Lactobacillus rhamnosus fermentation liquor for inhibiting norovirus proliferation and adsorption protein

Abstract

The invention provides lactobacillus rhamnosus fermentation liquor for inhibiting proliferation of human norovirus and protein which is synthesized through a chemical path or genetic engineering and has a human norovirus adsorption effect. The preparation method of the fermentation liquor comprises the following steps: activating a lactobacillus rhamnosus YQ001 strain on a culture medium, standing and culturing at 37 DEG C for 24 hours, inoculating to a liquid culture medium, and standing and culturing at 37 DEG C; the protein (C2JVE6, C2JX39, C2K0J4) is a functional domain capable of binding to a capsid protein of a virus capable of causing diarrhea, and the virus capable of causing diarrhea comprises a human norovirus (HuNoVs). The fermentation liquor is applied to food additives, environment disinfectants and protection products. After the HuNoVs are treated by using the fermentation liquid supernatant, the replication level of the virus in the zebra fish body is obviously reduced, and an inhibition effect is shown.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a Lactobacillus rhamnosus fermentation liquid and a virus adsorption protein capable of inhibiting the proliferation of human-derived Norovirus, as well as applications thereof. Background Art

[0002] Noroviruses (NoVs) belong to the genus Norovirus of the family Caliciviridae. Genotypes that can infect humans are collectively referred to as human noroviruses (HuNoVs). HuNoVs are one of the main causes of non-bacterial gastroenteritis in people of all ages worldwide, and are associated with about one-fifth of gastroenteritis cases worldwide. According to statistics, HuNoVs cause about 685 million infections and 212,000 deaths each year. The disease burden caused by HuNoVs is as high as about 60 billion US dollars each year, causing a huge economic burden. Among them, GII.4 HuNoVs is the most important prevalent strain in recent decades.

[0003] The difficulties in culturing HuNoVs in vitro have hindered the development of vaccines or therapeutics, as well as the evaluation of antivirals and virucides. Currently available HuNoVs culture systems include B cell lines, stem cell-derived human intestinal epithelial cells, and zebrafish. Among them, zebrafish larvae produce high-titer and clean-background HuNoVs, which can serve as an ideal model for evaluating HuNoVs antivirals. GI and GII type HuNoVs can replicate in zebrafish larvae. After HuNoVs clinical samples were inoculated into zebrafish larvae for 2-3 days, the viral RNA increased by 2-3 logarithms.

[0004] In recent years, the interaction between intestinal microbiota and HuNoVs has attracted widespread attention. Studies have shown that certain intestinal microorganisms can inhibit the replication of HuNoVs and reduce their infectivity. Lactobacillus rhamnosus is a common intestinal microorganism that has attracted attention in recent years for its antiviral potential. Studies have shown that Lactobacillus rhamnosus can bind to P particles and viral particles of GII.3 and GII.4 HuNoVs to inhibit viral infection. In the germ-free pig model, co-colonization of Lactobacillus rhamnosus and Escherichia coli Nissle 1917 significantly reduced the shedding rate and diarrhea incidence of HuNoVs, showing its good effect in anti-HuNoVs.

[0005] Therefore, technicians in this field are committed to developing a Lactobacillus rhamnosus fermentation broth and virus adsorption protein for inhibiting the proliferation of human norovirus, as well as applications. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a Lactobacillus rhamnosus fermentation broth and virus adsorption protein for inhibiting the proliferation of human norovirus, as well as applications thereof.

[0007] To achieve the above object, the present invention provides a drug for inhibiting the replication of GII.4 type HuNoVs virus, wherein the drug is a fermentation broth of Lactobacillus rhamnosus.

[0008] Furthermore, the Lactobacillus rhamnosus is the Lactobacillus rhamnosus YQ001 strain.

