Lactic acid bacteria protein with antioxidant and anti-radiation activity

Abstract

The invention relates to the technical field of biology. The invention discloses a novel antioxidant and anti-radiation protein, which is a lactococcus lactis protein LlrG. The invention finds that the protein has an anti-oxidative damage effect and can reduce the harm of H2O2 to cells in the cells; animal experiments prove that the protein can significantly increase the number of leukocytes and platelets of a radiated mouse, the marrow DNA content and the spleen organ index, alleviate oxidative stress caused by radiation and play a role in radiation resistance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a lactic acid bacteria protein with antioxidant and anti-radiation activities and an application thereof. Background Art

[0002] Ionizing radiation refers to the general term for radiation that can cause matter to ionize. It has the characteristics of high frequency and high energy. It is often used in the diagnosis and treatment of some diseases and the sterilization of food, which has promoted the development of medicine and food industry. While ionizing radiation brings convenience to people, high-dose radiation can also cause certain damage to the body. Excessive free radicals produced by radiation can attack biological macromolecules such as DNA and proteins, causing varying degrees of oxidative damage to the body's hematopoietic system, reproductive system and immune system, and even induce cancer. With the rapid development of nuclear technology, ionizing radiation has been applied to various fields of life. Radiation-exposed workers are exposed to ionizing radiation for a long time, and their risk of radiation damage continues to increase. At present, radiation protection has become one of the important issues that need to be urgently addressed in the nuclear field.

[0003] In order to prevent and reduce radiation damage, the development of radiation protectors has become a research focus. Radiation protectors are a type of functional preparations used before radiation to reduce the degree of radiation damage to the body, and are one of the effective ways to prevent and treat ionizing radiation damage. At present, the developed radiation protectors are mainly divided into two categories. One category is chemical radiation protectors mainly based on artificial synthesis, including sulfur compounds, hormone compounds, cytokines, etc., which are relatively expensive and have certain toxic side effects. The other category is radiation stress adaptation regulators from natural product sources, including polyphenol compounds, proteins and active peptides, which can improve the body's radiation tolerance through dietary intervention. Its advantages are low cost, long duration of action and no obvious toxic side effects. Finding new natural radiation protectors that are highly effective, non-toxic and can be taken for a long time has become the current research focus.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a Lactococcus lactis protein LlrG with antioxidant and anti-radiation functions. This protein is a two-component regulatory factor, which forms a two-component system with the membrane protein KinG and plays an important role in microbial stress adaptation.

[0006] The second invention objective of the present invention is to provide the application of the lactic acid bacteria protein in anti-oxidation and anti-radiation.

[0007] Accordingly, the present invention also provides a composition containing the protein, which can be used as an anti-radiation drug.

[0008] Furthermore, the protein has at least one of the following effects:

[0009] (1) Reduce the damage of H2O2 to cells:

[0010] (2) Increased the number of white blood cells and platelets in the peripheral blood of irradiated mice;

[0011] (3) Increased bone marrow DNA content and spleen organ index in irradiated mice;

[0012] (4) Reduce the levels of MDA, AST, and ALT in the blood of irradiated mice;

[0013] (5) Reduced MDA content in the spleen and liver of irradiated mice;

[0014] (6) Increase the levels of SOD and GSH in the blood of irradiated mice;

[0015] (7) Increased SOD and GSH levels in the spleen and liver of irradiated mice.

[0016] In order to achieve the purpose of the invention, the technical solution adopted is:

[0017] The present invention provides a lactic acid bacteria protein sequence, the GenBanK accession number of which is AAK05845.1, and the sequence is shown in SEQ ID No: 1 below.

[0018] The present invention also provides a nucleic acid sequence encoding the protein sequence, and the sequence is shown in SEQ ID No: 2 below.

[0019] The present invention also provides an expression vector, which is a recombinant vector containing a nucleic acid sequence encoding the protein. Furthermore, the vector of the recombinant vector is pET-32a.

