Uropathogenic escherichia coli recombinant protein composition LS, construction, expression and purification method therefor, and use thereof

Abstract

An uropathogenic escherichia coli recombinant protein composition LS, a construction, expression and purification method therefor, and a use thereof, relating to the technical field of genetic engineering. The method comprises: carrying out PCR amplification on LpcA and SurA genes, and respectively inserting the LpcA and SurA genes into multiple cloning sites of a cloning vector to construct a recombinant vector; then converting the recombinant vector into an escherichia coli expression protein; and finally, carrying out purification to obtain a composition LS of an LpcA protein and an SurA protein, wherein the composition LS can be used in a urinary tract infection vaccine. The present invention has the following advantages: a protein vaccine can induce relatively strong cell and humoral immune response of animals; and animal challenge experiments show that the protein vaccine has the protection effect of reducing the bacterial loads of the bladder and the kidney.

Description

A uropathogenic Escherichia coli recombinant protein composition LS, its construction, expression, purification method and application Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a uropathogenic Escherichia coli recombinant protein composition LS, and methods for constructing, expressing, purifying, and applying the composition. Background Art

[0002] Urinary tract infection (UTI) is the second most serious infectious disease threatening human health. Uropathogenic Escherichia coli is the primary causative agent. Reportedly, 60% of women and 12% of men experience at least one UTI in their lifetime. 20%-30% of female patients develop a secondary infection within 3-4 months of their first UTI; some patients experience multiple infections, or even a lifelong infection. Postmenopausal women, pregnant women, children, and those with immunocompromised conditions are particularly susceptible to UTI. Furthermore, factors such as indwelling urinary catheters and anatomical abnormalities of the urinary tract contribute to increased risk of UTI. UTIs severely impact patients' quality of life and can even lead to life-threatening urosepsis. Preventing and reducing recurrence of UTIs remains a major challenge in this area of ​​research.

[0003] While clinical antibiotic treatment can alleviate acute urinary tract infections (UTIs), its effectiveness in preventing and treating recurrent UTIs is limited. Furthermore, overuse of antibiotics can not only lead to bacterial resistance and treatment failure, but can also induce refractory UTIs. UTI vaccines, which stimulate antigen-specific immune responses and thus play a dual role in preventing and treating infection, have become a research hotspot in recent years. Based on their composition and preparation methods, UTI vaccines primarily include whole-cell / lysate vaccines, live-attenuated vaccines, and subunit vaccines. Whole-cell / lysate vaccines, now available in Europe, offer new clinical approaches for the treatment and prevention of recurrent UTIs, but require long-term, continuous vaccination to achieve optimal results. The protective immune response induced by live-attenuated vaccines is limited in duration, leading to limited research on this type of vaccine. Protein subunit vaccines, a novel vaccine type that combines a bacterial protective protein antigen with a delivery adjuvant, have become a hot topic in UTI research due to their clear composition, high safety profile, and the ability to induce a strong immune memory response, resulting in prolonged protection.

[0004] SurA, a parvulin-like peptidyl-prolyl isomerase (PPIase), functions as a molecular chaperone in the periplasm of Escherichia coli (the space between the inner and outer membranes of bacteria), participating in the assembly, transport, and folding of outer membrane proteins. LpcA (also known as GmhA), a cytoplasmic phosphoheptose isomerase, is involved in the synthesis of triglycerides, a core component of the lipopolysaccharide (LPS) precursor. Previous studies have shown that knockout of the genes encoding LpcA and SurA in uropathogenic Escherichia coli significantly reduced the bacterial survival and persistence in mice. Therefore, we hypothesized that these two proteins could serve as candidate targets for drug treatment and vaccine development for urinary tract infections caused by uropathogenic Escherichia coli. However, how to genetically engineer these two proteins and validate their use in urinary tract vaccines remains a technical challenge.

[0005] Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a uropathogenic Escherichia coli recombinant protein composition LS, its construction, expression, purification method and application.

