Trichina cathepsin B as well as coding gene and application thereof

Abstract

The invention discloses trichina cathepsin B as well as a coding gene and application thereof, and belongs to the technical field of gene engineering. According to the invention, a food-grade L.lactis expression system is utilized to construct a lactic acid bacteria expression system of a TsCB gene, after the L.lactis recombinant strain pNZ8149-TsCB / L.lactis NZ3900 is subjected to intragastric administration on a mouse, the body can be induced to generate 2-type immune response, the recruitment of M2-type macrophages is increased, the infiltration of neutrophil is reduced, and the expression of proinflammatory factors and inflammatory mediators is inhibited, so that the colitis of the DSS mouse is improved; and a foundation is laid for subsequent research and development of food-grade oral insect-derived molecular medicines for treating colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a trichinella cathepsin B and a coding gene and application thereof. Background Art

[0002] Cathepsin B (CB) is a cysteine ​​protease in lysosomes. CB plays an indispensable and important role in the functional degradation of proteins in lysosomes. Due to the complex composition of the excretory-secretory protein (ES) of Trichinella spiralis, its mechanism for improving inflammatory diseases is still unclear.

[0003] The ability of Trichinella spiralis-derived cathepsin B to produce a Th2 immune response to alleviate DSS-induced colitis is closely related. Previous studies have all cloned exogenous proteins from Escherichia coli, but this protein contains endotoxins produced by Escherichia coli and cannot be used directly. Special methods are required to remove endotoxins before use. Currently, there are no reports on the use of Trichinella spiralis cathepsin B that can be directly taken orally to treat DSS-induced acute colitis. Summary of the invention

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

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

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

[0007] The invention also discloses the application of Trichinella spiralis cathepsin B or a gene encoding Trichinella spiralis cathepsin B in preparing a medicine for treating colitis.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows:

[0009] Furthermore, the application is to relieve colon inflammation by reducing the expression level of pro-inflammatory factors and increasing the expression level 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 invention also discloses a recombinant plasmid containing a gene encoding trichinella cathepsin B.

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

[0013] On the basis of the above technical solution, the present invention can also be improved as follows:

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

[0015] The beneficial effects of the present invention are as follows: the present invention utilizes a food-grade L. lactis expression system to construct a lactic acid bacteria expression system of the TsCB gene, and after orally gavaging mice with the L. lactis recombinant strain pNZ8149-TsCB / L. lactis NZ3900, the body can be induced to produce a type 2 immune response, increase the recruitment of M2 macrophages, reduce neutrophil infiltration, and inhibit the expression of proinflammatory factors and inflammatory mediators, thereby improving DSS mouse colitis, and laying a foundation for subsequent research and development of food-grade oral insect-derived molecules for treating colitis. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0020] Figure 5 The 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 The PCR identification result of the genetic stability of the pNZ8149-TsCB plasmid in the lactic acid bacteria pNZ8149-TsCB / L.lactis NZ3900;

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

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

[0024] Fig. 9 The weight changes of mice in each group;

[0025] Fig.10 The fecal occult blood scores of mice in each group;

[0026] Fig.11 Diarrhea evaluation for each group of mice;

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

[0028] Fig.13 The gross images of the colon of mice in each group;

[0029] Fig.14 Comparison of colon lengths among mice in each group;

[0030] Fig.15 The gross lesion scores of the colon of mice in each group were given;

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

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

[0033] Fig.18 Colon pathology scores of mice in each group;

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

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

[0036] Fig.21 The percentage of neutrophils in MLN of mice in each group;

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

[0038] Fig.23 The percentage of neutrophils in the colon LP of mice in each group;

[0039] Fig.24 The qPCR detection results of CXCL1 expression levels in colon tissues of mice in each group;

[0040] Fig.25The qPCR detection results of CXCL2 expression levels in colon tissues of mice in each group;

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

[0042] Fig. 27 The percentage of T cells in the colon LP of each group of mice;

[0043] Fig.28 The percentage of cells producing IL-4 in the colon LP of each group of mice;

