Clostridium cellulolyticum subunit protein, and preparation method and application thereof

Abstract

The application belongs to the field of bioengineering, and particularly relates to a Clostridium edematis subunit protein and a preparation method and application thereof. The Clostridium edematis subunit protein provided by the application is a protein formed by artificial design and combination of recombination expression optimization according to the sequences of main immunogenic proteins of Clostridium edematis, such as main core antigenic fragment sequences of cytotoxin CctA, flagellin FliA and cell wall surface anchoring protein, etc. The protein is prepared into a Clostridium edematis subunit vaccine as an antigen, can effectively resist the infection of Clostridium edematis, can maximize the safety and effectiveness of the vaccine, and can simplify the preparation process and reduce the production cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of bioengineering, and particularly relates to a Clostridium chauvoei subunit protein and a preparation method and application thereof. BACKGROUND

[0002] Clostridium chauvoei is a specific anaerobic bacteria belonging to the class of bacteria, the family of Bacillaceae and the genus of Clostridium, has no capsule and flagellum, can form spores in vivo and in vitro, and the spores can survive in soil for a long time and are widely distributed in nature. Because the genome of Clostridium chauvoei is smaller than other Clostridium species, the bacteria generally invade the body of ruminants through digestive tract wounds or deep body wounds, and then cause gas swelling and inflammatory exudation of subcutaneous and muscle tissues, and show acute, hot and septic fulminant muscle necrosis, which usually leads to death of the animals in a short time. Clostridium chauvoei is considered to be one of the most pathogenic Clostridium species, and causes huge economic losses to the animal husbandry.

[0003] Clostridial myonecrosis develops rapidly, and the main prevention means is regular injection of vaccines. However, the current vaccines have certain effects on prevention of gas gangrene, but have problems of safety, effectiveness and complex preparation process. Therefore, it is of great significance to develop a high-efficiency and safe Clostridium chauvoei subunit vaccine. SUMMARY

[0004] In view of the problems in the prior art, the application provides a Clostridium chauvoei subunit protein, which is a protein formed by artificial design and combination of recombinant expression and optimization according to sequences of main immunogenic proteins of Clostridium chauvoei, such as main core antigenic fragment sequences of cytotoxin CctA, flagellin FliA and cell wall surface anchor protein. The protein is used as an antigen to prepare a Clostridium chauvoei subunit vaccine, which can effectively resist infection of Clostridium chauvoei, and can maximize the safety and effectiveness of the vaccine, simplify the preparation process and reduce the production cost.

[0005] The object of the application can be achieved by the following technical scheme.

[0006] The application provides a Clostridium chauvoei subunit protein in a first aspect, and an amino acid sequence of the Clostridium chauvoei subunit protein is shown as SEQ ID NO. 1.

[0007] The application provides a gene encoding the Clostridium chauvoei subunit protein in a second aspect, and a nucleotide sequence of the gene is shown as SEQ ID NO. 2.

[0008] The third aspect of the present application provides a preparation method of the Clostridium septicum subunit protein, comprising the following steps: connecting a coding gene to a vector Pet-30a(+) by taking BamHI and HindIII as enzyme cutting sites to obtain a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli BL21-DE3, inducing expression of the target protein by IPTG, and obtaining the Clostridium septicum subunit protein after purification.

[0009] The fourth aspect of the present application provides an application of the Clostridium septicum subunit protein in preparing a vaccine.

[0010] The fifth aspect of the present application provides a Clostridium septicum subunit vaccine, wherein the antigen of the vaccine is the Clostridium septicum subunit protein.

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

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

[0013] Further, the vaccine further comprises aluminum hydroxide adjuvant and 0.01M PBS buffer, the mass percentage of the aluminum hydroxide adjuvant is 10-15%, and the 0.01M PBS buffer is used as a volume complementary solution for preparation of the vaccine.

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

[0015] The sixth aspect of the present application provides an application of the Clostridium septicum subunit vaccine in preparing a drug for preventing Clostridium septicum infection.

[0016] The one or more technical solutions of the present application have the following beneficial effects:

[0017] 1. High safety: the Clostridium septicum subunit vaccine prepared by the present application is a genetic engineering subunit vaccine, and does not contain a complete pathogen, so the safety is high.