[0007] The present invention is specifically implemented by the following technical solutions:

[0008] In a first aspect, the present invention provides a uropathogenic Escherichia coli recombinant protein composition LS, which contains recombinant protein LpcA and recombinant protein SurA. The amino acid sequence of the recombinant protein LpcA is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant protein SurA is shown in SEQ ID NO.4.

[0009] Furthermore, the mass ratio of the recombinant protein LpcA to the recombinant protein SurA is 1:1.

[0010] The second aspect of the present invention provides use of the above-mentioned uropathogenic Escherichia coli recombinant protein composition LS in the preparation of a drug for treating urinary tract infection caused by uropathogenic Escherichia coli.

[0011] The third aspect of the present invention provides the use of the above-mentioned uropathogenic Escherichia coli recombinant protein composition LS in the preparation of a vaccine for treating urinary tract infection caused by uropathogenic Escherichia coli.

[0012] A fourth aspect of the present invention provides a method for constructing, expressing and purifying a uropathogenic Escherichia coli recombinant protein composition LS, comprising the following steps:

[0013] 1) The LpcA and SurA genes were amplified by PCR and inserted into the multiple cloning site of the cloning vector pET30a(+) to construct the recombinant vectors pET30a(+)-LpcA and pET30a(+)-SurA, respectively;

[0014] 2) Transforming the recombinant vector into Escherichia coli to obtain a strain expressing the fusion proteins LpcA and SurA, and using the strain to express the recombinant proteins LpcA and SurA;

[0015] 3) Purification of recombinant proteins LpcA and SurA.

[0016] Furthermore, the step 1) specifically includes:

[0017] (1) Based on the gene sequences of LpcA GeneID 949134, 579 bp and SurA GeneID 944812, 1287 bp in E. coli str. K12 in GenBank and the restriction site sequence of the cloned vector, upstream and downstream primers containing nucleases were designed: surA F and surA R; LpcA F and LpcA R, respectively. The nucleotide sequence of surA F is shown in SEQ ID NO. 5, the nucleotide sequence of surA R is shown in SEQ ID NO. 6, the nucleotide sequence of LpcA F is shown in SEQ ID NO. 7, and the nucleotide sequence of LpcA R is shown in SEQ ID NO. 8;

[0018] (2) DNA of uropathogenic Escherichia coli UTI89 strain was extracted and used as a template, and the LpcA and surA gene sequences were amplified by PCR using the primers in step (1); the PCR products were recovered and purified by gel recovery and set aside to obtain LpcA PCR amplification products and surA PCR amplification products;

[0019] (3) Extraction of pET30a(+) plasmid: Amplify Escherichia coli containing pET30a(+) plasmid to 5 mL, and extract pET30a(+) using a plasmid extraction kit;

[0020] (4) Using EcoI and HindIII endonucleases, the LpcA PCR amplification product, the surA PCR amplification product in step (2) and the pET30a(+) plasmid extracted in step (3) were respectively digested; the LpcA PCR amplification product, the surA PCR amplification product and the pET30a(+) plasmid after digestion were purified by PCR product purification and gel recovery purification, respectively; the purified LpcA gene and the pET30a(+) plasmid, and the surA gene and the pET30a(+) plasmid were ligated under the action of T4 ligase; the ligation products were transformed into E. coli DH5α, and after cloning screening on an antibiotic plate, positive clones were picked, plasmids were extracted, and sequencing was performed to obtain recombinant vectors pET30a(+)-LpcA and pET30a(+)-SurA.

[0021] Further, the step 2) specifically includes:

[0022] (a) The successfully constructed pET30a(+)-LpcA and pET30a(+)-SurA plasmid vectors were respectively transformed into the expression strain E. coli BL21(DE3) to obtain recombinant protein-conserving strains;

[0023] (b) taking the bacterial strain preserved in step (a) and performing activation, shaking amplification culture, induced shaking culture and centrifugation in sequence to obtain bacterial cells of the recombinant protein-preserving bacterial strain;

[0024] (c) The cells obtained in step (b) were resuspended in PB buffer, ultrasonically disrupted under ice bath, and centrifuged. The supernatant and precipitate were collected, and the supernatant and precipitate were subjected to polyacrylamide gel electrophoresis to analyze protein expression.

