A Trichinella spiralis cathepsin B, its encoding gene, and its applications

Abstract

This invention discloses a Trichinella spiralis cathepsin B, its encoding gene, and its applications, belonging to the field of genetic engineering technology. This invention utilizes a food-grade L. lactis expression system to construct a lactic acid bacteria expression system for the TsCB gene. By orally administering the recombinant L. lactis strain pNZ8149-TsCB / L. lactis NZ3900 to mice, a type 2 immune response can be induced, increasing the recruitment of M2 macrophages, reducing neutrophil infiltration, and inhibiting the expression of pro-inflammatory factors and inflammatory mediators, thereby improving colitis in DSS mice. This lays the foundation for the subsequent development of food-grade oral parasite-derived molecular drugs for the treatment of colitis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a Trichinella spiralis cathepsin B, its encoding gene, and its applications. Background Technology

[0002] Catepsin B (CB) is a lysosomal cysteine ​​proteolytic enzyme. CB plays an indispensable role in the functional degradation of proteins in lysosomes. Due to the complex composition of Trichinella excretory-secretory (ES) antigens, the mechanism by which it improves inflammatory diseases is currently unclear.

[0003] The ability of Trichinella spiralis-derived cathepsin B to generate a Th2-type immune response to alleviate DSS-induced colitis is closely related to this. Previous studies have all cloned exogenous proteins from Escherichia coli, but these proteins contain endotoxins produced by E. coli and cannot be used directly. Special methods are needed to remove the endotoxins before they can be used. Currently, there are no reports of using oral Trichinella spiralis cathepsin B to treat DSS-induced acute colitis. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a Trichinella spiralis cathepsin B and its encoding gene and application, so as to solve the technical problem that existing drugs for treating colitis cannot be taken orally directly and have poor effects.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide a Trichinella spiralis cathepsin B, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0006] The present invention also discloses a gene encoding Trichinella spiralis cathepsin B, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0007] This invention also discloses the application of Trichinella spiralis cathepsin B or the gene encoding Trichinella spiralis cathepsin B in the preparation of drugs for treating colitis.

[0008] Based on the above technical solution, the present invention can be further improved as follows:

[0009] Furthermore, it can be applied to alleviate colonic inflammation by reducing the expression levels of pro-inflammatory factors and increasing the expression levels of anti-inflammatory factors.

[0010] Furthermore, the pro-inflammatory factors are TNF-α, IL-6, and IL-1β, and the anti-inflammatory factor is IL-10.

[0011] The present invention also discloses a recombinant plasmid containing a gene encoding Trichinella cathepsin B.

[0012] The present invention also discloses a drug for treating colitis, comprising Trichinella cathepsin B, a gene encoding Trichinella cathepsin B, or a recombinant plasmid.

[0013] Based on the above technical solution, the present invention can be further improved as follows:

[0014] Furthermore, the drug is an oral medication.

[0015] The beneficial effects of this invention are as follows: This invention utilizes a food-grade L. lactis expression system to construct a lactic acid bacteria expression system for the TsCB gene. By orally administering the recombinant L. lactis strain pNZ8149-TsCB / L. lactis NZ3900 to mice, a type 2 immune response can be induced, increasing the recruitment of M2 macrophages, reducing neutrophil infiltration, and inhibiting the expression of pro-inflammatory factors and inflammatory mediators, thereby improving colitis in DSS mice and laying the foundation for the subsequent development of food-grade oral parasite-derived molecular drugs for the treatment of colitis. Attached Figure Description

[0016] Figure 1 The construction status of the recombinant strain pMD19-T-TsCB / DH5α;

[0017] Figure 2 The construction status of recombinant strain pNZ8149-TsCB / L.lactis NZ3900;

[0018] Figure 3 SDS-PAGE results of TsCB gene expression in recombinant Lactococcus lactis;

[0019] Figure 4 Western blot results of TsCB gene expression in recombinant Lactococcus lactis;

[0020] Figure 5 Growth curves of L. lactis NZ3900, pNZ8149 / L. lactis NZ3900 and pNZ8149-TsCB / L. lactis NZ3900 lactic acid bacteria cultured for 11 h;

[0021] Figure 6 PCR results of genetic stability assessment of pNZ8149-TsCB plasmid in pNZ8149-TsCB / L. lactis NZ3900 lactic acid bacteria;

[0022] Figure 7 To assess the tolerance of recombinant lactococcus pNZ8149-TsCB / L. lactis NZ3900 to acidic environments;

[0023] Figure 8 The survival status of recombinant lactic acid bacteria pNZ8149-TsCB / L.lactis NZ3900 in feces;

[0024] Figure 9 The changes in body weight of mice in each group;

[0025] Figure 10 Fecal occult blood scores of mice in each group;

[0026] Figure 11 Evaluation of diarrhea in each group of mice;

[0027] Figure 12 The DAI scores of mice in each group;

[0028] Figure 13 Gross images of the colons of mice in each group;

[0029] Figure 14 The colon length of mice in each group was compared.

