CN103275228A - K99-987P-F41 recombinant protein and application thereof - Google Patents

Abstract

The invention relates to a K99-987P-F41 recombinant protein, the amino acid sequence of which is shown in SEQ ID No.2. The invention also provides the coding gene of the recombinant protein, its preparation method and its application in the preparation of enterotoxigenic Escherichia coli vaccine. In the present invention, the ETEC adhesin K99, 987P, and F41 target genes are tandemly subcloned onto the Escherichia coli prokaryotic expression vector pET30a(+), which can be used to prepare the Escherichia coli K99-987P-F41 tandem expression subunit vaccine, and one immunization can obtain A variety of antibodies are easy to use and overcome the shortcomings of monovalent vaccines. At the same time, because they only contain a small number of surface proteins, they can eliminate many irrelevant antigenic determinants of bacteria and side reactions caused by crude extraction or semi-purified preparations. It has good safety and The characteristics of good stability can overcome the shortcomings of whole-bacteria inactivated vaccines, and have the advantage of high specificity of subunit vaccines.

Description

K99-987P-F41 recombinant protein and its application

technical field

The invention belongs to the fields of animal molecular biology and genetic engineering, and relates to a K99-987P-F41 recombinant protein and its application.

Background technique

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of calf diarrhea, usually causing acute diarrhea in cattle, pigs and other animals. ETEC mainly acts on the jejunum and ileum, especially the ileum. The virulence factors of ETEC include adhesins (also known as pili) and enterotoxins. The pathogenic mechanism of ETEC is that the specific pili bind to the specific receptors of the host small intestinal mucosal epithelial cells, and then colonize the small intestinal mucosa to prevent its loss due to intestinal peristalsis and intestinal content flow. ETEC proliferates in large numbers in the intestinal tract. secrete enterotoxin. Enterotoxin can lead to enhanced secretory function and decreased absorption function of small intestinal mucosal cells, causing large amounts of water and electrolytes to be retained in the intestinal tract, resulting in diarrhea. ETEC can often enter the environment through infected cattle and cause disease in newborn calves. Usually some calves will get natural immune protection, however, under the conditions of modern livestock environment, this protection is usually not able to protect calves, and even the infection rate of calves is increased. Due to the relatively weak ability of newborn calves to resist external pathogenic infection, the sudden death of ETEC is too late to be treated. For chronically ill cattle, antibiotics can be used for treatment. However, in clinical practice, the use of drugs is not standardized or long-term medication leads to ETEC drug resistance. If the sex is enhanced or drug-resistant strains appear, the clinical treatment effect is not significant, and the treatment cost will be increased. Effective vaccines need to be developed for prevention. At the same time, ETEC is also a zoonotic pathogen that can cause neonatal diarrhea. Therefore, it is also of great public health significance to prevent and control calf colibacillosis.

K99, 987P and F41 are three important adhesins of enterotoxigenic Escherichia coli, and these three adhesins will be described in detail below

(1) K99: In 1972, Smith and Linggood first reported the isolation of E. coli K99 adhesin from calves and lambs with diarrhea. Under the electron microscope, the purified K99 adhesin has a rod-like microfilament structure and a strong tendency to aggregate. In SDS-PAGE electrophoresis, it presents a protein band with a molecular weight of about 18ku, and its isoelectric point is determined to be 9.75. K99 is found in bovine, ovine, and porcine-derived ETEC, and its adhesion is limited to the posterior small intestine of young animals. It has mannose resistance and coagulation activity. Pili type F5 (K99) mediates ETEC-specific mucosa on the intestinal mucosal surface of calves, lambs, and piglets. The genes involved in the biosynthesis of K99 adhesin have been cloned and located on a 87.8 kb non-conjugative plasmid that also encodes ST and some antibiotic resistance genes. The synthesis of K99 pili requires the expression of 8 unique proteins: fanA-H. fanC, the major subunit and immunogenic polypeptide of the K99 pilus is the most highly expressed K99 gene. The results showed that some ETEC K99+ strains detected variable expression of K99. Therefore, using Minca medium can improve the identification rate of K99. These experiments showed that glucose inhibits K99 expression. The synthesis of several bacterial toxins appears to be inhibited by glucose, including Escherichia coli heat-stable enterotoxins and Staphylococcus aureus enterotoxins A, B, and C. Adding exogenous cyclic adenosine monophosphate (cAMP) can make thermostable enterotoxigenic Escherichia coli overcome glucose-mediated inhibition of thermostable enterotoxin synthesis. However, exogenous cAMP did not enable K99 E. coli to overcome glucose-mediated K99 repression. In addition to glucose-mediated regulation, K99 expression and detection are also regulated by other mechanisms. For example, it was found that the detection rate of K99 in K99+ETEC could be improved by increasing the aerobic growth of ETEC in liquid medium before plating.