[0044] Fig.29 The percentage of cells producing IFN-γ in the colon LP of each group of mice;

[0045] Fig.30 The percentage of cells producing IL-10 in the colon LP of each group of mice;

[0046] Fig.31 The percentage of M2 macrophages in the colon LP of mice in each group;

[0047] Fig.32 is the transcription level of Fizz in the colon of each group of mice;

[0048] Fig.33 is the transcription level of iNOS in the colon of each group of mice;

[0049] Fig.34 is the transcription level of TNF-α in the colon tissue of each group of mice;

[0050] Fig.35 is the transcription level of IL-10 in the colon tissue of each group of mice. DETAILED DESCRIPTION

[0051] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present 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] GATGGGCGTTCGGAGAAGTTCAAAGCTCGAGACCAATTCAATACCGTTGCCTGTCATCT

[0063] CTTACGAACATATCGATATGGAAATTCCCGTCGAATTTGATTCCAGGAAACAATGGCCCT

[0064] ATTGTCCAACAATTGGGGAAATACGTGATCAGTCGAACTGTGGTTCCTGTTGGGCATTTG

[0065] 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] Example 1 Preparation of Plasmid

[0078] 1. Extraction of total RNA of Trichinella spiralis: The total RNA of Trichinella spiralis was extracted using Trizol reagent provided by Shanghai Bioengineering, and the extracted total RNA of Trichinella spiralis was subjected to 1% agarose gel electrophoresis.

[0079] 2. Synthesis of cDNA: Using the obtained Trichinella spiralis RNA as a template, according to the TsCB gene sequence provided on the NCBI website, Pst I and Xba I were selected as restriction sites, and 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 instruction manual.

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

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

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

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

[0084] The reaction system was as follows: 2×PCR Mix 10 μL, cDNA 0.5 μL, TsCB F 1 μL, TsCB R 1 μL, ddH2O 7.5 μL. The PCR reaction parameters were: 94°C pre-denaturation for 3 min, 94°C denaturation for 45 s, 60°C annealing for 45 s, 72°C extension for 1 min, a total of 35 cycles, and finally renaturation at 72°C for 7 min, and stored at -20°C.

[0085] 4. Gel recovery: Detect the PCR product by 1% agarose gel electrophoresis, recover the amplified band of about 984 bp from the gel, and obtain the target gene fragment.

[0086] 5. Ligation and transformation: Ligate the target gene fragment to the pMD19-T vector, transform E. coli DH5α competent cells, and send the positive clones to the company for sequencing to confirm the correctness of the amplified sequence.

[0087] Figure 1Figure 1 is the construction of pMD19-T-TsCB / DH5α, where M is DM2000, 1 is the double restriction digestion product of recombinant cloning plasmid pMD19-T-TsCB by Pst I and Xba I, 2 is the PCR amplification product of recombinant plasmid pMD19-T-TsCB, 3 is the PCR amplification product of recombinant bacteria pMD19-T-TsCB / DH5α, and 4 is the recombinant plasmid pMD19-T-TsCB. It can be seen from the figure that there is a clear band around 984bp in the PCR product; the double restriction digestion identification results show a 984bp target band and a linear vector band. After identification, the bacterial solution was sent to Bio-Tech for sequencing, and the sequencing results showed that the amplified TsCB gene had a 99% similarity with the TsCB gene sequence on NCBI.

[0088] 6. Preparation of pMD19-T-TsCB plasmid: inoculate the transformed bacterial solution pMD19-T-TsCB / DH5α on a solid LB plate (composed of 1wt% tryptone, 0.5wt% yeast extract powder, 1wt% sodium chloride and 1.5wt% agar powder) containing ampicillin (50 mg / mL) resistance, and culture at 37°C for 14 h. Then pick a single colony into LB liquid culture medium (composed of 1wt% tryptone, 0.5wt% yeast extract powder and 1wt% sodium chloride) containing ampicillin (50 mg / mL), and culture with shaking at 37°C for 14 h. Finally, extract the pMD19-T-TsCB plasmid using the SanPrep column plasmid small-scale extraction kit.