[0009] The present invention also provides a method for preparing a fermentation broth of Lactobacillus rhamnosus, comprising the following steps:

[0010] Step 1: Take a small amount of bacterial solution from a frozen tube of Lactobacillus rhamnosus YQ001 strain using an inoculation loop and activate the strain on an MRS solid culture medium by streaking;

[0011] Step 2: After static culture at 37° C. for 24 h, pick a single colony and inoculate it into MRS liquid culture medium, and static culture at 37° C. for 24 h to obtain a fermentation broth.

[0012] The present invention also provides a method for preparing Lactobacillus rhamnosus bacterial membrane protein, comprising the following steps:

[0013] Step 1), extracting the whole genome DNA of Lactobacillus rhamnosus YQ001;

[0014] Step 2), find the protein gene coding sequences corresponding to the membrane proteins C2JVE6, C2JX39 and C2K0J4 from the Uniprot database. Use an online prediction website to analyze the signal peptide and transmembrane domain of the protein (signal peptide prediction website: https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0; transmembrane domain prediction website: https: / / services.healthtech.dtu.dk / service.php?TMHMM-2.0), and truncate the protein according to the analysis results. Design primers based on the gene sequence of the truncated membrane protein and introduce corresponding restriction sites, see Table 1. Perform PCR using the whole genome DNA obtained in step 1 as a template to obtain gene fragments of C2JVE6, C2JX39, and C2K0J4.

[0015] Table 1 Target gene amplification primer sequences and restriction endonuclease sites inserted

[0016]

[0017] Step 3), after the PCR reaction is completed, the reaction sample is added to the 1% agarose gel sample well for detection, and the gel containing the target DNA fragment is cut on a gel cutter and the gel is recovered.

[0018] Step 4), the gene fragments of C2JVE6, C2JX39 and C2KOJ4 obtained by gel recovery in step 3 are double-digested, and the empty pET-22b plasmid is double-digested at the same time, and then detected by agarose gel electrophoresis and gel recovery is performed.

[0019] Step 5), the target DNA fragment recovered by double restriction digestion in step 4 was ligated with the pET-22b plasmid using T4 ligase to obtain recombinant plasmids pET22b-C2JVE6, pET22b-C2JX39 and pET22b-C2KOJ4, and immediately transformed into E. coli DH5α competent cells.

[0020] Step 6), the pET22b-C2JVE6 / DH5α, pET22b-C2JX39 / DH5α and pET22b-C2K0J4 / DH5α monoclones successfully constructed by sequencing verification in step 5 were expanded and cultured in Amp-LB broth medium for plasmid extraction.

[0021] Step 7), the pET22b-C2JVE6, pET22b-C2JX39 and pET22b-C2K0J4 recombinant plasmids extracted above were transformed into E. coli BL21 (DE3) competent cells, to obtain pET22b-C2JVE6 / BL21 (DE3), pET22b-C2JX39 / BL21 (DE3) and pET22b-C2K0J4 / BL21 (DE3).

[0022] Step 8) Use the three recombinant bacteria obtained in step 7 to culture, induce expression and purify proteins. The induction conditions are as follows: pick a single clone of the recombinant bacteria, inoculate it into 5 mL of Kan-LB broth, and culture it at 37°C and 200 rpm for about 6 hours to activate the bacteria. Inoculate the bacterial solution into 5 mL of Kan-LB broth at a 1% inoculation rate. Culture it at 37°C and 200 rpm until the bacterial solution OD reaches 0. 600 When the pH value is 0.4-0.6, add IPTG with a final concentration of 0.4 mM and induce at 25°C for 12-16 hours. For protein purification steps, refer to the NiNTA Beads instruction manual.

[0023] The invention also provides an application of the fermentation liquid of Lactobacillus rhamnosus as a food additive.

[0024] Furthermore, the above-mentioned food additives can effectively block the spread of HuNoVs during food processing and storage.

[0025] Furthermore, the above-mentioned food includes seafood and frozen food.