K99+ strains recently isolated from clinical specimens do not usually appear to synthesize K99 in vitro; however, after several passages in liquid media, K99 is detectable. Results appearing in liquid media correlate with capsular mutants. It is speculated that the bursa hides some of ETEC K99, therefore, the capsular mutant is more suitable for detection of K99. The expression of K99 is also dependent on the temperature of cell growth. At 18°C, Escherichia coli cells can express K99 pili, but at 37°C, neither express nor other pili. The combination of K99 pili and cells requires a receptor, namely N-hydroxyacid GM3, which is located in the host cells in the small intestine. This site is the place where bacteria multiply and secrete toxins. When K99 pili binds to the receptor, it causes diarrhea. When ETEC host cell attachment is inhibited, the bacteria cannot accumulate in the gut without causing disease.

(2) 987P: Nagy et al. discovered 987P adhesin in 1976. Observed under an electron microscope, the diameter is 7nm and the length is about 2-4μm. Morphologically very similar to type I pili, it is a long, hard filament with a hollow structure. The molecular weight of its protein subunit is about 18.9ku. In the SDS-PAGE electrophoresis, only one protein band with a molecular weight of about 20ku appears in the purified antigen, and its isoelectric point is 3.7. 987P has no hemagglutination activity. Adsorbed to small intestinal epithelial cells and brush border, not inhibited by mannose. Easily lost in laboratory storage. 987P is found in some bovine and porcine-derived ETECs, and its adhesion is limited to epithelial cells of the posterior small intestine of young animals.

The gene encoding 987P adhesin is located on a plasmid. 987P biosynthesis requires fasA～fasH8 genes. In addition to the positive regulator fasH, there are three structural proteins including FasA, FasF, and FasG, fasA is the only adhesin subunit determinant with adhesion properties, and four accessory proteins that are biologically necessary for pili, such as FasD outer membrane protein The guide protein, FasB is a periplasmic chaperone of the main subunit FasA, FasC can stably and specifically interact with the adhesive FasG, and the chaperone FasE. The discovery of different chaperone proteins of 987P subunit provides a new idea for the study of bacterial pili biology.

(3) F41: In 1978, Morris et al. first proposed that the K99 antigen of the bovine B41 strain contained two protein antigens, anion and cation. In 1980, scientists proved that the cationic antigen was K99 pili, and the anionic antigen was a new pili. In 1982, De Graaf et al. purified the anionic antigen from the mutant strain of the B41 strain: F41 pili, and the coding gene exists on the bacterial chromosome. It is a filamentous structure with a diameter of 3.2nm. According to SDS-PAGE gel electrophoresis analysis, the molecular weight is about 29.5ku and the isoelectric point is 4.6. Adding alanine can inhibit the formation of pili. The genetic nature of F41 depends on chromosomal DNA rather than plasmid DNA. At present, the F41 pili of ETEC strains from different sources are only related to the strains containing two serotypes O9 or O101. In the strains with these two O antigens, the F41 pili can be expressed alone or with K99 on one strain. The pili appear simultaneously. F41 pilus usually only produces ST, but not LT. Compared with K99 pilus, it has stronger adhesion ability and can be inhibited by appropriate antibodies and their Fab fragments, but it is not affected by mannose. Immunization of animals with antigens carrying F41 pili can produce high titers of antibodies.