[0025] Further, the step 3) specifically includes:

[0026] (A) Bacteria expressing the fusion proteins LpcA and SurA were amplified, the harvested cells were resuspended in PB buffer, sonicated on ice, and the supernatant containing the recombinant proteins LpcA and SurA was collected by centrifugation and sterilized by filtration for later use.

[0027] (B) Purify LpcA and SurA from the supernatant obtained in step (A), perform affinity chromatography, and elute to obtain recombinant proteins LpcA and SurA.

[0028] Furthermore, the affinity chromatography and elution in step (B) are specifically as follows: first, the empty chromatography column is cleaned with sterile water until the water flow is smooth, 1 ml of nickel ion metal chelate affinity chromatography medium Ni-NTA is added close to the column wall, and after equilibrating the medium with 10 ml of imidazole binding buffer, the supernatant containing recombinant proteins LpcA and SurA is added to the medium for binding; after washing with 10 ml of imidazole binding buffer and 10 mL of imidazole rinsing buffer, the sample is gradient eluted with 50 mM, 100 mM, 200 mM, and 300 mM imidazole elution buffer; LpcA recombinant protein is eluted in 200-300 mM imidazole elution buffer, and SurA recombinant protein is eluted in 200-300 mM imidazole elution buffer, and protein quantitative detection is performed and stored for future use.

[0029] The present invention has the beneficial effects of successfully constructing a strain expressing LpcA and SurA proteins using genetic engineering techniques, and establishing a chromatographic purification method for these proteins. The combination of LpcA and SurA can induce strong cellular and humoral immune responses and exhibits a certain degree of protection against uropathogenic Escherichia coli in animals, potentially enabling the development of vaccines against uropathogenic Escherichia coli. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the expression and purification results of the recombinant proteins LpcA and SurA. (A) Expression results of the recombinant proteins LpcA and SurA. Lane M, marker; Lane 1, LpcA-expressing bacterial cell fragmentation; Lane 2, supernatant of LpcA-expressing bacterial cell fragmentation; Lane 3, precipitate of LpcA-expressing bacterial cell fragmentation; Lane 4, SurA-expressing bacterial cell fragmentation; Lane 5, supernatant of SurA-expressing bacterial cell fragmentation; Lane 6, precipitate of SurA-expressing bacterial cell fragmentation. (B) Purification results of the recombinant proteins LpcA and SurA. Lane M, marker; Lanes 1-2, purified LpcA; Lanes 3-4, purified SurA.

[0031] Figure 2 shows antigen-specific IFN-γ, IL-2, and IL-7 levels induced by the subunit vaccine LS / DP 4 weeks after the last immunization. (A) LpcA- and SurA-specific IFN-γ levels; (B) LpcA- and SurA-specific IL-2 levels; (C) LpcA- and SurA-specific IL-17 levels.

[0032] Figure 3 shows the levels of antigen-specific IFN-γ, IL-2, and IL-7 induced by the subunit vaccine LS / DP 8 weeks after the last immunization. (A) LpcA-specific IFN-γ level; (B) SurA-specific IFN-γ level; (C) LpcA-specific IL-2 level; (D) SurA-specific IL-2 level; (E) LpcA-specific IL-17 level; (F) SurA-specific IL-17 level.

[0033] Figure 4 shows the levels of antigen-specific IgG and IgG2c antibodies induced by the subunit vaccine LS / DDA-PolyI:C 4 weeks after the last immunization. (A) LpcA-specific IgG2c level; (B) LpcA-specific IgG2c level; (C) SurA-specific IgG level; (D) SurA-specific IgG2c level.

[0034] Figure 5 shows the levels of antigen-specific IgG and IgG2c antibodies induced by the subunit vaccine LS / DDA-PolyI:C 8 weeks after the last immunization. (A) LpcA-specific IgG2c level; (B) LpcA-specific IgG2c level; (C) SurA-specific IgG level; (D) SurA-specific IgG2c level.