[0026] The invention also provides an application of the fermentation liquid of Lactobacillus rhamnosus as an environmental disinfectant.

[0027] Furthermore, the above application location is a public place, including schools, restaurants and / or hospitals.

[0028] The present invention also provides an application of the fermentation liquid of Lactobacillus rhamnosus as a protective product.

[0029] The present invention also provides a Lactobacillus rhamnosus bacterial membrane protein, including C2JVE6, C2JX39 and / or C2K0J4, which has a functional domain capable of binding to the capsid protein of a virus that can cause diarrhea, wherein the virus that can cause diarrhea includes norovirus.

[0030] Furthermore, the amino acid sequence of membrane protein C2JVE6 is shown in SEQ ID NO: 1, the amino acid sequence of membrane protein C2K0J4 is shown in SEQ ID NO: 2, and the amino acid sequence of membrane protein C2JX39 is shown in SEQ ID NO: 3.

[0031] In the preferred embodiment 1 of the present invention, it is described in detail that the Lactobacillus rhamnosus fermentation liquid inhibits the replication of HuNoVs in zebrafish;

[0032] In another preferred embodiment 2 of the present invention, the process of Lactobacillus rhamnosus binding to the P protein of HuNoVsv is described in detail;

[0033] In another preferred embodiment 3 of the present invention, the process of adsorption of HuNoVs by Lactobacillus rhamnosus membrane protein is described in detail.

[0034] The beneficial technical effects of the present invention are as follows:

[0035] 1. Technical effect:

[0036] The present invention forms an intelligent capture and inhibition system by separating and fixing the membrane protein that specifically binds to the norovirus, which can accurately capture human norovirus and inhibit the replication of the virus using fermentation broth. By specifically binding to the P particles of HuNoVs, the virus can be efficiently adsorbed while reducing the proliferation of the virus in the host cells.

[0037] Low cost and easy production: By positioning the key components on the membrane protein, the dependence on the complete bacterial system is avoided and the production process is simplified. The membrane protein is small in size and easy to mass produce in a variety of expression systems (such as E. coli, yeast, etc.), which significantly reduces the production cost.

[0038] 2. Performance indicators:

[0039] ELISA and Western blot experiments verified that the binding efficiency of specific membrane proteins to Norovirus P particles was significantly higher, and efficient virus capture could be achieved at lower virus concentrations.

[0040] The Lactobacillus rhamnosus fermentation broth and supernatant in the present invention can effectively inhibit the proliferation of human norovirus in the body. Experiments show that in the virus infection experiment after being treated with the fermentation broth, the number of viral RNA copies is significantly reduced.

[0041] 3. Production implementation:

[0042] The membrane protein of the present invention is expressed in Escherichia coli through genetic engineering technology and is suitable for industrial production. The controllability and small molecular weight of membrane proteins make them suitable for a variety of product forms, such as antiviral sprays, food additives or pharmaceutical preparations, and have a wide range of application scenarios. At the same time, membrane proteins are easy to combine with other antiviral ingredients to form multiple protective barriers and enhance the antiviral effect.

[0043] 4. Prospects for industrial application:

[0044] The present invention has broad application prospects in food protection and can effectively block the spread of HuNoVs in food processing and storage, especially in high-risk foods such as seafood and frozen foods.

[0045] Due to the high infectivity of HuNoVs, the intelligent capture system of the present invention can be applied to public places such as schools, restaurants, hospitals, etc. as a core component of environmental disinfectants or protective products to reduce the risk of virus transmission.

[0046] The present invention provides a new antiviral mechanism, which can be further developed into an innovative drug for treating HuNoVs infection, meeting the market demand for highly effective antiviral preparations.

[0047] The present invention has the advantages of advanced technology, easy production, low cost and wide application potential, and is suitable for large-scale industrial promotion. Through innovative combination mechanism and stable production process, the present invention provides an efficient and low-cost solution for the prevention and control of HuNoVs, with extremely high conversion value and market competitiveness.