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

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

[0020] Figure 1 It is a verification diagram of SDS-PAGE for induction expression of the Clostridium septicum subunit protein.

[0021] Figure 2 Figure 4 is a SDS-PAGE verification chart of the Clostridium chauvoei subunit protein after purification.

[0022] Figure 3 Figure 5 is a chart of the antibody titer change of the serum of the mice immunized with the Clostridium chauvoei subunit vaccine.

[0023] Figure 4 Figure 6 is a chart of the IL-2 level change of the mice immunized with the Clostridium chauvoei subunit vaccine.

[0024] Figure 5 Figure 7 is a chart of the IFN-γ level change of the mice immunized with the Clostridium chauvoei subunit vaccine. DETAILED DESCRIPTION

[0025] In order to enable a person skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples.

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

[0027] Example 1

[0028] A Clostridium chauvoei subunit protein, the amino acid sequence (SEQ ID NO. 1) of which is as follows:

[0029] EYGKEGPKAGGGINGSYTAQRSVTYDQPDYRTLLMKDSVNSASWEVAFNATKDGYDRDSGGGGSAPKGTEESTVKVEYNRFNDQYRLRWSGTEWYGENNRNSRIDSSSGGGGSTSSPGEGADKAIDNNTSTDAGNRSNGTGGGGSSGIINYPKGGGGSKGTEESTVKGGGGSSIEYGKEGPKAGGGINGSYTAWYGENNRNSRIDSGGGGSIEGGNRVVLTTENRGDEEGAYKGGGGSRLEHTINNLNTSSENLTAAESRVRDVDATAGKSMEKLSSGLRINRAGDGGGGSTKVDSTNPNYYIYAEDGPGSIKGGGGSTTTIFSLTLGGGGSTVGDHELTFGGGGSDIAGVTITLELNGYSFEIGGGGSENAEPGYTMRVGLGFRIKGGGGSNLAGATFEIGKLRFLPY

[0030] by targeting the gene sequence (SEQ ID NO. 2) optimized for the expression codon of Escherichia coli:

[0031]

[0032] The preparation method of the Clostridium cellulolyticum subunit protein is as follows:

[0033] Based on the above gene sequence, restriction enzyme sites BamHI and Hind III were introduced, and the gene sequence was synthesized by GenScript Biotech (Shanghai) Co., Ltd. and cloned into the corresponding enzyme cutting sites of the vector pET-30a(+) through the restriction enzyme cutting sites BamHI and Hind III at both ends of the gene sequence, and transformed into the Escherichia coli Top10 cloning strain by heat shock method, and then coated on LB solid medium containing 50 μg / ml Kan+ and grown, and the transformants containing the recombinant plasmid 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, and the recombinant plasmid was extracted by the ordinary plasmid extraction kit of Genomics (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, and the transformed cells were coated on LB solid medium containing 50 μg / ml Kan+ and grown, and the BL21-DE3 transformants containing the recombinant plasmid were screened.

[0035] A single colony of BL21-DE3 containing the recombinant plasmid was picked up with a sterilized toothpick from the Kan+ resistant plate, inoculated in 10 ml of LB liquid medium containing 50 μg / ml Kan+, and incubated at 37°C with shaking at 200 rpm overnight. The next day, the bacterial solution was transferred to LB liquid medium containing 50 μg / ml Kan+ at a ratio of 1:100. When the OD600 value was about 0.6, IPTG stock solution (100 mmol / L) was added at a ratio of 1:200, and the culture was continued for 5 h to induce the expression of the target protein. The SDS-PAGE diagram of the induction expression of Clostridium cellulolyticum subunit target protein is shown in Figure 1 .

[0036] Purification of Clostridium cellulolyticum subunit protein:

