Clostridium swollen subunit protein as well as preparation method and application thereof

Abstract

The invention belongs to the field of bioengineering, and particularly relates to a clostridium gangrene subunit protein as well as a preparation method and application thereof. The subunit protein of clostridium gangrene provided by the invention is a protein formed by artificially designing, combining, recombining, expressing and optimizing according to main core antigenic fragment sequences of various main immunogenic proteins of clostridium gangrene, such as cytotoxin CctA, flagellin FliA, cell wall surface anchoring protein and the like; when the protein is used as an antigen to prepare the clostridium gangrene subunit vaccine, the clostridium gangrene subunit vaccine can effectively resist the infection of clostridium gangrene, the safety and effectiveness of the vaccine can be ensured to the greatest extent, the preparation process is simplified, and the production cost is reduced.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to a subunit protein of Clostridium emphysema and anthracis, and a preparation method and application thereof. Background Art

[0002] Clostridium emphysema is an obligate anaerobic bacterium belonging to the class Bacteria, family Bacillaceae, genus Clostridium. It has no capsule but flagella, can form spores both inside and outside the body, and the spores can survive in the soil for a long time, and are widely distributed in nature. Because its genome is smaller than that of other Clostridia, Clostridium emphysema is generally caused by ruminants eating contaminated forage or water sources, invading the body through digestive tract trauma, or invading through deep trauma infection, causing gas swelling and inflammatory exudation in subcutaneous and muscle tissues, showing acute, febrile and septic fulminant myonecrosis, which usually leads to the death of animals in a short period of time. Clostridium emphysema is considered to be one of the most pathogenic Clostridium species, causing huge economic losses to the animal husbandry industry.

[0003] The course of emphysematous gangrene develops rapidly, and the main means of prevention is regular vaccination. However, although the current vaccines have a certain effect in preventing emphysematous gangrene, there are problems such as safety, effectiveness and complex preparation process. Therefore, it is of great significance to develop an efficient and safe Clostridium subunit vaccine for emphysematous gangrene. Summary of the invention

[0004] In view of the problems in the prior art, the present invention provides a Clostridium difficile subunit protein, which is a protein formed by artificially designing, combining and recombining and expressing the main core antigenic fragment sequences of multiple main immunogenic proteins of Clostridium difficile, such as cytotoxin CctA, flagellar protein FliA and cell wall surface anchor protein. The protein is used as an antigen to prepare a Clostridium difficile subunit vaccine, which can effectively resist the infection of Clostridium difficile and can also maximize the safety and effectiveness of the vaccine, while simplifying the preparation process and reducing production costs.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A first aspect of the present invention provides a subunit protein of Clostridium emphysema, wherein the amino acid sequence of the subunit protein of Clostridium emphysema is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides a gene encoding the above-mentioned Clostridium emphysema subunit protein, and the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0008] The third aspect of the present invention provides a method for preparing the above-mentioned Clostridium emphysema subunit protein, comprising: connecting the coding gene to the vector Pet-30a(+) using BamHI and HindⅢ as restriction sites to obtain a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli BL21-DE3, inducing the expression of the target protein by IPTG, and obtaining the Clostridium emphysema subunit protein after purification.

[0009] A fourth aspect of the present invention provides a use of the above-mentioned Clostridium emphysema subunit protein in the preparation of a vaccine.

[0010] A fifth aspect of the present invention provides a Clostridium difficile subunit vaccine, wherein the antigen of the vaccine is the above-mentioned Clostridium difficile subunit protein.

[0011] Furthermore, the concentration of the subunit protein of Clostridium emphysema in the vaccine is 150-200 μg / mL.

[0012] Furthermore, the concentration of the subunit protein of Clostridium difficile in the vaccine is 200 μg / mL.

[0013] Furthermore, the vaccine also includes aluminum hydroxide gel adjuvant and 0.01M PBS buffer, the mass percentage of aluminum hydroxide gel adjuvant is 10-15%, and 0.01M PBS buffer is used as the volume supplement solution for vaccine preparation.

[0014] Furthermore, the mass percentage of aluminum hydroxide gel adjuvant in the vaccine is 10%.