[0035] Figure 6 shows the protective effect of the subunit vaccine LS / DDA-PolyI:C in mice.

[0036] FIG7 is a diagram showing the construction of recombinant gene vectors pET30a(+)-LpcA and pET30a(+)-SurA. DETAILED DESCRIPTION

[0037] The present invention is further described below with reference to the following examples.

[0038] Example 1: Construction, expression and purification of uropathogenic Escherichia coli recombinant protein composition LS

[0039] First, the pET30a(+)-LpcA and pET30a(+)-SurA plasmid vectors were constructed, respectively. Figure 7 shows the construction diagram of the recombinant gene vectors pET30a(+)-LpcA and pET30a(+)-SurA. Second, the successfully constructed plasmid vectors were transformed into E. coli BL21(DE3) to express the recombinant proteins LpcA and SurA. Then, based on the recombinant His tags on the proteins, the two recombinant proteins were successfully purified by affinity chromatography. Finally, the recombinant proteins LpcA and SurA were mixed in a 1:1 ratio to construct the recombinant protein composition LS.

[0040] The specific steps are as follows:

[0041] 1. Construction of recombinant gene vector pET30a(+)-LpcA

[0042] (1) Based on the gene sequence of LpcA (GeneID 949134, 579 bp) in Escherichia coli (E. coli str. K12) in GenBank and the restriction site sequence of the cloned vector, upstream and downstream primers were designed, which are:

[0043] Upstream primer lpcA F:

[0044] (The underlined italics are the BamHI restriction enzyme cleavage site sequence), the nucleotide sequence of which is shown in SEQ ID NO.5;

[0045] Downstream primer lpcA R:

[0046] (The underlined italics indicate the EcoRI restriction enzyme cleavage site sequence), and its nucleotide sequence is shown in SEQ ID NO.6.

[0047] (2) PCR amplification of LpcA gene: Using DNA of uropathogenic Escherichia coli as a template, the LpcA gene sequence was amplified by PCR using the two primers mentioned above; the PCR product was recovered and purified by gel recovery and then used for later use.

[0048] (3) Construction of the pET30a(+)-LpcA recombinant vector: The LpcA PCR amplification product and the pET30a(+) plasmid were digested with BamHI and EcoRI endonucleases, respectively. The LpcA PCR amplification product and the pET30a(+) plasmid digestion products were purified by PCR product purification and gel recovery, respectively. The purified LpcA gene and the pET30a(+) plasmid were ligated with T4 ligase. The ligation product was transformed into E. coli DH5α. After colony screening on antibiotic plates, positive clones were selected, plasmids were extracted, and sequencing was performed for identification.

[0049] (4) The identification results showed that the LpcA gene sequence in the plasmid extracted from the recombinant strain E. coli DH5 had no mutation and was consistent with the theoretical sequence in GenBank. The nucleotide sequence of the LpcA gene is shown in SEQ ID NO.1.

[0050] 2. Construction of recombinant gene vector pET30a(+)-SurA

[0051] (1) Based on the gene sequence of SurA (GeneID944812, 1287 bp) in Escherichia coli (E. coli str. K12) in GenBank and the restriction site sequence of the cloned vector, upstream and downstream primers were designed, which are:

[0052] Upstream primer surA F:

[0053] (The underlined italics indicate the EcoRI restriction enzyme cleavage site sequence), the nucleotide sequence of which is shown in SEQ ID NO.7;

[0054] Downstream primer surA R:

[0055] (The underlined italics indicate the HindⅢ restriction enzyme cleavage site sequence), and its nucleotide sequence is shown in SEQ ID NO.8.

[0056] (2) PCR amplification of surA gene: DNA of uropathogenic Escherichia coli UTI89 strain was extracted as a template, and the surA gene sequence was amplified by PCR using the two primers mentioned above; the PCR product was recovered and purified by gel recovery and then used for later use.

[0057] (3) Extraction of pET30a(+) plasmid: Amplify the Escherichia coli strain containing the pET30a(+) plasmid to 5 mL, and extract pET30a(+) using a plasmid extraction kit.