[0037] The E. coli fermentation broth expressing recombinant protein was centrifuged at 5000 rpm for 8 minutes to collect the bacterial cells, which were resuspended in 1 / 20 volume of PBS of the fermentation broth. The bacterial cells were broken by a high-pressure homogenizer, and appropriate protease inhibitors were added during the breaking process to prevent protein degradation. The broken liquid mixture was centrifuged at 13000 rpm for 10 minutes at 4°C to collect the precipitate. The target protein (precipitate) was resuspended in PBS containing 0.1% Triton-X100 and 0.5M urea, and centrifuged at 10000 rpm after 20 minutes of shaking at room temperature to collect the precipitate. This washing process was repeated twice to remove most of the soluble impurities. The washed precipitate was dissolved in PBS containing 8M urea, and shaken at room temperature for 30 minutes until the precipitate was completely dissolved without white particles. The mixture was centrifuged at 13000 rpm at 4°C to remove cell debris and unbroken cells, and the supernatant was collected to obtain the purified protein. The SDS-PAGE diagram of the purified Clostridium chauvoei subunit protein is shown in Figure 2 The purified protein was filtered through a 0.22μm filter to remove bacteria, and the protein concentration was determined by BCA. The purified protein was stored at -80°C for long-term preservation.

[0038] Example 2

[0039] This example provides a Clostridium chauvoei subunit vaccine, and the specific preparation method is as follows:

[0040] The purified Clostridium chauvoei subunit protein was used as an antigen to prepare a vaccine. The Clostridium chauvoei subunit protein that passed the sterile test was mixed with aluminum hydroxide adjuvant in a certain proportion to prepare a vaccine, wherein the concentration of the Clostridium chauvoei subunit protein was 200ug / ml, the mass percentage of the aluminum hydroxide adjuvant was 10%, and 0.01M PBS buffer was used as a volume supplement solution for the preparation of the vaccine.

[0041] Example 3

[0042] Performance evaluation:

[0043] Case 1:

[0044] 1. Test animals and grouping

[0045] Select the weight of 350-450 g guinea pigs 15, divided into A, B, C group 3 groups, 5 each group, A group as a blank control group, each guinea pig muscle injection 1 ml PBS; B group for clostridium chauvaei commercial inactivated vaccine immunization group, the immunization dose is 1 ml each guinea pig muscle injection; C group for clostridium chauvaei subunit vaccine once immunization group, the immunization dose is 1 ml each guinea pig muscle injection; D group for clostridium chauvaei subunit vaccine twice immunization group, the immunization dose is 1 ml each guinea pig muscle injection, 21 days after the first immunization, 14 days before the immunization of B group and C group.

[0046] 2. Preparation of clostridium chauvaei virulent bacteria liquid

[0047] Clostridium chauvaei C54-2 strain freeze-dried bacteria powder was dissolved in culture medium and inoculated into clostridium chauvaei subunit vaccine culture medium, cultured at 37℃ in CO2 incubator for 24h, harvested, and the bacteria liquid was fully mixed and counted.

[0048] 3. Challenge experiment

[0049] B group and C group guinea pigs were immunized for 21 days, and D group guinea pigs were immunized for 14 days after the second immunization. Each immunization group was challenged with fresh clostridium chauvaei virulent bacteria liquid 0.5 ml each muscle injection, observed for 10 days, and the immune protection of guinea pigs was recorded. The number of surviving guinea pigs in each group was counted every day after challenge, as shown in Table 1, and the challenge protection test results are shown in Table 2. The protection rate of clostridium chauvaei inactivated vaccine immunization group (B group) for guinea pigs was 80%; the protection rate of clostridium chauvaei subunit vaccine once immunization group (C group) for guinea pigs was 60%, and the protection rate of clostridium chauvaei subunit vaccine twice immunization group (D group) for guinea pigs was 80%.

[0050] The control group guinea pigs died within 72h after challenge. The injection site and changes after dissection of the control group guinea pigs were observed, the leg muscles were dark red, necrotic, and obviously swollen, and there was an acid smell after dissection, and dark red liquid was visible in the chest and abdominal cavity, and the pericardial fluid was dark red and increased. The immune effect of the subunit vaccine twice immunization in this experiment was equivalent to the immune protection effect of clostridium chauvaei inactivated vaccine, and the clostridium chauvaei subunit vaccine once immunization could also stimulate the guinea pigs to produce certain antibodies, and had certain resistance to clostridium chauvaei infection.

[0051] Table 1 Number of surviving guinea pigs in challenge protection test

[0052]

[0053] Table 2 Results of guinea pig challenge protection test

[0054]

[0055] Case 2:

[0056] 1. Test animals and grouping

[0057] Select 20 SPF KM mice, randomly divided into 2 groups, 10 in each group. The first group was immunized with PBS, the immunization amount was 200 μl / each / time, the second group was immunized with Clostridium chauvoei subunit vaccine, the immunization amount was 200 μl / each / time, the injection method was subcutaneous multiple point injection, inoculated once every two weeks, a total of 3 times of immunization.