At present, ETEC's vaccines are mainly inactivated whole-bacteria vaccines. Inactivated vaccines are pathogens that have been treated to kill them. They are safe and will not cause primary diseases themselves. The inactivated vaccine is relatively stable and not easy to fail due to improper handling. It has achieved good results in the prevention of diarrhea in pigs, chickens, ducks, cattle, and sheep in actual production. Wang Zhaojun et al prepared oil adjuvant inactivated vaccines from chicken Escherichia coli isolated from local clinics with serotypes O32, O68 and O78. The test results show that the vaccine is stable, safe and reliable, can induce a good immune response in the body, and can effectively protect chickens against local epidemic Escherichia coli infection. Liu Diqin et al prepared an inactivated vaccine from the local Escherichia coli epidemic strains rejuvenated by the virulence of piglets, and immunized pregnant sows. It is confirmed that the inactivated vaccine is safe and has no side effects, and can produce a good immune effect on pregnant sows. Solan Sizhu et al. took the number of yak Escherichia coli inactivated vaccine immunizations as the research object for the protection of yaks, and the results confirmed that the antibody titer and protection rate of yaks after 2 immunizations were significantly better than that of 1 immunization. According to the local Escherichia coli prevalence situation, Li Zhenqing used clinically isolated O78 Escherichia coli and O92 L. anatipestifer to prepare a dual inactivated vaccine. After immunizing meat ducks and ducklings, it was found that the vaccine stimulated the body to produce higher antibodies. Level, can prevent the attack of Escherichia coli and L. anatipestifer. Gao Rui screened three strains of bacteria with serotypes O2, O86 and O101 from the moderately virulent bacteria of calf diarrhea in Shanxi Province, prepared a trivalent inactivated vaccine with oil adjuvant, and immunized rabbits, and achieved good immune effects . Cui Yudong et al. used bovine Escherichia coli isolates with both K99 and F41 pili antigens as strains to prepare inactivated vaccines to immunize animals, showing that inactivated vaccines can induce a good immune response in the body. There are many serotypes of ETEC, and conventional whole-cell inactivated vaccines can only inhibit certain serotypes of ETEC. Although the immune effect of self-made vaccines is relatively good, it can only be applied in a small range. In addition, inactivated vaccines are expensive to produce and require adjuvants to enhance the body's immune response. In addition, important antigens are lost during inactivation and immunity is incomplete. Therefore, novel vaccines need to be invented to overcome the defects of whole bacterial inactivated vaccines.

Subunit vaccines are vaccines that induce the body to produce protective antibodies. There are only a few major surface proteins, thus eliminating many irrelevant epitopes in bacteria and crude or semi-purified preparations leading to side reactions. Its advantage is that the vaccine only contains the immunogen composition necessary for the protective immune response, does not contain infectious materials, and has good safety. Acute, persistent or latent infection does not occur after vaccination and can be used in some cases. Shifting to nonpathogenic, but harmless microorganisms for mass production of immunogenic vaccines also improves safety. Immunosuppression of allergens and other deleterious reactions reduces adverse effects. The vaccine has good stability and is convenient for storage and transportation. Disease control vaccines can be used for pathogens of exotic diseases and pathogens that cannot be cultured, expanding the scope of use. It induces an immune response in the body, and can distinguish immune response from infection, so it is more suitable for controlling and eliminating infectious disease outbreaks.

Contents of the invention

The first object of the present invention is to provide a K99-987P-F41 recombinant protein and its coding gene.

The second object of the present invention is to provide a method for preparing the above-mentioned recombinant protein.

The third object of the present invention is to provide the use of the above-mentioned recombinant protein.

The present invention is realized through the following technical solutions:

1. A K99-987P-F41 recombinant protein, the amino acid sequence of which is shown in SEQ ID No.2.

2. The gene encoding the above-mentioned K99-987P-F41 recombinant protein, the nucleotide sequence of which is shown in SEQ ID No.1.

Three, a kind of preparation method of K99-987P-F41 recombinant protein, this method comprises the following steps:

(1) Design primers based on the complete sequences of enterotoxigenic Escherichia coli adhesin K99, 987P, and F41 genes published in GenBank (accession numbers are M35282.1, M35257.1, and M21788.1, respectively), and use Escherichia coli C83912, Escherichia coli The genomes of C83916 and Escherichia coli C83919 were used as templates, and target fragments were respectively amplified by PCR; the primer sequences used were as follows:

P1: K99(F): CGG GGTACC ATGACTATTAACTTCAATGGCAAA (KpnI) (SEQ ID No. 3);

P2: K99(R): CGC GGATCC TGAAGTAGTAAATACGCCAGC (BamHI) (SEQ ID No. 4);

P3: 987P(F): CGC GGATCC GGTGGTGGTGGTTCTGCTGAAAACAACACCAGCCAG (BamHI) (SEQ ID No. 5);

P4:987P(R):C GAGCTC ACTAGCAGTGACAGTACCGGC(SacI) (SEQ ID No. 6);

P5: F41 (F): C GAGCTC GGCGGTGGCGGCAGCGTATCTGGTTCAGTGATGGCT (SacI) (SEQ ID No. 7);

P6: F41 (R): AAGGAAAAAA GCGGCCGC ACTGAGGTCATCCCAATTGTG (NotI) (SEQ ID No. 8).