[0015] A sixth aspect of the present invention provides a use of the above-mentioned Clostridium difficile subunit vaccine in the preparation of a drug for preventing Clostridium difficile infection.

[0016] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0017] 1. High safety: The Clostridium emphysema and anthracis subunit vaccine prepared by the present invention is a genetically engineered subunit vaccine, does not contain complete pathogens, and has high safety.

[0018] 2. Good effectiveness: By optimizing the gene sequence and constructing fusion proteins, the immunogenicity of the vaccine is improved, and it can induce the body to produce a strong immune response at a lower dose.

[0019] 3. Simple preparation process: The vaccine antigen is expressed in Escherichia coli, which simplifies the preparation process and production process and reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the SDS-PAGE verification diagram of the induced expression of subunit protein of Clostridium emphysema.

[0021] Figure 2 This is the SDS-PAGE verification diagram of the purified Clostridium emphysema subunit protein.

[0022] Figure 3 This is a graph showing the changes in serum antibody titers in mice immunized with Clostridium difficile subunit vaccine.

[0023] Figure 4 This is a graph showing the changes in IL-2 levels in mice immunized with Clostridium difficile subunit vaccine.

[0024] Figure 5 This is a graph showing the changes in IFN-γ levels in mice immunized with Clostridium difficile subunit vaccine. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0026] Unless otherwise specified, the materials, reagents and experimental equipment involved in the embodiments of the present invention are all commercially available products.

[0027] Example 1

[0028] A subunit protein of Clostridium emphysema, whose amino acid sequence (SEQ ID NO.1) is:

[0029] EYGKEGPKAGGGINGSYTAQRSVTYDQPDYRTLLMKDSVNSASWEVAFNATKDGYDRDSGGGGSAPKGTEESTVKVEYNRFNDQYRLRWSGTEWYGENNRNSRIDSSSGGGGSTSSPGEGADKAIDNNTSTDAGNRSNGTGGGGSSGIINYPKGGGGSKGTEESTVKGGGGSSIEYGKEGPKAGGGINGSYTAWYGENNRNSRI DSGGGGSIEGGNRVVLTTENRGDEEGAYKGGGGSRLEHTINNLNTSSENLTAAESRVRDVDATAGKSMEKLSSGLRINRAGDGGGGSTKVDSTNPNYYIYAEDGPGSIKGGGGSTTTIFSLTLGGGGSTVGDHELTFGGGGSDIAGVTITLELNGYSFEIGGGGSENAEPGYTMRVGLGFRIKGGGGSNLAGATFEIGKLRFLPY

[0030] Gene sequence optimized for E. coli expression (SEQ ID NO. 2):

[0031]

[0032] The preparation method of the emphysema and anthracis clostridium subunit protein is as follows:

[0033] Based on the above gene sequence, restriction enzyme sites BamHI and HindⅢ were introduced, which were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. and cloned into the corresponding restriction enzyme sites of vector pET-30a(+) through the restriction enzyme sites BamHI and HindⅢ at both ends of the gene sequence. The strain was transformed into Escherichia coli Top10 clone strain by heat shock method, and grown on LB solid medium containing 50 μg / ml Kan+. Transformants containing recombinant plasmids were screened by colony PCR method, inoculated in LB liquid medium containing 50 μg / ml Kan+, and cultured at 37°C with shaking at 200 rpm for 14 hours. The recombinant plasmid was extracted by a common plasmid extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0034] The obtained recombinant plasmid containing the target gene fragment was transformed into Escherichia coli BL21-DE3 competent cells by heat shock method, the transformed cells were spread on LB solid medium containing 50 μg / ml Kan+ for growth, and BL21-DE3 transformants containing the recombinant plasmid were screened.