[0058] (4) Construction of the pET30a(+)-LpcA recombinant vector: The surA PCR amplification product and the pET30a(+) plasmid were digested with EcoRI and HindIII endonucleases, respectively. The surA PCR amplification product and the pET30a(+) plasmid digestion products were purified by PCR and gel recovery, respectively. The purified surA gene and the pET30a(+) plasmid were ligated with T4 ligase. The ligation product was transformed into E. coli DH5α. After colony screening on antibiotic plates, positive clones were selected, plasmids were extracted, and sequencing was performed for identification.

[0059] (5) The identification results showed that the surA gene sequence in the plasmid extracted from the recombinant strain E. coli DH5 had no mutation and was consistent with the theoretical sequence in GenBank. The nucleotide sequence of the surA gene was shown in SEQ ID NO.3.

[0060] 3. Expression of recombinant proteins LpcA (192aa) and SurA (428aa)

[0061] (1) The successfully constructed pET30a(+)-LpcA and pET30a(+)-SurA plasmid vectors were transformed into the expression strain E. coli BL21 (DE3), respectively. Positive clones were screened by PCR verification and sequenced. The identified suitable strains were stored in 80% glycerol, which was the recombinant protein storage strain.

[0062] (2) First, 10 μL of the bacterial strain was added to 5 ml of LB liquid medium for activation; then, 1 mL of the activated bacterial cells was added to 200 mL of LB liquid medium and cultured with shaking until the OD600 nm reached 0.6. 100 μL of IPTG (1.0 mmol / l) was added and the culture was induced with shaking at 25°C for 12 h; the bacterial cells were collected by centrifugation at 10,000 rpm / min at 4°C for 10 min.

[0063] (3) The cells were resuspended in PB buffer (Na2HPO4·12H2O 20 mMol / L, NaH2PO4·2H2O 20 mMol / L, pH 7.4) at 10 ml / g wet bacteria and disrupted by ultrasonication for 1 h on ice (180-200 W, 4 s on, 5 s off). The supernatant and precipitate were collected after centrifugation at 10,000 RPM / min for 20 min and subjected to polyacrylamide gel electrophoresis for protein expression analysis.

[0064] (4) The results showed (as shown in Figure 1A) that compared with the E. coli BL21 (DE3) empty bacteria, the LpcA expression strain had obvious specific protein expression bands at a molecular weight of 17 kD and the SurA expression strain had a molecular weight of about 29 kD. The proteins mainly appeared in the supernatant, and relatively less proteins were found in the precipitate.

[0065] 4. Purification of recombinant proteins LpcA and SurA

[0066] (1) The strain expressing the fusion proteins LpcA and SurA was amplified in large quantities. The collected strains were resuspended in 20 mM PB buffer, ultrasonically disrupted for about 1 hour in an ice bath, and centrifuged at 4°C / 10,000 rpm for 10 minutes. After centrifugation, the supernatant containing the recombinant proteins LpcA and SurA was collected and sterilized by filtering with a 0.45 μm filter for later use.

[0067] (2) First, the empty chromatography column was cleaned with sterile water until the water flow was smooth. 1 ml of nickel ion metal chelate affinity chromatography medium (Ni-NTA) was added close to the column wall. After the medium was equilibrated with 10 ml of imidazole binding buffer, the supernatant containing the recombinant proteins LpcA and SurA was added to the medium for binding. Secondly, after washing with 10 ml of imidazole binding buffer and 10 mL of imidazole rinse buffer, the sample was gradient eluted with 50 mM, 100 mM, 200 mM, and 300 mM imidazole elution buffer. Finally, the elution concentration of the target protein was determined based on the results of polyacrylamide gel (SDS-PAGE) electrophoresis.