[0030] Figure 15 Gross lesion scores of the colon in each group of mice;

[0031] Figure 16 HE staining of the colon of mice in each group after lactic acid bacteria immunotherapy;

[0032] Figure 17 PAS staining of mouse colon before and after lactic acid bacteria immunotherapy;

[0033] Figure 18 The colon pathological scores of mice in each group;

[0034] Figure 19 Comparison of the number of goblet cells infiltrating the colon of mice in each group;

[0035] Figure 20 The percentage of neutrophils in the spleen of mice in each group;

[0036] Figure 21 The percentage of neutrophils in the MLN of each group of mice;

[0037] Figure 22 The percentage of neutrophils in the peripheral blood of mice in each group;

[0038] Figure 23 The percentage of neutrophils in the colonic LP of mice in each group;

[0039] Figure 24 The expression level of CXCL1 in the colon tissue of mice in each group was detected by qPCR.

[0040] Figure 25The expression level of CXCL2 in the colon tissue of mice in each group was detected by qPCR.

[0041] Figure 26 The results of colorimetric determination of MPO content in the colon of mice in each group;

[0042] Figure 27 The percentage of T cells in the colonic LP of mice in each group;

[0043] Figure 28 The percentage of IL-4-producing cells in the colonic LP of mice in each group;

[0044] Figure 29 The percentage of IFN-γ-producing cells in the colonic LP of mice in each group;

[0045] Figure 30 The percentage of IL-10-producing cells in the colonic LP of mice in each group;

[0046] Figure 31 The percentage of M2 macrophages in the colonic LP of mice in each group;

[0047] Figure 32 The transcriptional level of Fizz in the colon of mice in each group;

[0048] Figure 33 The transcriptional level of iNOS in the colon of mice in each group;

[0049] Figure 34 The transcriptional level of TNF-α in the colon tissue of mice in each group;

[0050] Figure 35 The transcriptional level of IL-10 in the colon tissue of mice in each group. Detailed Implementation

[0051] The specific embodiments of the present invention are described below to facilitate understanding of the invention by those skilled in the art. Unless otherwise specified, specific conditions are applied according to conventional conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various modifications are obvious as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims. All inventions utilizing the concept of this invention are protected.

[0052] The amino acid sequence of Trichinella spiralis cathepsin B (TsCB) is as follows:

[0053] NQRLYFNKMATYINNLQTTWKAGRNPYFETVPSHVIQGMMGVRRSSKLETNSIPLPVI

[0054] SYEHIDMEIPVEFDSRKQWPYCPTIGEIRDQSNCGSCWAFGAVEAISDRICIATDGRQKPHISS

[0055] TDLLSCCKICGFGCQGGDPHQAWSFWVKYGLVTGGNYTTHDGCRPYPFAPCNHHSNGTY

[0056] GPCSHDLEPTPVCKKACQSTYKIQYNKDKYYGLKAYSLHNKASDLQKELMMNGPMEVAF

[0057] EVYEDFLLYKTGVYQHHTGSVLGGHAVRLLGWGEENGVPYWLLANSWNTEWGDKGFFK

[0058] IYRGRNECGIESEAVAGX(SEQ ID NO: 1);

[0059] The nucleotide sequence of the gene encoding Trichinella spiralis cathepsin B (TsCB) is as follows:

[0060] AACCAAAGGCTGTATTTCAACAAAATGGCCACCTACATCAACAATTTGCAAACAAC

[0061] ATGGAAGGCTGGTCGAAATCCATACTTCGAAACTGTCCCTTCGCACGTCATACAAGGTAT

[0062] [[ID=二十七]]GATGGGCGTTCGGAGAAGTTCAAAGCTCGAGACCAATTCAATACCGTTGCCTGTCATCT

[0063] CTTACGAACATATCGATATGGAAATTCCCGTCGAATTTGATTCCAGGAAACAATGGCCCT

[0064] ATTGTCCAACAATTGGGGAAATACGTGATCAGTCGAACTGTGGTTCCTGTTGGGCATTTG

[0065] It should be noted that there seems to be an error in the tag "[[ID=二十七]]", which should probably be a correct ID number. Please check and correct it if necessary.GAGCAGTTGAAGCAATAAGTGATCGAATATGCATTGCTACTGATGGACGTCAGAAGCCT

[0066] CACATTTCATCCACCGATTTGTTGAGCTGTTGCAAAATTTGTGGTTTCGGATGCCAAGGT

[0067] GGTGATCCTCACCAAGCTTGGTCGTTTTGGGTTAAGTATGGTCTTGTCACTGGTGGAAAT

[0068] TACACAACGCACGACGGTTGTCGTCCATATCCGTTTGCCCCGTGCAATCACCACAGCAA

[0069] CGGCACGTATGGACCGTGCAGTCACGATTTGGAGCCTACTCCGGTTTGTAAAAAAGCAT

[0070] GCCAGTCAACTTACAAGATTCAGTACAACAAAGATAAATATTATGGCTTGAAAGCGTACT

[0071] CATTGCATAATAAGGCATCGGATTTGCAAAAGGAGCTGATGATGAACGGTCCAATGGAA

[0072] GTGGCATTTGAAGTTTACGAAGATTTTCTTCTGTACAAAACGGGAGTTTACCAACATCAC

[0073] ACTGGCTCAGTATTGGGTGGTCACGCAGTGAGGCTACTTGGATGGGGAGAAGAGAATG

[0074] GCGTTCCTTATTGGCTTTTGGCCAATTCTTGGAATACTGAATGGGGCGATAAGGGATTTTT

[0075] CAAAATTTACAGAGGTCGTAATGAATGCGGCATAGAATCAGAGGCAGTTGCTGGACT

[0076] (SEQ ID NO: 2).