[0035] Use a sterile toothpick to pick up a single colony of BL21-DE3 bacteria containing the recombinant plasmid grown on the Kan+ resistant plate, inoculate it into 10 ml of LB liquid culture medium containing 50 μg / ml Kan+, and culture it overnight in a constant temperature shaker at 37°C and 200 rpm. The next day, transfer the bacterial liquid to LB liquid culture medium containing 50 μg / ml Kan+ at a ratio of 1:100. When the OD600 value is about 0.6, add IPTG storage solution (100m mol / L) at a ratio of 1:200, and continue to culture for 5 hours to induce the expression of the target protein. The SDS-PAGE image of the induced expression of the target protein of Clostridium emphysema subunit is shown in the figure below. Figure 1 As shown (wherein M: 180 kDa color pre-stained protein molecular weight standard; lane 1: IPTG-induced bacterial solution; lane 2: uninduced bacterial solution).

[0036] Purification of Subunit Proteins from Clostridium emphysema:

[0037] The E. coli fermentation broth expressing the recombinant protein was centrifuged at 5000rpm for 8 minutes to collect the cells, and the cells were resuspended in PBS with 1 / 20 volume of the fermentation broth, and the cells were homogenized and broken by a high-pressure homogenizer. Appropriate protease inhibitors were added during the breaking process to prevent protein degradation. The broken liquid mixture was centrifuged again at 13000rpm, 4°C, for 10 minutes, and the precipitate was collected. The target protein (precipitate) was resuspended in PBS containing 0.1% Triton-X100 and 0.5M urea, and the precipitate was collected by centrifugation at 10000rpm after oscillation at room temperature for 20 minutes. This was repeated twice to remove most of the soluble impurities. The washed precipitated protein was dissolved in a PBS solution containing 8M urea, and oscillated at room temperature for 30 minutes until the precipitated protein was fully dissolved and free of white particles. Cell debris and unbroken cells were removed by centrifugation at 13000rpm at 4°C, and the supernatant was collected to obtain the purified protein. The SDS-PAGE image of the purified emphysema and anthracis subunit protein purification verification is shown below Figure 2 As shown (wherein, M: 180 kDa color pre-stained protein molecular weight standard; lane 1: purified protein solution collected in tube 1; lane 2: purified protein solution collected in tube 2), the purified protein obtained was sterilized by 0.22 μm filtration, the protein concentration was determined by BCA, and the protein was stored at -80°C for long-term use.

[0038] Example 2

[0039] This embodiment provides a subunit vaccine of Clostridium emphysema, and the specific preparation method is as follows:

[0040] Purified subunit protein of emphysema ulcers is used as an antigen to prepare a vaccine. Subunit protein of emphysema ulcers that has passed the sterility test is mixed with aluminum hydroxide gel adjuvant in proportion to prepare a vaccine, wherein the concentration of subunit protein of emphysema ulcers is 200ug / ml, the mass percentage of aluminum hydroxide gel adjuvant is 10%, and 0.01M PBS buffer is used as a volume supplement solution for vaccine preparation.

[0041] Example 3

[0042] Performance evaluation:

[0043] Case 1:

[0044] 1. Experimental animals and groups

[0045] Fifteen guinea pigs weighing 350-450g were selected and divided into 3 groups, A, B and C, with 5 in each group. Group A was the blank control group, and each guinea pig was injected intramuscularly with 1ml PBS; Group B was the immunization group with a commercial inactivated vaccine of Clostridium carbendazim, and the immunization dose was 1ml for each guinea pig intramuscularly; Group C was the group immunized once with a subunit vaccine of Clostridium carbendazim, and the immunization dose was 1ml for each guinea pig intramuscularly; Group D was the group immunized twice with a subunit vaccine of Clostridium carbendazim, and the immunization dose was 1ml for each guinea pig intramuscularly. The second immunization was carried out 21 days after the first immunization, 14 days earlier than Group B and Group C.

[0046] 2. Preparation of Clostridium difficile toxic liquid

[0047] The lyophilized powder of Clostridium emphysema C54-2 strain was dissolved in the culture medium and inoculated into the Clostridium emphysema subunit vaccine culture medium. The culture was placed in a CO2 incubator at 37°C for 24 hours and then harvested. The bacterial solution was fully mixed and the viable bacteria were counted. A Clostridium emphysema virulent bacterial solution with a concentration of 1×106 CFU / ml was prepared with PBS.