[0048] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a bar graph showing the inhibitory effects of different components of the Lactobacillus rhamnosus source on GII.4 type HuNoVs in zebrafish according to a preferred embodiment 1 of the present invention;

[0050] Figure 2 This is an SDS-PAGE and Western Blot electrophoresis diagram of the adhesion of the GⅡ.4 type HuNoVs P protein to bacteria according to a preferred embodiment 2 of the present invention;

[0051] Figure 3 It is a bar graph of the binding ability between the Lactobacillus rhamnosus membrane protein and the GII.4 type HuNoVs P protein according to a preferred embodiment 3 of the present invention;

[0052] Figure 4 It is a bar graph showing the binding ability between the membrane protein of a preferred embodiment 3 of the present invention and the GI.1 type HuNoVs P protein. DETAILED DESCRIPTION

[0053] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0054] Example 1 Lactobacillus rhamnosus inhibits the replication of GII.4 type HuNoVs in zebrafish

[0055] 1.1 Cultivation of Lactobacillus rhamnosus

[0056] 1) Take out the cryopreserved tube of Lactobacillus rhamnosus YQ001 strain from the -80℃ ultra-low temperature freezer, use an inoculation loop to dip a small amount of bacterial solution, and then activate the strain on MRS solid medium by plate streaking method. After static culture at 37℃ for 24 hours, pick a single colony and inoculate it into MRS liquid medium, and static culture at 37℃ for 24 hours to obtain fermentation liquid. Take 1mL of fermentation liquid for later use.

[0057] 2) The remaining Lactobacillus rhamnosus fermentation liquid was centrifuged at 10000 g for 10 minutes. The supernatant was taken and filtered with a 0.22 μm filter membrane to obtain a sterile supernatant. The bacterial precipitate was washed twice with PBS, resuspended in an equal volume, and 1 mL was taken for standby use, which was the Lactobacillus rhamnosus bacterial cell.

[0058] 3) Ultrasonicate the remaining bacterial cell suspension for 1 h until the bacterial liquid is basically clear to obtain a broken bacterial cell suspension, and take 1 mL for later use.

[0059] 4) The remaining broken bacterial suspension was centrifuged at 10,000 g for 10 minutes, the precipitate was washed twice with PBS, and resuspended in an equal volume to obtain an insoluble protein suspension.

[0060] 1.2 Interaction between different components of Lactobacillus rhamnosus and GII.4 HuNoVs

[0061] Mix different components of Lactobacillus rhamnosus and GII.4 HuNoVs in equal volumes at room temperature and let stand for 1 hour. If any precipitate appears during this period, resuspend it again.

[0062] 1.3 Evaluation of the antiviral effects of different fractions of Lactobacillus rhamnosus using the zebrafish system

[0063] 1) Collect 0 dpf zebrafish embryos, place 80-100 embryos in each culture dish (90×15 mm), add 15 mL of Danio solution, and store in an incubator at 28°C with a 14 / 10 h light / dark cycle.

[0064] 2) On the day of infection, transfer 72 hours post-fertilization (hpf) zebrafish larvae to a 6-well plate containing 5 mL of Danil solution per well (50-60 per well). Add 500 μL of tricaine stock solution (final concentration 5×) to anesthetize the zebrafish larvae.

[0065] 3) Use a plastic transfer pipette to transfer the zebrafish larvae to a clean Petri dish. Use a plastic transfer pipette to remove excess liquid.

[0066] 4) Fill 4 μL of virus solution into the microinjection needle, break the needle, and adjust the injection dose to 3 nL / time.

[0067] 5) Insert the microinjection needle into the yolk. Inject the droplet by pressing the pedal and retract the needle from the zebrafish larva.