[0077] [[ID=,36]]Example 1 Preparation of Plasmid

[0078] 1. Extraction of total RNA from Trichinella spiralis: Total RNA from Trichinella spiralis was extracted using Trizol reagent provided by Shanghai Sangon Biotech. The extracted total RNA was subjected to 1% agarose gel electrophoresis.

[0079] 2. cDNA synthesis: Using the obtained Trichinella spiralis RNA as a template, Pst I and Xba I were selected as restriction enzyme sites according to the TsCB gene sequence provided on the NCBI website. Protective bases and corresponding restriction enzyme sites were added respectively. Specific upstream and downstream primers were designed using Primer 5, and cDNA was obtained by reverse transcription according to the reverse transcriptase instructions.

[0080] The sequences of the specific upstream primer TsCB F (Pst I) and the specific downstream primer TsCB R (Xba I) are as follows:

[0081] TsCB F (Pst I): GCCTGCAGACATTCCTTTTGGTTCCAGA (SEQ ID NO: 3);

[0082] TsCB R(Xba I):ATTCTAGATCACGTTGGCTTCTTGTAC (SEQ ID NO: 4).

[0083] 3. PCR reaction: Using the cDNA obtained by reverse transcription as a template, PCR reaction was performed using TsCB F (Pst I) and TsCB R (Xba I) as primers.

[0084] The reaction mixture consisted of 10 μL of 2×PCR Mix, 0.5 μL of cDNA, 1 μL of TsCB F, 1 μL of TsCB R, and 7.5 μL of ddH2O. The PCR parameters were: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 45 s, 60℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 35 cycles. Finally, annealing was performed at 72℃ for 7 min, and the mixture was stored at -20℃.

[0085] 4. Gel recovery: The PCR products were detected by 1% agarose gel electrophoresis, and the amplified band of about 984 bp was recovered to obtain the target gene fragment.

[0086] 5. Ligation and transformation: The target gene fragment is ligated into the pMD19-T vector and transformed into E. coli DH5α competent cells. Positive clones are sent to the company for sequencing to confirm the correctness of the amplified sequence.

[0087] Figure 1The figure shows the construction of pMD19-T-TsCB / DH5α, where M represents DM2000, 1 is the product of double digestion of the recombinant cloning plasmid pMD19-T-TsCB with Pst I and Xba I, 2 is the PCR amplification product of the recombinant plasmid pMD19-T-TsCB, 3 is the PCR amplification product of the recombinant bacterial pMD19-T-TsCB / DH5α, and 4 is the recombinant plasmid pMD19-T-TsCB. As can be seen from the figure, a clear band exists at approximately 984 bp in the PCR product; the double enzyme digestion identification results show a target band of 984 bp and a linear vector band. After identification, the bacterial culture was sent to Sangon Biotech for sequencing. The sequencing results showed that the amplified TsCB gene had a 99% sequence similarity to the TsCB gene sequence on NCBI.

[0088] 6. Preparation of pMD19-T-TsCB plasmid: The transformed bacterial culture pMD19-T-TsCB / DH5α was inoculated into solid LB agar plates containing ampicillin (50 mg / mL) resistance (components: 1 wt% tryptone, 0.5 wt% yeast extract, 1 wt% sodium chloride, and 1.5 wt% agar powder) and cultured at 37°C for 14 h. Then, single colonies were picked and cultured in LB liquid medium containing ampicillin (50 mg / mL) (components: 1 wt% tryptone, 0.5 wt% yeast extract, and 1 wt% sodium chloride) and cultured with shaking at 37°C for 14 h. Finally, the pMD19-T-TsCB plasmid was extracted using the SanPrep column-based plasmid mini-extraction kit.

[0089] 7. Preparation of pNZ8149-TsCB plasmid: The gel-recovered fragment of the target gene was ligated into the L. lactis expression vector pNZ8149, and then electroporated into L. lactis NZ3900 competent cells. The transformation solution was inoculated on Elliker selective solid medium (composed of 2 wt% tryptone, 0.4 wt% sodium chloride, 1.5 wt% agar powder, 0.05 wt% ascorbic acid, 0.5 wt% yeast extract, 0.15 wt% anhydrous sodium acetate, 0.004 wt% bromocresol purple and 0.5 wt% lactose) for activation. Plasmid was extracted from the Gram-positive bacterial cultures, specifically using the Gram-positive plasmid miniprep kit provided by Solarbio to extract the pNZ8149-TsCB plasmid.