[0089] 7. Preparation of pNZ8149-TsCB plasmid: Connect the target gene gel recovery fragment to the L. lactis expression vector pNZ8149, and then electrotransform into L. lactis NZ3900 competent cells. The transformation liquid is inoculated on Elliker selection solid medium (composed of 2wt% tryptone, 0.4wt% sodium chloride, 1.5wt% agar powder, 0.05wt% ascorbic acid, 0.5wt% yeast extract powder, 0.15wt% anhydrous sodium acetate, 0.004wt% bromocresol purple and 0.5wt% lactose) for activation. The plasmid is extracted from the bacterial solution tested positive, specifically, the pNZ8149-TsCB plasmid is extracted using the Gram-positive plasmid mini-extraction kit provided by Solebo.

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

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

[0092] Table 1 pNZ8149 vector and TsCB gene fragment ligation reaction system

[0093] Element Volume (10 μL) T8hB 3.3 pNZ8149 2.0 T4 Ligase 1.0 10×T Buffer 1.0 <![CDATA[ddH2O]]> 2.7

[0094] A total of 10 μL system was connected in a 16°C metal bath for 16 hours. Subsequently, the above-mentioned connection solution was electrotransformed (electrotransformation conditions were 2500V, 200Ω, 25μF) into the competent cells of lactic acid bacteria L.lactis NZ3900, coated with Elliker solid selective medium and cultured at 37°C, 5% CO2 for 72 hours, and positive transformants were screened. After the culture was completed, a single colony was picked in GM17 liquid medium (components were 4.23wt% M17 broth (Qingdao Haibo Biological, item number HB0391) and 0.5wt% glucose), and cultured at 37°C, 5% CO2 for 72 hours, and then the bacterial liquid and plasmid PCR identification was performed, and the plasmid was extracted for double enzyme digestion identification. Finally, the correct partial transformants were sequenced to ensure that pNZ8149-TsCB was successfully transformed into lactic acid bacteria L.lactisNZ3900.

[0095] Figure 2 The figure shows the construction of pNZ8149-TsCB / L.lactis NZ3900, where M is DM10000, 1 is double restriction enzyme digestion (Pst I+Xba I) of recombinant plasmid pNZ8149-TsCB, 2 is the purified product of target gene TsCB, 3 is the purified product of expression vector pNZ8149, 4 is the PCR product of recombinant plasmid pNZ8149-TsCB, 5 is the PCR product of recombinant bacteria pNZ8149-TsCB / L.lactis NZ3900, and 6 is the recombinant plasmid pNZ8149-TsCB. It can be seen from the figure that the expected target gene was successfully amplified by PCR, and the size of the double restriction enzyme digestion fragment was consistent with the expected result. The restriction enzyme digestion sites were Pst I and Xba I, which further confirmed that the target gene TsCB was inserted into the promoter downstream of the expression vector pNZ8149 with the correct reading frame orientation, indicating that the recombinant plasmid was successfully constructed.

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

[0097] 1. Induce target protein expression

[0098] A single colony of pNZ8149-TsCB / L.lactis NZ3900 on GM17 solid medium was picked up and inoculated into 10 mL GM17 liquid medium, and cultured at 30°C and 5% CO2 for 14 h; overnight bacteria were added to GM17 liquid medium at a volume ratio of 1:4, and cultured at 30°C and 5% CO2 until OD 600 =0.3, add inducer nisin to a final concentration of 20ng / mL (Sigma, catalog number SBR00021), and then induce for 6h; at the same time, 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 the induction, the bacteria were collected by low-temperature centrifugation, the precipitate was washed with sterile PBS, and the precipitate was resuspended with 200μL PBS buffer. Destroy the cell wall: add lysozyme to the prepared bacterial solution to a final concentration of 10mg / mL (Solarbio, catalog number L1080), break the cell wall in a water bath at 37℃ for 1h, obtain 8μL of whole bacterial protein, add 2μL of 5×SDS-PAGE loading buffer, boil for 5min, and store at -20℃ for later use.