[0068] 6) After injection, rinse the zebrafish larvae with a plastic pipette containing Danio solution and transfer the zebrafish larvae to a 6-well plate with 50-60 larvae per well containing Danio solution. Keep the plate in an incubator at 32°C with a 14 / 10 h light / dark cycle.

[0069] 7) Collect samples 48 hours after injection. Collect 10 zebrafish larvae into a group, put them into a 1.5 mL EP tube, and store them at -80°C. Extract RNA and detect the virus titer.

[0070] 1.4 Technical Effect

[0071] The inhibitory effects of different components of Lactobacillus rhamnosus on GII.4 type HuNoVs in zebrafish Figure 1 As shown, the fermentation broth, supernatant and insoluble protein of Lactobacillus rhamnosus inhibited the replication of HuNoVs in zebrafish, with an inhibition effect of 1 log.

[0072] Example 2 Lactobacillus rhamnosus binds to the P protein of GII.4 type HuNoVsv

[0073] Western Blot Verification of Adhesion of 2.1GⅡ.4 HuNoVs P Protein to Lactobacillus rhamnosus

[0074] 1) Take out the cryopreserved tube of Lactobacillus rhamnosus YQ001 strain from the -80℃ ultra-low temperature freezer, use an inoculation loop to dip a small amount of bacterial liquid, and then activate the strain on MRS solid medium by plate streaking method. After static culture at 37℃ for 24 hours, pick a single colony and inoculate it into MRS liquid medium, and static culture at 37℃ for 24 hours to obtain fermentation liquid.

[0075] 2) Wash the bacteria three times with PBS and resuspend to an OD600 of 1.0.

[0076] 3) 100 μL of 500 μg / mL P protein was incubated with 1 ml of bacterial solution with OD600 of 1.0 at 37°C for 1 h. The mixture was centrifuged at 7500×g for 2 min and washed three times with PBS.

[0077] 4) Take 40.0 μL PBS to resuspend the bacteria in a 1 ml centrifuge tube, add 10.0 μL 5× protein loading buffer, and perform SDS-PAGE detection and verification.

[0078] 5) After the SDS-PAGE electrophoresis is completed, collect the separation gel that has not been stained with Coomassie Brilliant Blue, measure the length and width and record them.

[0079] 6) Cut 2 pieces of filter paper and 1 piece of PVDF membrane of the same size as the separation gel. Immerse the filter paper and PVDF membrane in the transfer solution until they are completely wet.

[0080] 7) Place the sponge pad on the black plate of the transfer splint according to the precise alignment, and use a glass rod to remove bubbles; place filter paper, separation gel, PVDF membrane in sequence, and finally place another piece of filter paper, and use a glass rod to carefully remove bubbles; clamp the splint and put in the transfer electrode core; put the transfer electrode core into the electrophoresis tank and add transfer solution.

[0081] 8) Connect the power supply, perform electroporation at a constant voltage of 90 V for 80 min, and then disconnect the power supply.

[0082] 9) Take out the PVDF membrane and immerse it in 5% skim milk, and block it at 37° C. for 60 minutes. After blocking, rinse the PVDF membrane with 0.5% (v / v) TBST washing solution for 3 times, each time for 10 minutes.

[0083] 10) The PVDF membrane was taken out and immersed in HuNoVs P protein polyclonal antibody diluted 1:2000 (v / v) with 5% skim milk, and incubated overnight at 4°C.

[0084] 11) Take out the PVDF membrane, rinse it with 0.5% (v / v) TBST washing solution for 3 times, 10 min each time, and then immerse it in the secondary antibody diluted with 5% skim milk at 1:2000 (v / v), and incubate it at 37°C for 1 h.

[0085] 12) Take out the PVDF membrane and rinse it with 0.5% (v / v) TBST washing solution for 3 times, 10 minutes each time. Use absorbent paper to slightly absorb excess liquid (do not touch the protein side of the membrane), and then place it on a clean plastic wrap. According to the size of the membrane, add 1 ml of BeyoECL Star working solution per 10 cm2 of membrane, drop BeyoECL Star working solution onto the membrane, ensure that the working solution is evenly covered on the membrane, and leave it for 1-2 minutes. Take out the membrane, use absorbent paper to slightly absorb excess liquid, and use chemiluminescence imager for detection.