(2) The three fragments were connected in series into the vector pET30a to construct the expression vector pET30a-K99-987P-F41;

(3) The expression vector pET30a-K99-987P-F41 was transformed into Escherichia coli competent cells, and the expression was induced by IPTG to obtain the recombinant protein.

4. Application of K99-987P-F41 recombinant protein in preparation of enterotoxigenic Escherichia coli vaccine.

Positive effects of adopting the above-mentioned technical scheme: the present invention subclones the three target genes of ETEC adhesin K99, 987P and F41 in tandem onto the Escherichia coli prokaryotic expression vector pET30a(+), and introduces a flexible Linker between the two adjacent genes , to avoid the influence of the interaction between the target proteins, it can be used to prepare Escherichia coli K99-987P-F41 tandem expression subunit vaccine, a variety of antibodies can be obtained in one immunization, it is easy to use, overcomes the shortcomings of monovalent vaccines, and because it only contains A small number of surface proteins, thereby eliminating many irrelevant antigenic determinants of bacteria and side reactions caused by crude extraction or semi-purified preparations, has the characteristics of good safety and good stability, can overcome the shortcomings of whole-bacteria inactivated vaccines, and has sub- The advantage of high specificity of unit vaccine.

Description of drawings

Fig. 1 is genome PCR amplification result figure;

In the figure, M.DL2000DNA Marker; 1. Negative control; 2. K99PCR product; 3.987P PCR product; 4. F41PCR product;

Fig. 2 is the result figure of PCR amplification of recombinant plasmid;

In the figure, M.DL2000DNA marker; 1.k99 amplification result; 2.987P amplification result; 3.F41 amplification result; 4.K99-987P-F41 amplification result;

Figure 3 is the result of enzyme digestion and identification of pET30a-K99-987P-F41;

In the figure, M. DL2000DNA marker; 1.K99; 2.987P; 3.F41; 4.K99-987P-F41;

Fig. 4 is the result of recombinant protein SDS-PAGE analysis;

In the figure, M. Pre-stained protein marker; 1. After pET30a induction; 2. Uninduced recombinant bacteria; 3. Supernatant of recombinant bacteria after induction; 4. Recombinant sonicated precipitate after induction

Figure 5 is the recombinant protein Western blot detection results;

In the figure, M. Pre-stained protein standard; 1. K99 natural pilus polyclonal antibody is the primary antibody; 2. 987P natural pilus polyclonal antibody is the primary antibody; 3. F41 natural pilus polyclonal antibody is the primary antibody; 4. K99 recombinant pilus polyclonal antibody was the primary antibody; 5.987P recombinant pilus polyclonal antibody was the primary antibody; 6. F41 recombinant pilus polyclonal antibody was the primary antibody; 7. PBS control group.

Detailed ways

Below in conjunction with embodiment and comparative example technical scheme of the present invention will be further described, but should not be interpreted as limiting the present invention:

Source of biological material in the present invention:

1. The standard strains Escherichia coli C83912, Escherichia coli C83916, and Escherichia coli C83919 were purchased from China Veterinary Microbiology Culture Collection Management Center;

2. The vectors pMD-18T and pET30a used were purchased from Takara Company;

3. The primers used were designed by ourselves and synthesized by Harbin Boshi Biological Company.

Example 1

Primers were designed according to the complete sequences of enterotoxigenic Escherichia coli adhesin K99, 987P, and F41 genes published in GenBank (accession numbers M35282.1, M35257.1, and M21788.1, respectively). The genome of Bacillus C83919 was used as a template, and target fragments were amplified by PCR respectively; among them, Escherichia coli C83912, Escherichia coli C83916, and Escherichia coli C83919 contained adhesin K99, adhesin 987P, and adhesin F41 respectively, and the sequences of the primers used were as follows:

P1: K99(F): CGG GGTACC ATGACTATTAACTTCAATGGCAAA (KpnI) (SEQ ID No. 3);

P2: K99(R): CGC GGATCC TGAAGTAGTAAATACGCCAGC (BamHI) (SEQ ID No. 4);

P3: 987P(F): CGC GGATCC GGTGGTGGTGGTTCTGCTGAAAACAACACCAGCCAG (BamHI) (SEQ ID No. 5);

P4:987P(R):C GAGCTC ACTAGCAGTGACAGTACCGGC(SacI) (SEQ ID No. 6);

P5: F41(F): C GAGCTC GGCGGTGGCGGCAGCGTATCTGGTTCAGTGATGGCT (SacI) (SEQ ID No. 7);

P6: F41 (R): AAGGAAAAAA GCGGCCGC ACTGAGGTCATCCCAATTGTG (NotI) (SEQ ID No. 8).