[0068] (3) The results showed that the recombinant protein LpcA was eluted in 200-300 mM imidazole elution buffer; the recombinant protein SurA was eluted in 200-300 mM imidazole elution buffer. After affinity chromatography purification, both recombinant proteins were obtained with a purity greater than 90%. As shown in Figure 1B, the purification results of the recombinant proteins LpcA and SurA. The purified LpcA and SurA proteins were mixed in a mass ratio of 1:1 to obtain the recombinant protein composition LS. After determination, the amino acid sequence of the recombinant protein LpcA is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant protein SurA is shown in SEQ ID NO.4.

[0069] Example 2: Preparation of subunit vaccines LS / DDA-PolyI:C (LS / DP), L / DP and S / DP

[0070] The recombinant protein composition LS and the individual proteins L and S obtained in Example 1 were diluted to 0.4 mg / mL with PBS (phosphate buffered saline). PolyI:C was dissolved in PBS to 0.5 mg / mL. The cationic liposome dimethyltridecyl ammonium bromide (DDA) was prepared at 2.5 mg / mL with sterile distilled water. The mixture was incubated at 80°C for 10 minutes and then cooled to room temperature. 50 μL of the PolyI:C solution was thoroughly mixed with an equal amount of the protein solution and allowed to stand at room temperature for 1 minute. 100 μL of the DDA solution was added dropwise to the mixed solution and then fully emulsified to a uniform emulsion. This yielded the subunit vaccines LS / DP, L / DP, and S / DP. For immunization, the vaccine dosage was 200 μL per mouse.

[0071] Example 3: Evaluation of the immunological activity of subunit vaccines LS / DP, L / DP and S / DP

[0072] 1. Experimental materials: subunit vaccine LS / DDA-PolyI:C obtained in Example 1; phosphate buffered saline (PBS).

[0073] 2. Experimental animals: C57BL / 6 mice.

[0074] 3. Experimental animal grouping (three groups in total):

[0075] (1) PBS (negative control); (2) LS / DP; (3) L / DP; (4) S / DP; (5) DDA-PolyI:C (DP adjuvant control).

[0076] 4. Immunized animals:

[0077] In week 0, the experimental group animals were immunized once with the prepared subunit vaccine (200 μL / animal) subcutaneously in the groin, and the control group was immunized at the same time. After the experimental group animals were immunized in week 0, they were boosted with the same dose twice in week 2 and week 4 respectively.

[0078] 5. Immune index determination method:

[0079] (1) ELISA technique was used to detect the levels of antigen-specific IFN-γ, IL-2, and IL-7 secreted by mouse spleen

[0080] Four and eight weeks after the last immunization, spleen lymphocytes were aseptically isolated from mice, and the secretion levels of IFN-γ, IL-2, and IL-7 by spleen lymphocytes after LpcA and SurA stimulation were detected by enzyme-linked immunosorbent assay (ELISA).

[0081] Specific steps: The spleen was removed aseptically, ground and filtered through a 200-mesh nylon mesh, and lymphocytes were separated using lymphocyte separation medium. The separated lymphocytes were added to a 24-well plate (5×10 6 Each well was then supplemented with the stimulatory proteins LpcA (final concentration 5 μg / mL) and SurA (final concentration 5 μg / mL). The cells and proteins were incubated at 37°C, 5% CO2 for 72 hours. After incubation, the cell culture supernatant was collected and cytokine secretion levels were quantitatively analyzed by ELISA.

[0082] The results showed that immunization of mice with the subunit vaccine LS / DDA-PolyI:C of the present invention induced spleen cells to produce high levels of IFN-γ, IL-2, and IL-7 against specific antigens, which were significantly higher than those in the PBS and DP groups, as shown in Figures 2 and 3. This result suggests that immunization with the subunit vaccine LS / DP can induce high levels of cellular immune responses (Th1, Th2, and Th17) in mice.

[0083] (2) ELISA method to detect the level of antigen-specific antibodies (IgG and IgG2c) secreted by mice

[0084] Four and eight weeks after the last immunization, peripheral blood was collected from the mice through the orbital cavity, and the serum was separated. The secretion levels of LpcA and SurA-specific antibodies IgG and IgG2c in the serum were detected by enzyme-linked immunosorbent assay (ELISA).