[0086] 2.2 Technical Effect

[0087] SDS-PAGE and Western Blot verification of the adhesion of GⅡ.4 type HuNoVs P protein to bacteria Figure 2 As shown, the upper figure is SDS-PAGE and the lower figure is Western Blot. The target protein is marked with a red box. Western Blot detection showed that the polyclonal antibody of P protein could identify proteins at about 35kDa and about 70kDa in Lactobacillus rhamnosus after interacting with P protein, confirming that they were P protein and P protein dimer. The results showed that Lactobacillus rhamnosus could bind to P protein and its dimer.

[0088] Example 3 Lactobacillus rhamnosus membrane protein captures GII.4 type HuNoVs

[0089] 3.1 Purification of membrane proteins

[0090] After mass spectrometry identification and molecular docking, three membrane proteins that can bind to the GII.4 HuNoVs P protein were screened, with protein IDs of C2JVE6, C2JX39, and C2K0J4. Recombinant plasmids pET28a-C2JVE6, pET28a-C2JX39, and pET28a-C2K0J4 were constructed and prokaryotic expression was performed using E. coli as an expression vector. The protein purification steps refer to the Ni NTA Beads instruction manual.

[0091] 3.2 Elisa experiments to verify the binding ability of membrane proteins to HuNoVs

[0092] 1) Use carbonate / bicarbonate coating buffer to dilute membrane protein as coating solution, with a final concentration of 142.86 pmol / mL. Use 1.0% BSA as negative control, and use 1.0 mg / mL PGM and 142.86 pmol / mL heat shock protein 70 (HSP 70) as positive controls.

[0093] 2) Add 100 μL of coating solution to each ELISA well and coat overnight at 4°C.

[0094] 3) The next day, the coating solution was discarded. The ELISA plate was washed 4 times with 150 μL PBS / well, and after washing, 120 μL 1.0% BSA / well was added and placed in a 37°C constant temperature incubator for 1 hour for blocking.

[0095] 4) The next day, discard the blocking solution. Wash the ELISA plate 4 times with 150 μL PBS / well, add 100 μL 10 μg / mL P protein / well after washing, and incubate in a 37°C constant temperature incubator for 1 hour.

[0096] 5) The solution was discarded and the ELISA plate was washed 5 times with 150 μL TBST / well. After washing, 100 μL of primary antibody diluted with blocking solution (1:2000) was added / well and the plate was incubated in a 37°C constant temperature incubator for 1 hour.

[0097] 6) Discard the solution, wash the ELISA plate 5 times with 150 μL TBST / well, add 100 μL of secondary antibody diluted with blocking solution (1:2000) / well, and incubate in a 37°C constant temperature incubator for 1 hour. Take out a certain volume of TMB solution from the 4°C refrigerator and place it at room temperature for use.

[0098] 7) Discard the solution, wash the ELISA plate 5 times with 150 μL TBST / well, add 100 μL TMB / well, and react for about 15 minutes in the dark. Then add 50 μL 2M H2SO4 / well to the ELISA plate to terminate the reaction.

[0099] 8) Use an ELISA reader to read the absorbance of each well on the ELISA plate at a wavelength of 450 nm and record it for analysis.

[0100] 9) Technical Effects

[0101] The binding ability of membrane protein to GII.4 HuNoVs P protein is as follows Figure 3 As shown in Figure 2, the ability of membrane proteins to bind to the P protein of GI.1 HuNoVs is as follows Figure 4 As shown, it can be seen that the three membrane proteins can bind to the P proteins of GII.4 type HuNoVs and GI.1 type HuNoVs viruses.

