197 protein self-aggregates into particles larger than 10 nm.

[0077] (4.2) The protein particle size was measured using a dynamic light scattering instrument.

[0078] Figure 5 The particle size diagram of the fatty acid-modified CRM197 protein according to the present invention is shown in the figure. As can be seen from the figure: the particle size of the Lipo-CRM197 protein is about 27.77 nm, indicating that the Lipo-CRM197 protein self-polymerizes into particles larger than 10 nm.

[0079] (4.3) The protein particle size was detected using transmission electron microscopy (TEM).

[0080] Figure 6 The transmission electron micrograph of the fatty acid-modified CRM197 protein according to the present invention is shown in FIG. As can be seen from the figure, the Lipo-CRM197 protein self-polymerizes into nanoparticles.

[0081] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fatty acid-modified diphtheria toxin antigen.

2. The fatty acid-modified diphtheria toxin antigen according to claim 1, characterized in that: It contains a fatty acid-modified N-terminal cysteine ​​(Cys).

3. The fatty acid-modified diphtheria toxin antigen according to claim 2, characterized in that: The fatty acid modification is that the N-terminal cysteine ​​Cys is specifically modified by the thiol group of Cys with diacylglycerol, or the thiol group of Cys is modified by diacylglycerol and the α-amino group is modified by an acyl group.

4. The fatty acid-modified diphtheria toxin antigen according to claim 3, characterized in that: The carbon chain length of the fatty acyl group is C16 to C18.

5. The fatty acid-modified diphtheria toxin antigen according to claim 1, characterized in that: The fatty acid-modified diphtheria toxin antigen is a non-toxic mutant CRM197.

6. The fatty acid-modified diphtheria toxin antigen according to claim 1, characterized in that: The fatty acid-modified diphtheria toxin antigen includes variants that retain the same function as diphtheria toxin.

7. The fatty acid-modified diphtheria toxin antigen according to claim 1, characterized in that: The fatty acid-modified diphtheria toxin antigen includes the amino acid sequence 33-566 as shown in SEQ ID NO.

1.

8. The fatty acid-modified diphtheria toxin antigen according to any one of claims 1 to 7, characterized in that: The fatty acid-modified diphtheria toxin antigen is a polymer.

9. A method for preparing the fatty acid-modified diphtheria toxin antigen according to any one of claims 1 to 7, characterized in that: The method comprises the steps of expressing the diphtheria toxin protein gene containing a fatty acid modified signal peptide in Escherichia coli, and purifying and preparing the diphtheria toxin protein containing fatty acid modified signal peptide.

10. The method according to claim 9, characterized in that The signal peptide contains three regions: an N-terminal positive charge region, a middle hydrophobic region and a C-terminal lipid conservative sequence [LVI][ASTVI][GAS][C].

11. The method according to claim 9, characterized in that The signal peptide is the amino acid sequence of SEQ ID NO.

3.

12. The method according to claim 9, characterized in that The C-terminal cysteine ​​is a fatty acid modification site.

13. Use of the fatty acid modified diphtheria toxin antigen as claimed in any one of claims 1 to 8 in the preparation of a product capable of inducing animals to produce antigen-specific antibodies, wherein the immune composition can be administered by intramuscular injection, subcutaneous injection, nasal drop immunization, or aerosol immunization.

14. The use according to claim 13, characterized in that The product is a vaccine.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fatty acid-modified diphtheria toxin antigen according to any one of claims 1-8.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a nucleic acid encoding the fatty acid-modified diphtheria toxin antigen according to any one of claims 1 to 8.

17. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a vector encoding the fatty acid-modified diphtheria toxin antigen according to any one of claims 1-8.

18. Biomaterial, characterized in that The biological material is any of the following: C1) a nucleic acid molecule encoding the fatty acid-modified diphtheria toxin antigen according to any one of claims 1 to 8; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant cell containing the nucleic acid molecule described in C1), or a recombinant cell containing the expression cassette described in C2), or a recombinant cell containing the recombinant vector described in C3).

19. A method for preparing a vaccine using the fatty acid-modified diphtheria toxin antigen according to any one of claims 1 to 8.

20. A method for preparing a vaccine using the biological material of claim 18.

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