[0020] The present invention also provides a method for expressing the protein, the steps of which include:

[0021] The lactic acid bacteria DNA group was extracted, and the nucleic acid sequence shown in SEQ ID No: 2 was obtained by specific amplification. The nucleic acid sequence was connected to the pET-32a vector by homologous recombination. The constructed expression vector was transferred into the competent Escherichia coli DHα for amplification. The recombinant plasmid was extracted and transferred into Escherichia coli BL21 to construct an expression strain. The positive clones were selected by ampicillin resistance screening, and the strains were expanded and cultured. When the OD 600 When the value reaches 0.4-0.8, IPTG is added to induce expression for 4 hours, and the bacteria are collected and broken; the precipitate is collected by centrifugation, resuspended in a buffer, and centrifuged after an ice bath to obtain a supernatant, and the supernatant is purified by nickel column affinity chromatography to prepare the protein shown in SEQ ID No: 1, and the active protein is obtained by gradient urea dialysis and renaturation.

[0022] The above protein GenBanK number is AAK05845.1, and the sequence is SEQ ID No: 1:

[0023] MTKIFIVEDDEIIVKAIKIALEKEFQVRSVSNFRAVKQEIMEFEADLVLMDIGLPFYSGFYWTNELRKLSQIPIIFISSASDDMNQVTAMNQGADDFVTKPFSLEILNAKI KALLRRSYSFSGIEKLEFAGYVLSENTLISTQEGEREEIELTSSENKILTLLFRGNGEVVTKEKILQELWQTDEFIDANTLNVKMTRLRKKLGEIGFDKHIMTKRGSGYALV

[0024] The corresponding nucleic acid sequence is SEQ ID No: 2:

[0025] ATGACTAAAATATTTATTGTAGAAGATGATGAAATTATTGTGAAAGCAATTAAGATTGCCTTAGAAAAAGAATTTCAAGTCAGAAGTGTTTCCAATTTTCGAGCAGTAAAACAAGAAATTATGGAATTTGAAGCAGATTTAGTATTAATGGATATTGGGCTACCATTT TATAGTGGTTTTATTGGACAAATGAATTAAGAAAGCTTTTCCCAAATTCCTATTATCTTTATTTCTTCGGCATCGGATGACATGAATCAAGTAACCGCTATGAATCAAGGAGCTGATGATTTTGTAACCAAGCCGTTTTCATTAGAAATTTTAAATGCAAAAATAAAA GCTTTACTGCGCAGAAGTTATTCTTTTCAGGGATTGAAAAGTTAGAATTTGCTGGTTATGTTTTATCTGAAAATACTTTGATTTCAACTCAAGAAGGTGAACGCGAAGAAATTGAGCTTACAAGTTCTGAAAATAAAATTTTAACTCTTCTTTTTCGTGGAAATGGT GAAGTGGTGACCAAGGAAAAAATTCTACAAGAGTTGTGGCAAACAGATGAATTTATTGATGCAAATACTTTAAATGTAAAAATGACTCGTTTACGCAAAAAACTTGGTGAAATTGGCTTTGATAAGCATATTATGACAAAAAGAGGAAGTGGTTATGCTCTGGTTTAA

[0026] The present invention also relates to the use of the lactic acid bacteria protein LlrG in the preparation of products for resisting oxidative damage and preventing oxidative damage.

[0027] The present invention also relates to the use of the lactic acid bacteria protein LlrG in the preparation of products for resisting and preventing radiation damage.

[0028] The present invention has at least the following beneficial effects:

[0029] The lactic acid bacteria protein LlrG sequence raw material of the invention is novel, has high purity, has high antioxidant damage and radiation damage resistance effects, and has strong application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1The protective effect of protein LlrG on oxidative damage of HL-7702 cells;

[0031] Figure 2 The effect of protein LlrG on peripheral blood leukocytes of irradiated mice;

[0032] Figure 3 The effect of protein LlrG on peripheral blood platelets of irradiated mice;

[0033] Figure 4 The effect of protein LlrG on spleen organ index of irradiated mice;

[0034] Figure 5 The effect of protein LlrG on the DNA content in the bone marrow of irradiated mice;

[0035] Figure 6 The effect of protein LlrG on SOD in the serum of irradiated mice;

[0036] Figure 7 The effect of protein LlrG on MDA in the serum of irradiated mice;

[0037] Figure 8 The effect of protein LlrG on GSH in the serum of irradiated mice;

[0038] Fig. 9 The effect of protein LlrG on SOD in spleen of irradiated mice;

[0039] Fig.10 The effect of protein LlrG on MDA in spleen of irradiated mice;

[0040] Fig.11 The effect of protein LlrG on GSH in spleen of irradiated mice;

[0041] Fig.12 The effect of protein LlrG on SOD in the liver of irradiated mice;

[0042] Fig.13 The effect of protein LlrG on MDA in the liver of irradiated mice;

[0043] Fig.14 The effect of protein LlrG on GSH in the liver of irradiated mice. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0045] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Lactic acid bacteria is a general term for bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. The present invention has found through research that the lactic acid bacteria protein LlrG has the effect of resisting oxidative damage and radiation damage.