[0090] Example 2: Construction and transformation of pNZ8149-TsCB expression vector into Lactococcus lactis NZ3900

[0091] The amplified TsCB gene sequence and pNZ8149 vector were digested with Pst I and Xba I, respectively, to obtain fragments with the same sticky ends. The two fragments were ligated with T4 DNA ligase to obtain the ligated recombinant plasmid. The reaction system and reaction conditions are shown in Table 1.

[0092] Table 1. Ligation reaction system of pNZ8149 vector and TsCB gene fragment.

[0093] Element Volume (10 μL) TsCB 3.3 pNZ8149 2.0 T4 ligase 1.0 10×T Buffer 1.0 <![CDATA[ddH2O]]> 2.7

[0094] A total of 10 μL of the ligation solution was incubated in a 16℃ metal bath for 16 h. Subsequently, the ligation solution was electroporated (at 2500 V, 200 Ω, and 25 μF) into competent *L. lactis* NZ3900 cells, plated on Elliker solid selective medium, and incubated at 37℃ and 5% CO2 for 72 h. Positive transformants were then screened. After incubation, single colonies were picked and cultured in GM17 liquid medium (composed of 4.23 wt% M17 broth (Qingdao Haibo Biotechnology, catalog number HB0391) and 0.5 wt% glucose) at 37℃ and 5% CO2 for 72 h. PCR identification of the bacterial culture and plasmid was performed, and the plasmid was extracted and subjected to double enzyme digestion for identification. Finally, the partially correct transformants were sequenced to ensure successful transformation of pNZ8149-TsCB into *L. lactis* NZ3900.

[0095] Figure 2 The figure shows the construction of pNZ8149-TsCB / L.lactis NZ3900, where M represents DM10000, 1 is the recombinant plasmid pNZ8149-TsCB double digestion (Pst I + Xba I), 2 is the purified product of the target gene TsCB, 3 is the purified product of the expression vector pNZ8149, 4 is the PCR product of the recombinant plasmid pNZ8149-TsCB, 5 is the PCR product of the recombinant bacterium pNZ8149-TsCB / L.lactis NZ3900, and 6 is the recombinant plasmid pNZ8149-TsCB. The figure shows that the expected target gene was successfully amplified by PCR, and the size of the double-digested fragment is consistent with the expected result. The digestion sites are Pst I and Xba I, further confirming that the target gene TsCB was inserted downstream of the promoter of the expression vector pNZ8149 in the correct reading frame, indicating that the recombinant plasmid was successfully constructed.

[0096] Example 3: Protein expression of the transgenic TsCB gene in L. lactis strain

[0097] 1. Inducing the expression of the target protein

[0098] A single colony of pNZ8149-TsCB / L. lactis NZ3900 was picked from GM17 solid medium and inoculated into 10 mL of GM17 liquid medium. The culture was then incubated statically at 30°C and 5% CO2 for 14 h. The overnight colony was added to the GM17 liquid medium at a volume ratio of 1:4 and incubated statically at 30°C and 5% CO2 until OD500 was reached. 600 =0.3, add nisin (Sigma, product number SBR00021) to the recombinant strain to a final concentration of 20 ng / mL, and then induce for 6 h; pNZ8149 / L.lactisNZ3900 was used as a control, and the nisin induction concentration and induction time were the same as those of the recombinant strain. After induction, the bacterial cells were collected by low-temperature centrifugation, the precipitate was washed with sterile PBS, and the precipitate was resuspended in 200 μL PBS buffer. Cell wall disruption: lysozyme (Solarbio, product number L1080) was added to the prepared bacterial culture to a final concentration of 10 mg / mL, and the cell walls were disrupted in a water bath at 37 °C for 1 h to obtain 8 μL of whole bacterial protein. 2 μL of 5×SDS-PAGE loading buffer was added, boiled for 5 min, and stored at -20 °C for later use.

[0099] 2. Western blot analysis of the induced expression product

[0100] (1) Electrophoresis: The treated TsCB protein was loaded at a rate of 15-30 μg per well and subjected to SDS-PAGE (5% stacking gel and 10% separating gel). The electrophoresis results are as follows: Figure 3 As shown in the figure, TsCB protein was successfully induced.

[0101] (2) Transfer: After SDS-PAGE electrophoresis, remove the gel and place it in the transfer buffer for equilibration for 10 min; cut a PVDF membrane of the same size as the gel, immerse the cut PVDF membrane in methanol solution for 10 s, and when the PVDF membrane turns slightly blue, immediately rinse it in distilled water to renature; then perform "sandwich" transfer, placing the membrane in the order of filter paper, gel, PVDF membrane, and filter paper from bottom to top, and removing air bubbles between the layers to avoid affecting the transfer results; use a wet electrophoresis apparatus for transfer, with the transfer parameters being a constant current of 250 mA and ice bath transfer for 1.5 h.

[0102] (3) Pre-development: After the transfer is completed, place the membrane in Ponceau S staining solution for a few seconds until the protein bands are observed. Then rinse the PVDF membrane in distilled water, cut the PVDF membrane along the protein lanes with a blade and mark it, and wash off the Ponceau S on the bands with TBST.