1. First, specific primers P1/P2 are used, and the genome of Escherichia coli C83912 is used as a template to carry out PCR amplification respectively. The PCR reaction system is a conventional reaction system in the prior art. PCR reaction program: pre-denaturation at 98°C for 10 s, denaturation at 98°C for 30 s, annealing at 51°C for 30 s, extension at 72°C for 1 min, 25 cycles, final extension at 72°C for 10 min, and storage at 4°C.

2. Use PCR product gel recovery kit to recover K99 PCR product, the steps are as follows:

1) Use a scalpel blade to quickly cut off the agarose gel of the target fragment under ultraviolet light, and put it into a 1.5mL centrifuge tube.

2) Add Extraction Buffer at a ratio of 1:3.

3) Place in a constant temperature water bath at 50°C for 10 minutes, and gently invert every 2 minutes.

4) Add the mixture in the previous step to the Spin column, centrifuge at 6000×g for 1 min, and discard the liquid in the collection tube.

5) Add 500μL Extraction Buffer to the Spin column, centrifuge at 12000×g for 1min, and discard the liquid in the solution.

6) Add 750μL Wash Buffer to the spin column, centrifuge at 12000×g for 1min, and discard the liquid in the tube.

7) Put the spin column back into the collection tube and centrifuge at 12000×g for 1 min. Put the spin column into a sterile 1.5mL EP tube.

8) Add 50μL Elution Buffer to the Spin column and let stand at room temperature for 1min. Centrifuge at 12000×g for 1 min, and the solution in the microcentrifuge tube contains the target DNA fragment. Store at -20°C.

3. Add A kit according to the blunt-ended DNA fragment, the specific steps are as follows

1) Configure and add A reaction liquid system as follows:

2) Incubate at 72°C for 30 minutes.

3) Stand on ice for 2 min to cool the reaction.

4. pMD-18T vector ligation reaction

The reaction system is as follows:

Solution I 5μL

Add A product 4μL

PMD-18T 1μL

Ligation overnight at 16°C.

5. Transformation: Take out the competent cells from -70°C and bury them in ice cubes, take out the ligation products from the 16°C water bath, remove the parafilm, and put them on ice. After the competent cells melt, add all the ligation products 100 μL DH5α competent cells, mix well. On ice for 30 minutes. Heat shock at 42°C for 90s, and act on ice for 2min. Then add 800 μL LB, shake at 170 r/min for 1 h at 37 °C. The culture was centrifuged at 5000r/min for 5min. Discard part of the supernatant, keep about 100 μL, mix well and apply kana-resistant LB plate. Incubate at 37°C for 12-16 hours.

6. Mini-extraction of plasmid DNA

1) Take 2 mL of fresh bacterial liquid, centrifuge at 12000×g for 1 min, and discard the supernatant.

2) Add 250μL Buffer S1 to suspend the bacterial pellet, and mix the suspension evenly, and no small bacterial clumps should be left.

3) Add 250 μL Buffer S2, gently and fully turn it up and down 4-6 times to mix evenly to fully lyse the bacteria.

4) Add 350μL Buffer S3, gently and fully turn up and down to mix 6-8 times, and centrifuge at 12000×g for 10min.

5) Aspirate the supernatant and transfer it to a preparation tube, centrifuge at 12000×g for 1 min, and discard the filtrate.

6) Add 500 μL Buffer W1 to the preparation tube, centrifuge at 12000×g for 1 min, and discard the filtrate.

7) Put the preparation tube back into the collection tube, add 700 μL Buffer W2, centrifuge at 12000×g for 1 min, discard the filtrate; wash once again with 700 μL Buffer W2 in the same way. Discard the filtrate.

8) Centrifuge at 12000×g for 1 min.