[0048] The embodiment of the present invention provides a lactic acid bacteria protein LlrG sequence, the amino acid sequence of the lactic acid bacteria protein LlrG is shown in SEQ ID No: 1, and the protein can be used as a radiation protectant and an antioxidant.

[0049] The present invention also relates to the use of the lactic acid bacteria protein LlrG in the preparation of products for resisting oxidative damage and preventing oxidative damage. The present invention has found through research that the lactic acid bacteria protein LlrG can significantly increase the relative activity of normal mouse liver cells AML-12 under H2O2 oxidative damage, indicating that the lactic acid bacteria protein LlrG can remove H2O2 and reduce the damage of H2O2 to cells.

[0050] The embodiment of the present invention also relates to the application of the lactic acid bacteria protein LlrG in the preparation of products for anti-oxidative damage and prevention of oxidative damage. In vivo experiments have found that the lactic acid bacteria protein LlrG of the embodiment of the present invention can increase the DNA content, organ index and the number of white blood cells and platelets in the peripheral blood of mice after radiation, and can reduce the oxidative stress level in the serum, liver and spleen of mice after radiation. Superoxide dismutase (SOD) is an important antioxidant enzyme in the body, which mainly removes superoxide anion free radicals. The damaged body produces excessive ROS, which triggers a series of lipid peroxidation reactions, thereby producing malondialdehyde (MDA). Reduced glutathione (GSH) is a substrate necessary for glutathione peroxidase (GSH-PX) to catalyze the decomposition of peroxides. It is one of the most important antioxidants in cells and plays an important role in maintaining cell biological functions. Radiation can significantly reduce the SOD enzyme activity and GSH content in mouse serum, liver and spleen, while increasing the MDA content, indicating that radiation can cause oxidative stress in mice. Intragastric administration of the lactic acid bacteria protein LlrG according to the embodiment of the present invention can alleviate the oxidative stress level in the serum, liver and spleen of mice after radiation, increase the activity of antioxidant enzymes, and reduce the content of MDA, a lipid peroxidation product.

[0051] Example 1

[0052] 1. Activate the lactic acid bacteria twice, culture the second generation to the logarithmic phase, extract the DNA, and amplify the target gene LlrG by PCR (the target gene sequence is shown in SEQ ID No: 2 above).

[0053] The vector pET-32a was double-digested with XhoI and EcoRI and recovered by gel electrophoresis. The amplified target gene was recovered by gel electrophoresis and ligated to the vector pET-32a after restriction digestion to obtain a recombinant expression plasmid containing the target gene fragment, which was recorded as pET-32a-llrG.

[0054] 2. Transform the obtained pET-32a-llrG into E. coli DH5a competent cells for amplification and plasmid extraction. Then transform the expression plasmid into E. coli BL21 competent cells, pick single colonies, and inoculate the recombinant expression strain into LB liquid culture medium containing Amp (100μg / mL) antibiotics. The culture conditions are 37°C, 200rpm overnight culture, extract the recombinant plasmid, and use double enzyme digestion to verify that the recombinant plasmid is successfully constructed. Inoculate the overnight cultured bacterial solution of the successfully constructed strain into 200mL LB liquid culture medium containing Amp antibiotics at a ratio of 1:100, and culture at 37°C, 200rpm for 3h. The OD 600 At 0.4-0.8, 0.2 mmol / L IPTG was added, and the cells were collected after 4 hours of induction culture. The SDS-PAGE electrophoresis results of the expression products were as follows: Figure 3As shown. The protein-expressing bacteria were induced in large quantities by the above method, and the precipitate after washing with Solusion Binding Buffer was collected, and the bacteria were resuspended with Solusion Binding Buffer, and the bacteria were broken by ultrasonic disruption, and the conditions were: power 250W, ultrasonic 3s, pause 5s, and the total working time was 20min. The bacterial solution after ultrasonication was centrifuged at 10000rpm and 10min. After centrifugation, the bacterial solution precipitate was collected and resuspended in Inclusion Body Binding Buffer, and ice bathed for 1h to dissolve the inclusion body. The recombinant protein was purified by Ni-NTA His Bind Resn affinity chromatography, and the purified recombinant protein LlrG was refolded by gradient urea dialysis, and the specific conditions were: dialyzed in 7mol / L, 6mol / L, 5mol / L, 4mol / L, 3mol / L, 2mol / L, 1mol / L, and 0mol / L PBS for 4h to obtain active recombinant protein llrG. The amino acid sequence of the recombinant protein is shown in the above SEQ ID No: 1.