[0099] 2. Western blot analysis of induced expression products

[0100] (1) Electrophoresis: 15-30 μg of the treated TsCB protein was loaded into each well and subjected to SDS-PAGE (5% stacking gel and 10% separation gel). The electrophoresis results were as follows: Figure 3 As shown, it can be seen from the figure that TsCB protein was successfully induced.

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

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

[0103] (4) Blocking: The cut strips were completely immersed in 5% skimmed milk powder and incubated in a shaker at 37°C for 2 h.

[0104] (5) Washing: Completely immerse the blocked strips with TBST and place them on a horizontal shaker for three washes, each for 10 min.

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

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

[0107] (8) DAB color development: Soak the strips in DAB color development solution, take them out immediately after color development for 1-9 seconds, and rinse them in distilled water to terminate the color development.

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

[0109] The whole bacterial protein of lactococcus pNZ8149 / L.lactis NZ3900 with vector and pNZ8149-TsCB / L.lactis NZ3900 after induction was subjected to SDS-PAGE electrophoresis and transferred to membrane. The bands after transfer were identified by normal serum and anti-TsCB serum respectively. Western blot results showed ( Figure 4 ), anti-TsCB serum recognized a band at 36.9 kDa, while normal serum could not, indicating that TsCB protein was successfully expressed in Lactococcus.

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

[0111] 1. Draw 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 / pNZ8149-TsCB were picked from the plate and placed in 5 mL of liquid GM17 medium, placed in a CO2 incubator, and cultured at 30°C and 5% CO2 for 14 hours. The overnight bacterial solution was inoculated into GM17 liquid medium at a volume ratio of 1:25, and cultured at 30°C and 5% CO2. One tube was taken every 1 hour, and the OD was measured on a UV spectrophotometer. 600 Each tube was measured 3 times and the average value was taken. 600The value is the vertical axis and the growth curve is drawn.

[0113] like Figure 5 As shown in the figure, the bacterial growth was relatively slow in the first 2 hours, then entered the logarithmic growth phase, and entered the plateau phase after about 6 hours. 600 pNZ8149 / L.lactis NZ3900 and pNZ8149-TsCB / L.lactisNZ3900 were similar in logarithmic growth phase, but grew slightly slower than L.lactis NZ3900 empty bacteria, and eventually reached the same plateau phase, indicating that the insertion of the TsCB gene had no effect on the growth of the strain.

[0114] 2. Genetic stability test

[0115] After the pNZ8149-TsCB / L.lactis NZ3900 strain preserved at -80℃ was cultured on a GM17 culture plate for 18-24h, a single colony was picked from the culture plate and inoculated into a GM17 liquid medium for overnight bacterial culture and used as the primary strain. The primary strain was inoculated into the GM17 liquid medium at a volume ratio of 1:100, placed in a 30℃ constant temperature incubator under 5% CO2 and cultured for 8h, and the resulting bacterial solution was used as the second generation of bacteria, and this was continued until the 20th generation was subcultured. The bacterial solution of the 20th generation seed was revived and cultured for 18-24h, and subcultured for 20 generations, and the bacterial solution of each generation was collected, centrifuged and ultrasonically broken for PCR, and electrophoresis was used to observe whether the pNZ8149-TsCB plasmid can be stably inherited in the pNZ8149-TsCB / L.lactis NZ3900 strain.

[0116] The pNZ8149-TsCB plasmid was extracted in large quantities and transformed into Lactococcus lactis NZ3900 by electroporation. Then, the Lactococcus lactis containing the pNZ8149-TsCB plasmid was obtained by plate-spreading culture. In order to detect the genetic stability of the pNZ8149-TsCB plasmid, the pNZ8149-TsCB / L. lactis NZ3900 bacteria were cultured for 20 generations and PCR was performed with TsCB primers. The results are shown in Figure 2. Figure 6 As shown, the TsCB gene can be amplified in each generation of bacteria.