[0103] (4) Sealing: Immerse the cut strips completely in 5% skim milk powder and incubate in a shaker at 37°C for 2 hours.

[0104] (5) Washing: The sealed strip is completely immersed in TBST and washed three times on a horizontal shaker for 10 minutes each time.

[0105] (6) Primary antibody: TsCB immune serum was diluted with 1×TBST at a volume ratio of 1:100, and identified with the corresponding bands. The serum was then incubated at 37°C for 2 hours.

[0106] (7) Secondary antibody: HRP-labeled goat anti-mouse IgG diluted at a volume ratio of 1:5000 was incubated at 37°C for 2 hours.

[0107] (8) DAB color development: Immerse the strip in DAB color development solution, develop color for 1-9 seconds, remove it immediately, rinse with distilled water, and stop the color development.

[0108] (9) Observe and record the results: Observe the corresponding results, take pictures with a gel scanner and analyze the results.

[0109] SDS-PAGE electrophoresis was performed on whole-cell proteins from *Lactococcus lactis* pNZ8149 / L. lactis NZ3900 with a carrier and *Lactococcus lactis* pNZ8149-TsCB / L. lactis NZ3900 after induction, followed by membrane transfer. The transferred bands were identified using normal serum and anti-TsCB serum, respectively. Western blot results showed ( Figure 4 Anti-TsCB serum showed a band at 36.9 kDa, while normal serum did not, indicating that TsCB protein was successfully expressed in Lactococcus lactis.

[0110] Example 4: Determination of biological characteristics of TsCB gene-transgenic lactococci

[0111] 1. Plot the growth curves of Lactococcus lactis NZ3900, pNZ8149 / L.lactis NZ3900, and pNZ8149-TsCB / L.lactis NZ3900.

[0112] Lactococcus lactis NZ3900, L. lactis NZ3900 / p NZ8149, and L. lactis NZ3900 / p NZ8149-TsCB were picked from the plates and added to 5 mL of GM17 liquid medium. The plates were then placed in a CO2 incubator and incubated statically at 30°C and 5% CO2 for 14 h. The overnight bacterial culture was then inoculated into GM17 liquid medium at a 1:25 volume ratio and incubated statically at 30°C and 5% CO2. One tube was taken every 1 h, and the OD values ​​were measured using a UV spectrophotometer. 600 Each tube was measured three times, and the average value was taken. OD was plotted on the x-axis as time. 600Use the vertical axis to plot the growth curve.

[0113] like Figure 5 As shown, bacterial growth is relatively slow in the first 2 hours, then enters the logarithmic growth phase, and simultaneously enters the plateau phase after about 6 hours, at which point the OD... 600 The value reached approximately 1.8. pNZ8149 / L.lactis NZ3900 and pNZ8149-TsCB / L.lactisNZ3900 were similar during the logarithmic growth phase, but the growth rate was slightly slower than that of the empty L.lactis NZ3900. Both eventually reached the same plateau phase, indicating that the insertion of the TsCB gene did not affect the growth of the strains.

[0114] 2. Genetic stability testing

[0115] The pNZ8149-TsCB / L. lactis NZ3900 strain preserved at -80℃ was cultured on GM17 plates for 18-24 hours. Single colonies were picked from the plates and inoculated into GM17 liquid medium for overnight enrichment culture, which was then used as the primary generation. The primary generation was inoculated into GM17 liquid medium at a volume ratio of 1:100 and cultured at 30℃ with 5% CO2 for 8 hours. The resulting bacterial culture was used as the second generation, and this process was repeated until the 20th generation. The bacterial culture from the 20th generation was revived and cultured for 18-24 hours, and then continuously subcultured for 20 generations. Each generation of bacterial culture was collected, centrifuged, and sonicated to disrupt the bacterial cells for PCR. Electrophoresis was used to observe whether the pNZ8149-TsCB plasmid could be stably inherited in pNZ8149-TsCB / L. lactis NZ3900.

[0116] The pNZ8149-TsCB plasmid was extracted in large quantities and transformed into *Lactococcus lactis* NZ3900 via electroporation. Lactococcus lactis containing the pNZ8149-TsCB plasmid was then obtained through plate culture. To assess the genetic stability of the pNZ8149-TsCB plasmid, the pNZ8149-TsCB / *L. lactis* NZ3900 bacteria were cultured for 20 generations, and PCR detection was performed using TsCB primers. The results are as follows: Figure 6 As shown, the TsCB gene can be amplified in each generation of bacteria.

[0117] 3. Tolerance of recombinant lactococci to acidity

[0118] A digestive solution (0.9 wt% sodium chloride, 0.75 wt% hydrochloric acid, and 1 wt% pepsin) was prepared using physiological saline, concentrated hydrochloric acid, and pepsin to simulate gastric juice. Different pH values ​​(pH = 1, 2, 3, and 4) were adjusted with concentrated hydrochloric acid, using physiological saline (pH = 7.2) as a control. 1 mL of recombinant bacteria (OD100) in the logarithmic growth phase was used. 600 =0.3-0.4), centrifuged at 4℃ and 12000g for 20 min, then resuspended in 1 mL PBS, and inoculated into 9 mL of liquids with different pH values. The mixture was then incubated statically at 30℃ in 5% CO2. 1 mL of bacterial culture was collected at 0, 1, 2, 3, and 4 h after incubation, and diluted with PBS to different concentration gradients (10⁻⁶). -4 10 -5 and 10 -6 CFU / mL), 100 μL was plated on GM17 solid medium and cultured for 48 h. Viable bacteria were counted by plate count method to observe the growth ability of recombinant bacteria under different acidic conditions.