[0048] 3. Virus challenge experiment

[0049] 21 days after immunization of guinea pigs in groups B and C, and 14 days after secondary immunization of guinea pigs in group D, the guinea pigs in each immune experimental group and the blank control group were challenged with 0.5 ml of freshly prepared Clostridium emphysema and sulphate virulent bacterial solution injected intramuscularly, and observed for 10 days, and the immune protection of the guinea pigs was recorded. The daily survival statistics of each experimental group after the challenge are shown in Table 1, and the results of the challenge protection test are shown in Table 2. The protection rate of the Clostridium emphysema and sulphate inactivated vaccine immunization group (group B) for guinea pigs is 80%; the protection rate of the Clostridium emphysema and sulphate subunit vaccine primary immunization group (group C) for guinea pigs is 60%, and the protection rate of the Clostridium emphysema and sulphate subunit vaccine secondary immunization group (group D) for guinea pigs is 80%.

[0050] All guinea pigs in the control group died within 72 hours after the virus attack. The changes in the injection site and dissection of the guinea pigs in the control group were observed. The leg muscles were dark red, necrotic, and obviously swollen. After dissection, there was a sour smell. Dark red fluid was seen in the chest and abdominal cavity, and the pericardial fluid was dark red and increased. The immune effect of the secondary immunization of the subunit vaccine in this experiment is equivalent to the immune protection effect of the inactivated vaccine of Clostridium emphysema. In addition, the immunization of Clostridium emphysema subunit vaccine once can also stimulate the guinea pig body to produce certain antibodies, and have a certain resistance to infection with Clostridium emphysema.

[0051] Table 1 Number of surviving guinea pigs in the challenge protection test (only)

[0052]

[0053] Table 2 Guinea pig challenge protection test results

[0054]

[0055] Case 2:

[0056] 1. Experimental animals and groups

[0057] Twenty SPF KM mice were selected and randomly divided into two groups, each with 10 mice. The first group was immunized with PBS, with an immunization dose of 200 μl / mouse / time, and the second group was immunized with Clostridium emphysema subunit vaccine, with an immunization dose of 200 μl / mouse / time, and the injection method was subcutaneous multiple injections, once every two weeks, for a total of 3 immunizations.

[0058] 2. Isolation of serum from immune animals

[0059] Orbital blood was collected one day before vaccination as a control, and then orbital blood was collected 14 days after the first vaccination, 14 days after the second vaccination, and 7 days, 14 days, and 21 days after the third vaccination. The serum was separated and stored at -20°C.

[0060] 3. Indirect ELISA to detect antibody levels

[0061] A. Dilute the Clostridium emphysema subunit protein to 5 μg / ml with coating solution, coat the ELISA plate with 100 μl / well, and incubate at 4°C overnight.

[0062] B. Discard the coating solution, add PBST at 150 μl / well, and wash three times, each time for 3 minutes.

[0063] C. Add 200 μl of blocking solution containing 0.15% BSA to each well and incubate at 37°C for 2 hours.

[0064] D. Washing: Add PBST at 200 μl / well and wash three times for 3-5 minutes each.

[0065] E. Dilute the serum samples of each group of experimental mice with blocking solution, with a dilution of 1:200, and add them to the ELISA plate in the order of sample numbers, 100 μl / well, and place at 37°C in the dark for 1 hour.

[0066] F. Wash the plate by adding 200 μl / well PBST and washing three times for 3-5 minutes each time.

[0067] G. Dilute the enzyme-labeled secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG, 5000 times with buffer, 100 μl / well, and incubate at 37° C. in the dark for 1 hour.

[0068] H. Wash the plate by adding 200 μl / well PBST and washing three times for 3-5 minutes each time.

[0069] I. Color development: Use TMB substrate solution for color development, 100 μl per well, and develop the color for 10 minutes at room temperature in the dark.

[0070] J. Stop the reaction: add 100 μl / well of stop solution (2 mol / L H2SO4 solution) and measure the OD value at a wavelength of 450 nm using an enzyme marker.