9) Transfer the preparation tube into a new 1.5mL centrifuge tube, add 70μL Eluent to the center of the preparation tube, and let stand at room temperature for 1min. Centrifuge at 12000×g for 1 min. Store at -20°C.

7. Identification of recombinant plasmids

1) PCR identification of recombinant plasmids

The extracted plasmid was amplified by PCR, electrophoresed on 1% agarose gel, and the results were observed.

2) Double enzyme digestion identification of the recombinant plasmid

Use KpnI and BamHI for enzyme digestion, and the double enzyme digestion system is as follows:

After mixing, 37°C for 5min, 80°C for 5min. 1% agarose gel electrophoresis identification results. The result was correctly named pMD-18T-K99-DH5α.

8. Enzyme digestion of the target fragment and vector: identify the correctly identified plasmid and pET30a by digestion with KpnI and BamHI, and the steps are the same as above. The digested product was purified with a PCR product purification kit, and the specific steps were as follows:

1) Analyze PCR products by agarose gel electrophoresis.

2) Determine the volume of the PCR product, transfer the product to a 1.5 mL centrifuge tube, and then add 4 to 5 times the volume of BufferCP.

3) Vortex to mix thoroughly, centrifuge briefly to remove capping liquid.

4) Put the HiBind DNA column into a 2mL collection tube.

5) Transfer the sample in step 3 into the HiBind DNA column, and centrifuge at 1000×g for 1 min at room temperature.

6) Discard the filtrate and put the HiBind DNA column back into the collection tube.

7) Add 700μL DNA Wash Buffer and centrifuge at 1000×g for 1min at room temperature.

8) Discard the filtrate. Repeat step 7, add 700 μL DNA Wash Buffer, and centrifuge at 1000×g for 1 min at room temperature.

9) Centrifuge at 13000×g for 2 minutes.

10) Transfer the HiBind DNA column into a new 1.5mL centrifuge tube, add 20 μL Lution buffer to the center of the preparation tube, and let stand at room temperature for 1 min. Centrifuge at 13000×g for 1 min. Store at -20°C.

9. Connection

The connection system is as follows:

Connect at 22°C for 20 minutes.

10. Take 10 μL of the ligation product, transform it into 100 μL DH5α competent cells, and send the positively identified plasmid pET30a-K99 for sequencing.

Example 2

Using the same method as in Example 1, 987P was amplified using P3/P4 and using the Escherichia coli C83916 genome as a template. The gel was recovered, connected to pMD-18T, and transformed into DH5α competent cells. After identification by PCR and double enzyme digestion, the positive plasmids were named pMD-18T-987P-DH5α respectively. pMD-18T-987P-DH5α and pET30a-K99 were digested with BamHI and SacI, the target fragments were recovered by gel cutting, and then ligated to construct pET30a-K99-987P, which was identified by PCR and double digestion. F41 was amplified using P5/P6 and using the Escherichia coli C83919 genome as a template. The gel was recovered, connected to pMD-18T, and transformed into DH5α competent cells. After identification by PCR and double enzyme digestion, the positive plasmids were named pMD-18T-F41-DH5α respectively. pMD-18T-F41-DH5α and pET30a-K99-987P were digested with SacI and NotI, the target fragments were recovered by gel cutting, and then ligated to construct pET30a-K99-987P-F41, which was identified by PCR and double digestion. The PCR amplification results of the three fragments are shown in Fig. 1 . The PCR detection of the recombinant plasmid is shown in Figure 2, and the enzyme digestion identification is shown in Figure 3. Positive plasmids were sent to Shanghai Sangon for sequencing. The positive plasmid transformed BL21(DE3) competent cells, named pET30a-K99-987P-F41-BL21(DE3).

Example 3

This example illustrates the induced expression of recombinant proteins.

The positive recombinant expression plasmid pET30a-K99-987P-F41-BL21(DE3) was transformed into Escherichia coli BL21(DE3), and a single colony was picked and cultured with shaking in LB liquid medium containing Kana at 37°C. When the OD600 value reached 0.6, IPTG was added to a final concentration of 0.5 mmol/L for induction. At the same time, the expression vector control group was set, and the bacterial liquid was taken 5 hours after induction of expression, centrifuged at 12000r/min for 1min, and the precipitate was preserved. The pellet was resuspended in PBS, ultrasonically lysed, centrifuged at 10,000 r/min for 20 min, and the supernatant and precipitate were collected for SDS-PAGE electrophoresis. The results are shown in Figure 4.