[0119] The acid resistance test results showed that ( Figure 7 Compared to a normal environment (pH=7.2), pNZ8149-TsCB / L.lactisNZ3900 cells survived for 2-3 hours at pH=1 and 2, and 4 hours at pH=3. However, there was no statistically significant difference (ns) in survival time between pH=4 and 7.2. This indicates that pNZ8149-TsCB / L.lactisNZ3900 cells have some tolerance to acidic environments, but these environments slightly reduce the cell count.

[0120] 4. Survival status of recombinant strain pNZ8149-TsCB / L.lactis NZ3900 in the host.

[0121] To verify the survival of recombinant bacteria pNZ8149-TsCB / L. lactis NZ3900 during colitis modeling, fecal samples were collected from mice 24 hours after gavage administration of the recombinant bacteria during DSS-induced acute colitis. Fecal genomic DNA was extracted using a fecal genomic DNA extraction kit (Solarbio, catalog number D2700) and amplified. Figure 8 As shown, the target gene was still detected in the fecal colon of mice 24 hours after gavage, indicating that the recombinant bacteria pNZ8149-TsCB / L.lactis NZ3900 survived well in mice for 24 hours.

[0122] Example 5: TsCB alleviates clinical symptoms in DSS-induced colitis mice

[0123] Eight-week-old female C57BL / 6J mice, raised for at least one week under specific pathogen-free conditions, were randomly divided into six groups: Control group, DSS group, pNZ8149 / L.lactis NZ3900 group (pNZ8149 group), pNZ8149 / L.lactis NZ3900-DSS group (pNZ8149-DSS group), pNZ8149-TsCB / L.lactis NZ3900 group (TsCB group), and pNZ8149-TsCB / L.lactis NZ3900-DSS group (TsCB-DSS group), with five mice in each group. Mice were fasted and deprived of water for 4 hours before the first gavage. Gastric acid was neutralized by gavage with 5% NaHCO3 solution 30 minutes before inoculation with the bacterial culture. The feeding details for each group are as follows:

[0124] (1) Control group: normal drinking water for 10 days, and 200μL PBS was administered by gavage on days 5-10.

[0125] (2) DSS group: Received 2.5% (w / v) DSS solution for 7 consecutive days, followed by normal drinking water for 3 days, and 200 μL PBS by gavage on days 5-10.

[0126] (3) pNZ8149 / L.lactis NZ3900 group (pNZ8149 group): normal drinking water for 10 days, and pNZ8149 / L.lactis NZ3900 (200μL, 5×10) was used on days 5-10. 10 Gavage with CFU / mL.

[0127] (4) pNZ8149 / L.lactis NZ3900-DSS group (pNZ8149-DSS group): Received 2.5% (w / v) DSS solution for 7 consecutive days, followed by normal drinking water for 3 days, while on days 5-10, pNZ8149 / L.lactis NZ3900 bacterial suspension (200 μL, 5 × 10⁻⁶ mcg) was administered. 10 Gavage with CFU / mL.

[0128] (5) pNZ8149-TsCB / L.lactis NZ3900 group (TsCB group): Normal drinking water for 10 days, and at the same time, pNZ8149-TsCB / L.lactis NZ3900 bacterial solution (200μL, 5×10) was used on days 5-10. 10 Gavage with CFU / mL.

[0129] (6) pNZ8149-TsCB / L.lactis NZ3900-DSS group (TsCB-DSS group): Received 2.5% (w / v) DSS solution for 7 consecutive days, followed by normal drinking water for 3 days, while on days 5-10, pNZ8149-TsCB / L.lactis NZ3900 bacterial suspension (200 μL, 5 × 10⁻⁶ mcg) was administered. 10 Gavage with CFU / mL.

[0130] 1. TsCB alleviates weight loss, diarrhea, and fecal occult blood in DSS-induced colitis mice.

[0131] The mice were weighed daily, and the condition of each mouse's feces was examined to assess the Disease Awareness Index (DAI).

[0132] Three important clinical indicators in the colitis model were varying degrees of weight loss, diarrhea, and fecal occult blood. Results were as follows: Figures 9-11 As shown, throughout the entire colitis trial, the body weight of mice in the Control, pNZ8149, and TsCB groups remained relatively stable, with no diarrhea or bloody stools. After DSS induction, the body weight of each group gradually decreased, and the severity of diarrhea and bloody stools worsened. This decreasing trend was mitigated after lactic acid bacteria treatment. Starting from day 6, after TsCB treatment, the weight loss in the colitis group mice decreased, and the shape of the stool changed, losing its original cylindrical shape and gradually becoming softer and looser until diarrhea symptoms became obvious. Simultaneously, fecal occult blood gradually decreased.