[0085] Specific steps: 96-well plates (100 μL / well) were coated with LpcA (5 μg / mL) and SurA (5 μg / mL) overnight at 4°C. The plates were washed three times with 300 μL / well of PBST (1 min / well). Serum samples were serially diluted from 1:400 to 1:20,480, and the diluted serum samples were added to the coated 96-well plates and incubated at 37°C for 1 hour. After washing the plates as described above, 200 μL / well of 1:50,000 dilutions of rabbit anti-mouse IgG1 and 1:50,000 dilutions of rabbit anti-mouse IgG2c were added and incubated at 37°C for 1 hour. After washing, 100 μL / well of TMB colorimetric solution was added and the color was developed at room temperature in the dark for 15 minutes. After development, 100 μL / well of stop solution (2N H₂SO₄) was added to terminate the reaction. The OD values ​​were measured at 450 nm.

[0086] The results showed that the antibody levels produced by mice immunized with the subunit vaccine LS / DP of the present invention were higher than those in the PBS group and the DDA-PolyI:C group, as shown in Figures 4 and 5. This result suggests that the subunit vaccine LS / DP can induce a high level of humoral immune response in mice.

[0087] Example 4: Evaluation of the protective effect of the subunit vaccine LS / DP

[0088] 1. Experimental materials: subunit vaccines LS / DP, L / DP, S / DP, phosphate buffered saline (PBS).

[0089] 2. Experimental animals: C57BL / 6 mice.

[0090] 3. Experimental animal grouping (five groups in total):

[0091] (1) PBS (negative control); (2) LS / DP; (3) L / DP; (4) S / DP; (5) DP (adjuvant control).

[0092] 4. The animal immunization method and time point are the same as in Example 3.

[0093] 5. Evaluation of protection effect:

[0094] After vaccination, the uropathogenic Escherichia coli UTI89 strain was challenged at week 4 by intraurethral catheterization with a challenge dose of 5×10 7 CFU / mouse.

[0095] One week after challenge with the strain, bacterial loads in the bladder and kidneys were assessed. Specific methods: Mouse bladders and kidneys were aseptically removed and ground in a tissue grinder. 1 mL of PBS buffer was added, and the resulting solution was serially diluted. 0.1 mL of each gradient solution was inoculated onto LB agar plates. The cultures were incubated at 37°C for 12 hours, and CFU counts were performed. The results showed that bacterial loads in the organs of mice immunized with the subunit vaccine LS / DP were significantly lower than those in the PBS and adjuvant DP control groups, as shown in Figure 6.

[0096] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A uropathogenic Escherichia coli recombinant protein composition LS, characterized in that It contains recombinant protein LpcA and recombinant protein SurA, the amino acid sequence of the recombinant protein LpcA is shown as SEQ ID NO.2, and the amino acid sequence of the recombinant protein SurA is shown as SEQ ID NO.

4.

2. A uropathogenic Escherichia coli recombinant protein composition LS according to claim 1, characterized in that The mass ratio of the recombinant protein LpcA to the recombinant protein SurA is 1:

1.

3. Use of a uropathogenic Escherichia coli recombinant protein composition LS as claimed in claim 1 or 2 in the preparation of a drug for treating urinary tract infection caused by uropathogenic Escherichia coli.

4. Use of a uropathogenic Escherichia coli recombinant protein composition LS as claimed in claim 1 or 2 in the preparation of a vaccine for treating urinary tract infection caused by uropathogenic Escherichia coli.

5. The method for constructing, expressing and purifying a uropathogenic Escherichia coli recombinant protein composition LS according to claim 1, characterized in that The following steps are involved: 1) The LpcA and SurA genes were amplified by PCR and inserted into the multiple cloning site of the cloning vector pET30a(+) to construct the recombinant vectors pET30a(+)-LpcA and pET30a(+)-SurA; 2) transforming the recombinant vector into Escherichia coli to obtain a strain expressing the fusion proteins LpcA and SurA, and using the strain to express the recombinant proteins LpcA and SurA; 3) Purification of recombinant proteins LpcA and SurA.