[0071] 4. Detection of Cytokines

[0072] The cytokine detection of mice was carried out according to the instructions of the ELISA kits for IL-2 and IFN-γ, and the serum extracted from the immunized mice was used for detection.

[0073] 5. Experimental results and analysis

[0074] As shown in Table 3, Figure 3 As shown in the figure, after immunization with the vaccine, the antibody titer level of mouse serum increased significantly. 14 days after the third immunization, the serum antibody titer value of mice in the vaccine immunization group reached the maximum value. The results show that the vaccine immunization group can induce humoral immunity in mice. There was no significant change in the data of the blank control group, i.e., the PBS group. The ordinate in the figure is the OD value, and the abscissa is the time. The results of the significance analysis show that the specific antibody levels of the vaccine immunization group and the blank control group were significantly different (P<0.01).

[0075] Table 3 Serum antibody titers of mice immunized with Clostridium emphysema subunit vaccine (x-±SD)

[0076]

[0077] The cytokine ELISA kit was used to detect IL-2 and IFN-γ in the serum of immunized mice, as shown in Table 4. Figure 4 As shown in the figure, the IL-2 level of mice immunized with Clostridium emphysema subunit vaccine increased significantly 14 days after the second immunization and reached the maximum 14 days after the third immunization; the IL-2 level of mice in the blank control group was relatively stable. The results of significance analysis showed that the IL-2 level of the vaccine immunization group was significantly different from that of the blank control group (P<0.01).

[0078] As shown in Table 5, Figure 5 As shown in the figure, the IFN-γ level in the vaccine immunization group increased significantly 14 days after the second immunization and reached the maximum value 14 days after the third immunization; the IFN-γ level in the blank control group was very stable. The results of the significance analysis showed that the IFN-γ level in the vaccine immunization group was significantly different from that in the blank control group (P<0.01). In summary, the Clostridium emphysema subunit vaccine can induce cellular immunity in the body.

[0079] Table 4 Changes in IL-2 levels in mice immunized with Clostridium difficile subunit vaccine (x-±SD)

[0080]

[0081] Table 5 Changes in IFN-γ levels in mice immunized with Clostridium difficile subunit vaccine (x-±SD)

[0082]

Claims

1. A subunit protein of Clostridium emphysema, characterized in that: The amino acid sequence of the subunit protein of Clostridium emphysema is shown in SEQ ID NO.

1.

2. A gene encoding the subunit protein of Clostridium emphysema according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A method for preparing the subunit protein of Clostridium emphysema according to claim 1, characterized in that: include: The coding gene was connected to the vector Pet-30a(+) using BamHI and HindⅢ as restriction sites to obtain a recombinant plasmid; the recombinant plasmid was transformed into Escherichia coli BL21-DE3, the expression of the target protein was induced by IPTG, and the subunit protein of Clostridium emphysema was obtained after purification.

4. Use of the Clostridium emphysema subunit protein according to claim 1 in preparing a vaccine.

5. A Clostridium difficile subunit vaccine, characterized in that: The antigen of the vaccine is the subunit protein of Clostridium emphysema according to claim 1.

6. The Clostridium emphysema subunit vaccine according to claim 5, characterized in that: The concentration of the subunit protein of Clostridium emphysema in the vaccine is 150-200 μg / ml.

7. The Clostridium emphysema subunit vaccine according to claim 6, characterized in that: The concentration of the subunit protein of Clostridium emphysema in the vaccine is 200 μg / ml.

8. The Clostridium emphysema subunit vaccine according to claim 5, characterized in that: The vaccine also includes aluminum hydroxide gel adjuvant and 0.01M PBS buffer solution. The mass percentage of the aluminum hydroxide gel adjuvant is 10-15%. The 0.01M PBS buffer solution is used as a volume supplement solution for vaccine preparation.

9. The Clostridium emphysema subunit vaccine according to claim 8, characterized in that: The mass percentage of aluminum hydroxide gel adjuvant in the vaccine is 10%.

10. Use of the Clostridium emphysema subunit vaccine according to any one of claims 5 to 9 in the preparation of a drug for preventing Clostridium emphysema infection.

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