[0133] The Disease Activity Index (DAI) score is a comprehensive reflection of three factors: weight loss, diarrhea, and fecal occult blood, indicating the extent of colonic damage in mice with colitis. Results are as follows... Figure 12 As shown, throughout the entire colitis trial, the DAI scores of mice in the Control group, pNZ8149 group, and TsCB group remained stable with no significant differences. After DSS induction, the DAI scores of each group gradually increased. However, after probiotic treatment, from day 6 onwards, there was a statistically significant difference in DAI scores between the TsCB-DSS group and the DSS group. Furthermore, compared with the DSS group, the DAI scores of mice in the pNZ8149-DSS group were smaller, but compared with the pNZ8149-DSS group, the DAI scores of mice in the TsCB-DSS group were significantly lower.

[0134] 2. The effect of TsCB on improving colonic pathological symptoms in mice with acute colitis

[0135] Mice in all six groups were sacrificed on day 10 after DSS treatment, and samples were collected to analyze changes in various indicators. After the experiment, blood was collected from the eyeballs, and the colons of the mice were dissected and their length measured. Figure 13 ), 0.5 cm of intestinal tissue above the anus was frozen at -80℃, and another 0.5 cm of intestinal tissue was frozen at -80℃ for measuring colonic myeloperoxidase activity. The remaining colonic tissue of one batch of mice was longitudinally cut with scissors and washed with PBS. A portion was stored in buffer for collecting LP cells, while the remaining colonic tissue of another batch of mice was rolled into Swiss rolls and fixed in 10% formalin solution.

[0136] like Figure 14 As shown, the normal colon length of mice is generally 7-8 cm. After DSS treatment, the colon length of mice shortened by 2-4 cm. Compared with the DSS group, the colons of the TsCB-DSS group and the pNZ8149-DSS group were longer, and the colon of the TsCB-DSS group was significantly longer than that of the pNZ8149-DSS group. In the control group, the colon of mice contained multiple formed fecal particles, and there was no congestion, swelling, or ulceration in the colon. However, after DSS induction, the colon wall of mice showed diffuse congestion, severe swelling, and multiple ulcers. In severely affected mice, the colon to the anus and rectum contained bloody, mucus-like contents. Gross pathological scoring of the colon revealed ( Figure 15 Lactic acid bacteria treatment of mice with colitis significantly reduced DSS-induced colonic damage, and TsCB showed better improvement in enteritis compared to pNZ8149.

[0137] 3. TsCB reduces pathological damage to colonic tissue in mice with acute colitis.

[0138] To better verify the therapeutic effect of TsCB on mice with colitis, colon tissue samples from each group of mice were subjected to HE (hematologic reaction). Figure 16 ) and PAS staining ( Figure 17 The results of tissue section staining showed that the DSS group mice had severe colonic lesions, including loss or partial loss of crypts, disappearance or partial disappearance of goblet cells, severe colonic mucosal edema, and inflammatory cell infiltration. These lesions were significantly improved in colitis mice treated with TsCB and pNZ8149. The number of colonic goblet cells in the TsCB inflammation treatment group was significantly increased compared with the inflammation group.

[0139] Histopathological scoring was performed on colonic sections of mice in each group. Figure 18 ) and goblet cell count ( Figure 19 Further, it was found that after DSS-induced colitis, the colonic pathological score increased and the number of goblet cells in the colon disappeared in large numbers; TsCB significantly reduced the colonic pathological damage in mice with DSS-induced acute colitis, and the number of colonic goblet cells increased, and the degree of reduction was higher than that in the pNZ8149-DSS group.

[0140] Example 6: TsCB reduces neutrophil infiltration in mice with acute colitis

[0141] In the early stages of acute inflammation, neutrophils are the main inflammatory cells infiltrating the affected area and are chemotactically attracted to the site of inflammation to participate in the inflammatory response. Flow cytometry was used to detect changes in neutrophils in the spleen, mesenteric lymph nodes (MLN), peripheral blood, and lamina propria (LP) of mice in the Control, pNZ8149, TsCB, DSS, pNZ8149-DSS, and TsCB-DSS groups. Gating diagrams of immune cells and typical scatter plots of neutrophils were used for each group. Figures 20-23 As can be seen, after DSS treatment, the number of Ly6G+CD11b+ neutrophils in the spleen, MLN, peripheral blood and LP of mice in each group increased significantly; compared with the DSS group, the number of neutrophils in the TsCB-DSS group decreased significantly.

[0142] qPCR analysis of neutrophil chemokine transcription levels in DSS-treated mouse colon tissue revealed that ( Figure 24 and Figure 25 Compared with the DSS group, the TsCB-DSS treatment group showed a significant decrease in CXCL1 and CXCL2, indicating that TsCB can inhibit the expression of CXCL1 and CXCL2 in the colon.