[0117] 3. Tolerance of recombinant Lactococcus lactis to acidity

[0118] The digestive juice prepared by physiological saline, concentrated hydrochloric acid and pepsin (0.9wt% sodium chloride, 0.75wt% hydrochloric acid and 1wt% pepsin) was used to simulate gastric juice, and concentrated hydrochloric acid was used to adjust the pH to different values ​​(pH=1, 2, 3, 4). With physiological saline (pH=7.2) as the control, 1 mL of the recombinant bacteria (OD 600 =0.3-0.4), centrifuged at 4°C and 12000g for 20 min, added with 1 mL PBS and resuspended, and inoculated into 9 mL of liquid with different pH values, and cultured at 30°C in 5% CO2. 1 mL of bacterial solution was taken at 0, 1, 2, 3, and 4 h after culture, and diluted with PBS to different concentration gradients (10 -4 , 10 -5 and 10 -6 CFU / mL), 100 μL was plated on GM17 solid medium for culture, and the live bacteria were counted by plate counting method after 48 h to observe the growth ability of the recombinant bacteria under different acidic conditions.

[0119] The acid resistance test results show that ( Figure 7 ), compared with the normal environment (pH = 7.2), pNZ8149-TsCB / L.lactisNZ3900 bacteria survived for 2-3h in the environment of pH = 1 and 2, and survived for 4h in the environment of pH = 3. However, there was no statistical difference in the survival time of pNZ8149-TsCB / L.lactis NZ3900 bacteria in the environment of pH = 4 and 7.2 (ns). This shows that pNZ8149-TsCB / L.lactis NZ3900 bacteria have a certain tolerance to acidic environment, but it has a slight effect on reducing the number of bacteria.

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

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

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

[0123] Eight-week-old C57BL / 6J female mice cultured 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), 5 mice in each group, the mice were fasted and deprived of water for 4 hours before the first oral gavage, and 5% NaHCO3 solution was gavaged to neutralize gastric acid 30 minutes before inoculation of bacterial solution. The feeding conditions of each group of mice are as follows:

[0124] (1) Control group: The mice were given normal drinking water for 10 days and gavage with 200 μL PBS on days 5-10.

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

[0126] (3) pNZ8149 / L.lactis NZ3900 group (pNZ8149 group): The mice were given normal drinking water for 10 days, and pNZ8149 / L.lactis NZ3900 (200 μL, 5×10 10 CFU / mL) by intragastric administration.

[0127] (4) pNZ8149 / L. lactis NZ3900-DSS group (pNZ8149-DSS group): The cells received 2.5% (w / v) DSS solution for 7 consecutive days, followed by normal drinking water for 3 days. On days 5 to 10, the cells were treated with pNZ8149 / L. lactis NZ3900 bacterial solution (200 μL, 5 × 10 10 CFU / mL) by intragastric administration.

[0128] (5) pNZ8149-TsCB / L.lactis NZ3900 group (TsCB group): The mice were given normal drinking water for 10 days, and pNZ8149-TsCB / L.lactis NZ3900 bacterial solution (200 μL, 5×10 10 CFU / mL) by intragastric administration.

[0129] (6) pNZ8149-TsCB / L. lactis NZ3900-DSS group (TsCB-DSS group): The cells received 2.5% (w / v) DSS solution for 7 consecutive days, followed by normal drinking water for 3 days. On days 5 to 10, the cells were treated with pNZ8149-TsCB / L. lactis NZ3900 bacterial solution (200 μL, 5 × 10 10 CFU / mL) by intragastric administration.

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

[0131] The body weight of mice was measured every day, and the feces of each mouse was checked to evaluate the disease activity index (DAI) of mice.

[0132] Three important clinical indicators of the colitis model are varying degrees of weight loss, diarrhea, and fecal occult blood. Figure 9-11 As shown in the figure, during the whole colitis test period, the weight of mice in the Control group, pNZ8149 group and TsCB group showed a relatively stable trend, without diarrhea and bloody stool symptoms; after DSS induction, the weight of each group gradually decreased, and the degree of diarrhea and bloody stool worsened. After lactic acid bacteria treatment, this downward trend improved. Starting from the 6th day, after TsCB treatment, the weight loss of mice in the colitis group decreased, and the shape of the mouse feces changed, losing the original cylindrical shape, gradually becoming softer and thinner, until the diarrhea symptoms were obvious, and the fecal occult blood gradually decreased.