[0102] 3.2 In situ capture method to verify the binding ability of membrane proteins to GII.4 HuNoVs

[0103] 1) Use carbonate / bicarbonate coating buffer to dilute the membrane protein as the coating solution, with a final concentration of 142.86pmol / mL. Use 1.0% BSA as the negative control, and 1.0mg / mL PGM and 142.86pmol / mL heat shock protein 70 (HSP 70) as the positive control. Add 100μL of coating solution to each NuncrmJopXieldrm8 strip well and coat overnight at 4℃.

[0104] 2) After discarding the solution, add 150.0 μL PBS (pH 7.4) to each well and wash three times.

[0105] 3) Add 1.0% BSA at 120.0 μL / well and block at 37°C for 1 h.

[0106] 4) After discarding the solution, add 150.0 μL PBS (pH 7.4) to each well and wash three times.

[0107] 5) Add 100 μL of HuNoVs clinical samples diluted 5-10 times with PBS (pH 7.4) into the wells and incubate at 37° C. for 1 h.

[0108] 6) After discarding the liquid, add 150.0 μL 0.5‰ TBST to each well and wash three times, and then wash once with DEPC water.

[0109] 7) Add 10 mL of DEPC water to each well and seal it with a sealing film (add it in the middle of the well plate to prevent it from sticking to the wall), thermally crack it at 95°C for 5 minutes, and cool it at 4°C.

[0110] 8) After the high temperature incubation, the 8-tube strip was centrifuged briefly, and the lysate was used as a template for RT-qPCR detection. Each sample was repeated 3 times.

[0111] 9) The technical effects are shown in Table 2. It can be seen that the membrane protein-mediated in situ capture method can effectively capture GII.4 type HuNoV.

[0112] Table 2 In situ capture method verifies the binding ability of membrane proteins to GII.4 type HuNoVs

[0113]

[0114] The above describes in detail the preferred specific embodiments of the present invention. It should be understood that ordinary technicians in this field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in this technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the existing technology should be within the scope of protection determined by the claims. abc indicates significant differences between groups.

Claims

1. A drug for inhibiting HuNoVs virus replication, characterized in that: The medicine is the fermentation liquid of Lactobacillus rhamnosus.

2. The use according to claim 1, characterized in that The Lactobacillus rhamnosus is the Lactobacillus rhamnosus YQ001 strain.

3. A method for preparing a fermentation broth of Lactobacillus rhamnosus, characterized in that: The method comprises the following steps: Step 1: Take a small amount of bacterial solution from a frozen tube of Lactobacillus rhamnosus YQ001 strain using an inoculation loop and activate the strain on an MRS solid culture medium by streaking; Step 2: After the activated strain obtained in step 1 is statically cultured at 37° C. for 24 hours, a single colony is picked and inoculated into MRS liquid culture medium, and statically cultured at 37° C. for 24 hours to obtain a fermentation broth.

4. Application of a fermentation broth of Lactobacillus rhamnosus as a food additive.

5. The use according to claim 4, characterized in that The application can effectively block the spread of HuNoVs during food processing and storage.

6. The use according to claim 4, characterized in that The food includes seafood and frozen food.

7. Use of a fermentation broth of Lactobacillus rhamnosus as an environmental disinfectant.

8. The use according to claim 6, characterized in that The application location is a public place, including schools, restaurants and / or hospitals.

9. Use of a fermentation liquid of Lactobacillus rhamnosus as a protective product.

10. A Lactobacillus rhamnosus bacterial membrane protein, characterized in that: The bacterial membrane protein is C2JVE6, C2JX39 or / and C2K0J4, the amino acid sequence of the membrane protein C2JVE6 is shown in SEQ ID NO: 1, the amino acid sequence of the membrane protein C2K0J4 is shown in SEQ ID NO: 2, and the amino acid sequence of the membrane protein C2JX39 is shown in SEQ ID NO: 3.

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