6. The method according to claim 5, characterized in that The step 1) specifically includes: (1) According to the gene sequences of LpcA GeneID 949134, 579 bp and SurAGeneID 944812, 1287 bp in Escherichia coli E. coli str.K12 in GenBank and the restriction site sequence of the cloned vector, upstream and downstream primers containing nucleases were designed respectively: surA F and surA R; LpcA F and LpcA R, wherein the nucleotide sequence of surA F is shown in SEQ ID NO.5, the nucleotide sequence of surA R is shown in SEQ ID NO.6, the nucleotide sequence of LpcA F is shown in SEQ ID NO.7, and the nucleotide sequence of LpcA R is shown in SEQ ID NO.8; (2) extracting DNA of uropathogenic Escherichia coli UTI89 strain as a template, using the primers in step (1) to amplify LpcA and surA gene sequences by PCR; the PCR products are recovered and purified by gel recovery and set aside to obtain LpcA PCR amplification products and surA PCR amplification products; (3) Extraction of pET30a(+) plasmid: Amplify the Escherichia coli strain containing the pET30a(+) plasmid to 5 mL, and use a plasmid extraction kit to extract pET30a(+); (4) using EcoI and HindIII endonucleases to digest the LpcA PCR amplification product in step (2), the surA PCR amplification product and the pET30a(+) plasmid extracted in step (3), respectively; the LpcA PCR amplification product, the surA PCR amplification product and the pET30a(+) plasmid after digestion were purified by PCR product purification and gel recovery purification, respectively; the purified LpcA gene and the pET30a(+) plasmid, the surA gene and the pET30a(+) plasmid were ligated under the action of T4 ligase; the ligation products were transformed into E. coli DH5α, cloned and screened on antibiotic plates, positive clones were picked, plasmids were extracted, and sequencing was performed to obtain recombinant vectors pET30a(+)-LpcA and pET30a(+)-SurA.

7. The method according to claim 5, characterized in that The step 2) specifically includes: (a) The successfully constructed pET30a(+)-LpcA and pET30a(+)-SurA plasmid vectors were transformed into the expression strain Escherichia coli BL21(DE3) to obtain recombinant protein storage strains; (b) taking the strain preserved in step (a), sequentially performing activation, shaking amplification culture, induced shaking culture and centrifugation to obtain bacterial cells of the strain preserved with the recombinant protein; (c) The cells obtained in step (b) were resuspended in PB buffer, ultrasonically disrupted in an ice bath, and centrifuged to collect the supernatant and precipitate, respectively. The supernatant and precipitate were subjected to polyacrylamide gel electrophoresis to analyze the protein expression.

8. The method according to claim 5, characterized in that The step 3) specifically includes: (A) The strains expressing the fusion proteins LpcA and SurA were amplified in large quantities, the collected strains were resuspended in PB buffer, ultrasonically disrupted in an ice bath, and the supernatant containing the recombinant proteins LpcA and SurA was collected by centrifugation and sterilized by filtration for later use; (B) Purify LpcA and SurA from the supernatant obtained in step (A), perform affinity chromatography, and elute to obtain recombinant proteins LpcA and SurA.

9. The method according to claim 8, characterized in that The affinity chromatography and elution in step (B) are specifically as follows: first, the empty chromatography column is cleaned with sterile water until the water flow is unobstructed, 1 ml of nickel ion metal chelate affinity chromatography medium Ni-NTA is added close to the column wall, the medium is equilibrated with 10 ml of imidazole binding buffer, and the supernatant containing recombinant proteins LpcA and SurA is added to the medium for binding; after washing with 10 ml of imidazole binding buffer and 10 mL of imidazole rinsing buffer, the sample is gradient eluted with 50 mM, 100 mM, 200 mM, and 300 mM imidazole elution buffer; LpcA recombinant protein is eluted in 200-300 mM imidazole elution buffer, and SurA recombinant protein is eluted in 200-300 mM imidazole elution buffer, and the protein quantitative detection is performed and the samples are stored for future use.

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