[0133] The DAI score is the disease activity index score, which is a comprehensive reflection of the degree of weight loss, diarrhea and fecal occult blood status, reflecting the degree of damage to the mouse colon during colitis. Fig.12 As shown in the figure, during the entire colitis test period, the DAI scores of mice in the Control group, pNZ8149 group, and TsCB group showed a stable trend of change, with no significant difference. After DSS induction, the DAI scores of each group gradually increased. After the action of probiotics, starting from the 6th day, the DAI scores of mice in the TsCB-DSS group and the DSS group were statistically different. In addition, compared with the DSS group, the DAI scores of mice in the pNZ8149-DSS group decreased, but compared with the pNZ8149-DSS group, the DAI scores of mice in the TsCB-DSS group were significantly smaller.

[0134] 2. Effect of TsCB on the improvement of colon pathological symptoms in mice with acute colitis

[0135] The mice in the above six groups were killed on the 10th day after DSS treatment and samples were collected to detect changes in various indicators. After the experiment, blood was collected from the eyeballs, and the colon of the mice was dissected and measured for length ( Fig.13 ), 0.5 cm intestinal tissue above the anus was taken and frozen at -80°C, another 0.5 cm intestinal tissue was taken and stored at -80°C for measuring colon myeloperoxidase activity, the remaining colon tissue of one group of mice was cut longitudinally with scissors and washed with PBS, one part was stored in buffer for collecting LP cells, and the remaining colon tissue of another group of mice was rolled into Swiss rolls and fixed in 10% formalin solution.

[0136] like Fig.14 As shown in the figure, the colon length of normal mice is generally 7-8cm. After DSS treatment, the colon length of mice shortened by 2-4cm. 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. The colon of the mice in the Control group had multiple pieces of formed feces, and the colon had no congestion, swelling, or ulcers. After DSS induction, the colon wall of the mice was diffusely congested, the intestinal wall was severely swollen, and multiple ulcers occurred. The colon to the anorectum of mice with severe symptoms was full of bloody mucus. The gross pathological scoring of the colon revealed that ( Fig.15 ), lactic acid bacteria treatment of colitis mice significantly alleviated DSS-induced colon damage in mice, and TsCB had a better effect in improving enteritis than pNZ8149.

[0137] 3. TsCB alleviates pathological damage of colon tissue in mice with acute colitis

[0138] In order to better verify the therapeutic effect of TsCB on colitis mice, the colon tissue samples of each group of mice were subjected to HE ( Fig.16 ) and PAS staining ( Fig.17 ). The results of section staining showed that the degree of colonic lesions in mice in the DSS group was severe, including the loss or partial loss of crypts, the disappearance or partial disappearance of goblet cells, severe colonic mucosal edema and infiltration of inflammatory cells. The above pathological conditions were significantly improved in the colitis mice treated with TsCB and pNZ8149. The number of colonic goblet cells in the mice treated with TsCB inflammation was significantly increased compared with that in the inflammation group.

[0139] The pathological histological scores of the colon pathological sections of mice in each group were calculated ( Fig.18 ) and goblet cell count ( Fig.19 ), further showed that after DSS-induced colitis, the colon pathology score increased and the number of goblet cells in the colon disappeared in large numbers; TsCB significantly alleviated the colon pathological damage of mice with DSS-induced acute colitis, and the number of colon goblet cells increased, and the degree of alleviation was higher than that of the pNZ8149-DSS group.

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

[0141] In the early stage of acute inflammation, neutrophils are the main inflammatory cells in local infiltration and are chemotactic to the inflammatory site to participate in the inflammatory response. Flow cytometry was used to detect the changes in neutrophils in the spleen, mesenteric lymph nodes (MLN), peripheral blood, and colonic lamina propria (LP) of mice in the Control group, pNZ8149 group, TsCB group, DSS group, pNZ8149-DSS group, and TsCB-DSS group. The gate diagram of immune cells in each group and the typical scatter diagram of neutrophils ( Figure 20-23 ) It can be seen that 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 was significantly reduced.