Example 4

This example illustrates the Western blot detection of recombinant proteins.

SDS-PAGE electrophoresis was performed on the protein sample of the recombinant strain pET30a-K99-987P-F41 expressing for 5 hours and the protein sample before induction, and the gel was taken out. Cut the filter paper and PVDF membrane according to the size of the gel. After soaking PVDF membrane in methanol solution for 15s, soak filter paper and gel in transfer buffer for 15min. Then carry out western blotting reaction in a semi-dry electroblotting apparatus, from the anode to the cathode in sequence is filter paper, PVDF membrane, gel, filter paper, each layer should be exhausted as much as possible of air bubbles, cover the anode cover, constant voltage 20V After energizing for 30 minutes, the protein in the gel will be transferred to the PVDF membrane. Block overnight at 4°C. Wash 3 times with PBST, 5min each time; immerse the PVDF membrane in 1:200 diluted natural pili K99 pili polyclonal antibody, 37°C for 1h, wash 3 times with PBST, 5min each time; then immerse the membrane in 1:5000 diluted In the HRP-labeled goat anti-mouse IgG antibody, 37 ° C for 1 h; add DAB solution to develop color, and observe the results.

The target protein was transferred to PVDF membrane after SDS-PAGE electrophoresis, and non-recombinant pilus polyclonal antibodies K99, 987P and F41 and recombinant pilus polyclonal antibodies K99, 987P and F41 were used as primary antibodies. Goat anti-mouse HRPIgG was used as the secondary antibody. The Western blot detection results are shown in Figure 5, and they all have a positive band around 60KDa. There was no reaction band in the control group. It was confirmed that the recombinant fusion protein K99-987p-F41 could be specifically recognized by both the non-recombinant antigen polyclonal antibody and the recombinant antigen polyclonal antibody. It shows that the fusion protein has good immunological activity.

Comparative example 1

Escherichia coli whole cell trivalent inactivated vaccine, Escherichia coli natural pili K99, 987P and F41 trivalent subunit vaccine, Escherichia coli recombinant pili K99, 987P and F41 trivalent subunit vaccine and Escherichia coli pET30a-K99- The 987P-F41-BL21(DE3) tandem expression subunit vaccine was used for comparative experiments.

(1) Preparation of Escherichia coli whole cell trivalent inactivated vaccine:

Three strains of Escherichia coli were selected as vaccine strains, studied according to the monovalent vaccine procedure first, and then mixed at a ratio of 1:1:1, with a final concentration of 2×10 ¹⁰ CFU, followed by the trivalent vaccine development procedure.

(2) Preparation of Escherichia coli natural pili K99, 987P and F41 trivalent subunit vaccines:

The natural pili K99, 987P and F41 were mixed in a ratio of 1:1:1 according to the concentration of the pili purified liquid measured. The final concentration was 50μg/0.1ml.

(3) Preparation of Escherichia coli recombinant pili K99, 987P and F41 trivalent subunit vaccines:

The recombinant proteins K99, 987P and F41 were mixed in a ratio of 1:1:1 according to the measured concentration, and the final concentration was 50 μg/0.1ml.

(4) Preparation of Escherichia coli pET30a-K99-987P-F41-BL21(DE3) tandem expression subunit vaccine:

According to the measured concentration, the purified pET30a-K99-987P-F41-BL21(DE3) protein was adjusted to a final concentration of 50 μg/0.1 ml.

The test is divided into 5 groups, namely, test group 1: Escherichia coli recombinant pilus K99-987P-F41 subunit vaccine, test group 2: Escherichia coli non-recombinant pilus K99, 987P and F41 trivalent subunit vaccine, third: Escherichia coli Whole cell trivalent inactivated vaccine, test four: Escherichia coli recombinant pili K99, 987P and F41 trivalent subunit vaccine, and control group: PBS. 10 mice per group.

Blood was collected from the tail vein before immunization to prepare serum. Experimental groups 1, 2 and 4 were mixed with 0.1mg/ml purified protein and Freund's complete adjuvant at a ratio of 1:1, emulsified, and 0.2ml/mouse were subcutaneously injected with experimental group 1 to immunize mice. Blood was collected from the tail vein 14 days after the first immunization to prepare serum. Experimental groups 1, 2 and 4 respectively immunized mice with 0.2ml/mouse subcutaneous injection of 0.1mg/ml purified protein and incomplete Freund's adjuvant at a ratio of 1:1 and experimental group 3 respectively. The second immunization was carried out, and 14 days after the second immunization, blood was collected from the tail vein to prepare serum. The third immunization was carried out, and blood was collected from the tail vein 14 days after the third immunization to prepare serum.