[0058] 2. Serum separation of immunized animals

[0059] Orbital blood was taken one day before inoculation as a control, and then orbital blood was taken at 14 days after the first immunization, 14 days after the second immunization, 7 days, 14 days and 21 days after the third immunization, serum was separated and stored at -20℃.

[0060] 3. Indirect ELISA detection of antibody levels

[0061] A. Dilute Clostridium chauvoei subunit protein to 5 μg / ml with coating solution, coat ELISA plate with 100 μl / well, at 4℃ overnight.

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

[0063] C. Add 200 μl of blocking solution containing 0.15% BSA to each well, block at 37℃ for 2 hours.

[0064] D. Wash, add PBST at 200 μl / well, wash 3 times, 3-5 minutes each time.

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

[0066] F. Wash the plate, add PBST at 200 μl / well, wash 3 times, 3-5 minutes each time.

[0067] G. Dilute the enzyme-labeled secondary antibody, i.e. horseradish peroxidase (HRP) labeled goat anti-mouse IgG, 5000 times with buffer, 100 μl / well, avoid light, 37℃ for 1 hour.

[0068] H. Wash the plate, add PBST at 200 μl / well, wash 3 times, 3-5 minutes each time.

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

[0070] J. Stop reaction: add 100 μl / well of stop solution (2 mol / L H2SO4 solution), and measure OD value at 450 nm wavelength with microplate reader.

[0071] 4. Detection of cytokines

[0072] Detection of cytokines in mice was performed according to the instruction of ELISA kit for IL-2 and IFN-γ. The serum of immunized mice was detected.

[0073] 5. Experimental results and analysis

[0074] As shown in Table 3, Figure 3 the antibody titer level of serum of mice immunized with vaccine was obviously improved. The antibody titer of serum of mice immunized with vaccine reached the maximum value 14 days after the third immunization. The results showed that the vaccine immunization group could induce the production of humoral immunity in mice. The data of the blank control group, i.e. PBS group, had no obvious change. The vertical coordinate in the figure was OD value, and the horizontal coordinate was time. The significance analysis results showed that the specific antibody level of the vaccine immunization group was extremely significantly different from that of the blank control group (P < 0.01).

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

[0076]

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

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

[0079] Table 4 IL-2 level change of mice immunized with Clostridium chauvoei subunit vaccine (x-±SD)

[0080]

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

[0082]

Claims

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

1.

2. A gene encoding the Clostridium cellulolyticum subunit protein of claim 1, characterized by: The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A method for preparing the Clostridium perfringens subunit protein according to claim 1, characterized by: The application also provides a vaccine containing the Clostridium chauvoei subunit protein. The coding gene is connected to the vector Pet-30a (+) with BamHI and HindIII as enzyme cutting sites to obtain a recombinant plasmid; the recombinant plasmid is transformed into E. coli BL21-DE3, the expression of the target protein is induced by IPTG, and the Clostridium chauvoei subunit protein is obtained after purification.

4. The Clostridium chauvoei subunit protein of claim 1 is used for preparing a vaccine for preventing Clostridium chauvoei infection.

5. A Clostridium gas genosus subunit vaccine, characterized in that: The antigen of the vaccine is the Clostridium chauvoei subunit protein of claim 1.

6. The Clostridium perfringens subunit vaccine of claim 5, wherein: The concentration of the Clostridium chauvoei subunit protein in the vaccine is 150-200 μg / ml.

7. The Clostridium perfringens subunit vaccine of claim 6, wherein: The concentration of the Clostridium chauvoei subunit protein in the vaccine is 200 μg / ml.

8. The Clostridium gas genitale subunit vaccine of claim 5, characterized by: The vaccine further comprises aluminum hydroxide adjuvant, 0.01M PBS buffer, and the mass percentage of the aluminum hydroxide adjuvant is 10-15%, and the 0.01M PBS buffer is used as a volume complementary solution for preparing the vaccine.

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

10. The Clostridium chauvoei subunit vaccine of any one of claims 5-9 is used for preparing a drug for preventing Clostridium chauvoei infection.

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