[0142] The transcription level of neutrophil chemokine was analyzed by qPCR in the colon tissue of mice treated with DSS. The results showed that ( Fig.24 and Fig.25 ), compared with the DSS group, the expression of CXCL1 and CXCL2 in the TsCB-DSS treatment group was significantly decreased, and TsCB could 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 measure the MPO activity in the colon of each group of mice by colorimetry to quantify the infiltration of inflammatory cells (polymorphonuclear neutrophils) in the colon. Fig.26 As shown, after DSS induction, the MPO activity of each group increased, and the MPO activity (neutrophil accumulation index) of each group after lactic acid bacteria treatment was significantly lower than that of 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 lower.

[0144] Example 7 TsCB induces Th2 immune response

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

[0146] Flow cytometry was used to detect the changes of CD4+T cells in colon LP cells in mice in the Control group, pNZ8149 group, TsCB group, DSS group, pNZ8149-DSS group and TsCB-DSS group. First, find out the cell population: remove cell debris under the FSC-A and SSC-A parameters, screen live cells from LP cells based on immunofluorescent dyes that stain dead and live cells, and then analyze them; circle single cells under the FSC-A and FSC-H parameters, remove dimer or polymer cells to prevent cell adhesion; circle CD45+ positive immune cells under the CD45+ and FSC-A parameters; analyze CD4+ labeled T cells in the CD45+ cell population, and compare the percentage of T cell populations in immune cells among the four groups. According to the results of quantitative analysis of T cells, ( Fig. 27 ), after DSS treatment, the number of T cells in the colonic lamina propria of each group of mice was significantly decreased; compared with the DSS group, the number of T cells in the TsCB-DSS group was significantly increased.

[0147] 2. TsCB increases the production of Th2 cytokines in the colonic lamina propria of mice

[0148] Flow cytometry was used to analyze the Th1 cytokine IFN-γ, Th2 cytokine IL-4, and anti-inflammatory factor IL-10 in the intestinal LP cells of each group of mice. Figure 28-Figure 30 As shown, compared with the mice in the Control group, pNZ8149 group, and TsCB group, the type 2 cytokine IL-4 and anti-inflammatory factor IL-10 produced in the colon LP cells of each group of mice were reduced after DSS treatment; compared with the DSS group, IFN-γ was significantly reduced after probiotic treatment. Data analysis found that compared with the pNZ8149-DSS group, the level of IFN-γ in the TsCB-DSS group was lower, while IL-4 was significantly increased. In addition, after TsCB treatment of colitis mice, the anti-inflammatory factor IL-10 was also significantly increased. 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 colonic lamina propria of mice

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

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

[0152] The transcription levels of inflammatory cytokines in colon tissue were analyzed by qPCR. Fig.34 and Fig.35 As shown, it was found that the transcription levels of proinflammatory 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 was improved after treatment with Lactococcus lactis; compared with the DSS group, the transcription levels of proinflammatory factors TNF-α, IL-6, and IL-1β were significantly decreased after TsCB treatment, while the transcription level of anti-inflammatory factor IL-10 was significantly increased; data analysis found that compared with pNZ8149, the expression level of IL-10 in the colon was increased after TsCB treatment.

Claims

1. A trichinella 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 according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

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

4. The use according to claim 3, characterized in that: The application is to relieve colon inflammation by reducing the expression level of pro-inflammatory factors and increasing the expression level of anti-inflammatory factors.

5. The use according to claim 4, characterized in that: The pro-inflammatory factors are TNF-α, IL-6 and IL-1β, and the anti-inflammatory factor is IL-10.

6. A recombinant plasmid, characterized in that: The invention comprises the gene encoding the Trichinella spiralis cathepsin B as described in claim 2.

7. A drug for treating colitis, characterized in that: It comprises the Trichinella spiralis cathepsin B according to claim 1, the gene according to claim 2 or the recombinant plasmid according to claim 6.

8. The drug for treating colitis according to claim 7, characterized in that: The medicine is an oral medicine.

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