14 days after the third immunization, 10 mice from the experimental group 1, 2, 3, 4 and the control group were respectively challenged with Escherichia coli, and the half-lethal dose was used, that is, the challenge dose of each mouse was 5.0×10 ⁸ CFU/mL, the challenge site is subcutaneous injection. Continuously observe for one week, count the number of infection and death of mice and calculate the protection rate. The results are shown in Table 1.

Table 1 Results of challenge experiment

It can be seen from Table 1 that all the control groups died after the challenge. Among them, the total protection rate of the test group was 60%-80%; the protection rate of the tandem expression K99-987P-F41 subunit vaccine and the whole bacterial inactivated vaccine were both 80%, and the recombinant K99, 987P and F41 pili trivalent subunit Unit vaccines followed, and finally non-recombinant pili K99, 987P and F41 trivalent subunit vaccines. It was found that the four vaccines all obtained immunity, and the immune effect was: Escherichia coli whole bacterial body trivalent inactivated vaccine > Escherichia coli recombinant tandem K99-987P-F41 protein subunit vaccine > Escherichia coli non-recombinant pilus K99, 987P and F41 three Valence subunit vaccine > Escherichia coli recombinant pili K99, 987P, F41 trivalent subunit vaccine. Although there is little difference between Escherichia coli recombinant tandem K99-987P-F41 protein subunit vaccine and E. The content of the antigen will be greatly reduced, and the conformation of the antigen protein may be changed after inactivation, and the vaccine protection rate is low, but these problems will not occur in the tandem K99-987P-F41 protein subunit vaccine. At the same time, the tandem K99-987P-F41 protein subunit vaccine can induce a variety of antibodies after one immunization, overcoming the E. coli non-recombinant pili K99, 987P and F41 trivalent subunit vaccines and E. The problem of inconvenient preparation of 987P and F41 trivalent subunit vaccines. Therefore, the Escherichia coli recombinant tandem K99-987P-F41 protein subunit vaccine has broad application prospects.

Claims (5)

1. K99-987P-F41 recombinant protein, its aminoacid sequence is shown in SEQ ID No.2.

2. the gene of coding claim 1 described K99-987P-F41 recombinant protein, its nucleotide sequence is shown in SEQ ID No.1.

3. the preparation method of the described K99-987P-F41 recombinant protein of claim 1, it is characterized in that: this method may further comprise the steps:

(1) enterotoxigenic escherichia coli adhesin K99, the 987P that has delivered according to GenBank, F41 gene complete sequence (accession number is respectively M35282.1, M35257.1, M21788.1) design primer, genome with intestinal bacteria C83912, intestinal bacteria C83916, intestinal bacteria C83919 is template, and PCR amplifies the purpose fragment respectively;

(2) three fragment series connection are connected into carrier pET30a, construction of expression vector pET30a-K99-987P-F41;

(3) expression vector pET30a-K99-987P-F41 is converted into competent escherichia coli cell, and the IPTG abduction delivering obtains recombinant protein.

4. preparation method according to claim 3, it is characterized in that: described primer sequence is as follows, and wherein underscore is restriction enzyme site:

P1：K99(F)：CGG GGTACCATGACTATTAACTTCAATGGCAAA（KpnI）（SEQ ID No.3）；

P2：K99(R)：CGC GGATCCTGAAGTAGTAAATACGCCAGC(BamHI)（SEQ ID No.4）；

P3：987P(F)：CGC GGATCCGGTGGTGGTGGTTCTGCTGAAAACAACACCAGCCAG(BamHI)（SEQ ID No.5）；

P4：987P(R)：C GAGCTCACTAGCAGTGACAGTACCGGC(SacI)（SEQ ID No.6）；

P5：F41(F)：C GAGCTCGGCGGTGGCGGCAGCGTATCTGGTTCAGTGATGGCT(SacI)（SEQ ID No.7）；

P6：F41(R)：AAGGAAAAAA GCGGCCGCACTGAGGTCATCCCAATTGTG(NotI)（SEQ ID No.8）。

5. the application of the described K99-987P-F41 recombinant protein of claim 1 in preparation enterotoxigenic escherichia coli vaccine.

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