[0143] The MPO detection kit (Nanjing Jiancheng, catalog number A044-1-1) was used to quantify the infiltration of inflammatory cells (polymorphonuclear neutrophils) in the colon of mice in each group by measuring MPO activity in a colorimetric method. Results are as follows: Figure 26 As shown, after DSS induction, MPO activity increased in all groups. After lactic acid bacteria treatment, the MPO activity (neutrophil accumulation index) in all groups was significantly lower than that in the DSS group. At the same time, compared with the pNZ8149-DSS group, the MPO activity in the colon of the TsCB-DSS group was even lower.

[0144] Example 7: TsCB induces Th2 immune response

[0145] 1. Effects of TsCB on T cells in the lamina propria of the intestine in mice with DSS-induced acute colitis

[0146] Flow cytometry was used to detect changes in CD4+ T cells in colonic LP cells of mice in the Control, pNZ8149, TsCB, DSS, pNZ8149-DSS, and TsCB-DSS groups. First, cell populations were identified: cell debris was removed under FSC-A and SSC-A parameters. Live cells were screened from LP cells using immunofluorescence dyes that stained dead cells for further analysis. Single cells were circled under FSC-A and FSC-H parameters, and dimer or multimerous cells were removed to prevent cell adhesion. CD45+ positive immune cells were circled under CD45+ and FSC-A parameters. CD4+ labeled T cells were analyzed within the CD45+ cell population, and the percentage of T cells among immune cells was compared across the four groups. The quantitative analysis of T cells showed (…). Figure 27 After DSS treatment, the number of T cells in the lamina propria of the colon of mice in each group decreased significantly; compared with the DSS group, the number of T cells in the TsCB-DSS group increased significantly.

[0147] 2. TsCB increases the production of Th2-type cytokines in the lamina propria of the mouse colon.

[0148] Flow cytometry was used to analyze the levels of Th1 cytokine IFN-γ, Th2 cytokine IL-4, and anti-inflammatory cytokine IL-10 in the intestinal LP cells of mice in each group. Results are as follows: Figures 28-30 As shown, compared with the Control group, pNZ8149 group, and TsCB group, DSS treatment reduced the production of type 2 cytokine IL-4 and anti-inflammatory factor IL-10 in the colonic LP cells of all groups. Compared with the DSS group, probiotic treatment significantly reduced IFN-γ. Data analysis revealed that compared with the pNZ8149-DSS group, the TsCB-DSS group had lower IFN-γ levels and significantly increased IL-4 levels. Furthermore, TsCB treatment significantly increased the anti-inflammatory factor IL-10 in colitis mice, and compared with the pNZ8149-DSS group, the TsCB-DSS group produced higher levels of IL-4 and lower levels of IFN-γ.

[0149] 3. TsCB increases the recruitment of M2 macrophages in the lamina propria of the mouse colon.

[0150] Flow cytometry analysis was performed on CD45+CD206+CD11b+M2 macrophages in the intestinal LP cells of mice in each group. The results showed that ( Figure 31 The level of M2 macrophages in the TsCB-DSS group was significantly higher than that in the DSS group. In addition, qPCR analysis was used to analyze the transcriptional levels of the macrophage cytokines iNOS and Fizz in mouse colon tissue. The results showed ( Figure 32 and Figure 33Compared with the DSS group, lactic acid bacteria treatment significantly reduced the transcriptional level of iNOS in the colon, while the transcriptional level of Fizz in the colon decreased. Meanwhile, compared with pNZ8149-treated enteritis mice, TsCB-treated enteritis mice showed increased Fizz expression and decreased iNOS expression in the colon.

[0151] Example 8: TsCB upregulates the level of the regulatory cytokine IL-10 in colitis.

[0152] The transcriptional levels of inflammatory cytokines in colon tissue were analyzed using qPCR. The results are as follows: Figure 34 and Figure 35 As shown, the transcriptional levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the DSS-treated group were significantly higher than those in the normal group, and this situation improved after treatment with Lactococcus lactis. Compared with the DSS group, the transcriptional levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β were significantly reduced after TsCB treatment, while the transcriptional level of anti-inflammatory factor IL-10 was significantly increased. Data analysis revealed that the expression level of IL-10 in the colon was increased after TsCB treatment compared with pNZ8149.

Claims

1. A Trichinella spiralis cathepsin B, characterized in that, The amino acid sequence of the Trichinella spiralis cathepsin B is shown in SEQ ID NO:

1.

2. A gene encoding the Trichinella spiralis cathepsin B of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. The use of the Trichinella spiralis cathepsin B of claim 1 or the gene of claim 2 in the preparation of a drug for treating colitis.

4. The application according to claim 3, characterized in that, The application is to alleviate colonic inflammation by reducing the expression levels of pro-inflammatory factors and increasing the expression levels of anti-inflammatory factors; the pro-inflammatory factors are TNF-α, IL-6 and IL-1β, and the anti-inflammatory factor is IL-10.

5. A recombinant plasmid, characterized in that, It includes the gene encoding Trichinella cathepsin B as described in claim 2.

6. A drug for treating colitis, characterized in that, It contains the Trichinella cathepsin B of claim 1, the gene of claim 2, or the recombinant plasmid of claim 5.

7. The medicament for treating colitis according to claim 6, characterized in that, The drug is an oral medication.

Images