[0055] Example 2

[0056] The cck-8 method was used to detect the protective effect of recombinant protein LlrG on oxidative damage of AML-12 cells. The specific steps are as follows:

[0057] 1. Prepare solutions of the recombinant protein llrG with concentrations of 0 ng / mL, 62.5 ng / mL, 125 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, and 2000 ng / mL respectively;

[0058] 2. Collect normal mouse liver cells AML-12 cells in the logarithmic growth phase and prepare single cell suspensions, inoculate in 96-well plates, with 400 cells per well, add 90 μL of culture medium to each well, culture in the incubator for 12 hours, and then add 10 μL of recombinant protein of different concentrations. The specific grouping is as follows:

[0059] Add 10 μL of recombinant protein of different concentrations to make the final concentrations 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL, and set 1 μL of PBS as a control well.

[0060] 3. Continue culturing for 12 h. Add 11 μL of H2O2 to each well except the control group (Control group). The final concentration of H2O2 is 200 μmol / L. An equal volume of PBS is added to the Control group.

[0061] 4. After 24 hours of culture, aspirate the culture medium, add 10 μL serum, 10 μL CCK-8 solution, and 90 μL culture medium to each well, continue to culture for 2 hours, and measure the absorbance at 450 nm using an enzyme reader.

[0062] The experimental results obtained are as follows Figure 1 As shown ( ### P < 0.001 compared with the Control group; * P<0.05, ** P<0.01, *** P<0.001 compared with H2O2 group).

[0063] Experimental Example 3

[0064] 1. Drugs and experimental equipment

[0065] Positive drugs: Licojun tablets, recombinant protein LlrG of Example 1;

[0066] Experimental equipment: 60 Co-gamma ray radiation source.

[0067] 2. Experimental animals and methods

[0068] Experimental subjects: Kunming mice, provided by Henan Animal Experiment Center.

[0069] Experimental methods: 90 mice were adaptively cultured for one week and randomly divided into 6 groups, 15 mice in each group. They were divided into control group (NC group), model group (IR group), positive control group (PC group), recombinant protein llrG low-dose group (LlrG-L group), recombinant protein LlrG medium-dose group (LlrG-M group), and recombinant protein LlrG high-dose group (LlrG-H group).

[0070] After continuous gavage for 30 days, all mice in each group except the control group were given 60 The mice were uniformly irradiated with Co-γ rays at one time, with a total irradiation dose of 6Gy and a dose rate of 2Gy / min. After the irradiation, the mice were deprived of food but not water, and the subsequent indicators were measured.

[0071] Control group (NC group): 10mL / kg normal saline, gavage for 30 consecutive days, no 60 Co-γ irradiation treatment.

[0072] Model group (IR group): 10mL / kg normal saline, gavage for 30 days 60 Co-γ irradiation treatment.

[0073] Positive control group (PC group): 12 mg / kg of Licojun tablets aqueous solution, gavage for 30 consecutive days 60 Co-γ irradiation treatment.

[0074] The recombinant protein LlrG low-dose group (LlrG-L group): 1.25 mg / kg recombinant protein LlrG (Example 1) aqueous solution, continuously gavaged for 30 days and then 60 Co-γ irradiation treatment.

[0075] The recombinant protein LlrG medium dose group (LlrG-M group): 2.5 mg / kg recombinant protein LlrG (Example 1) aqueous solution, continuously gavaged for 30 days and then 60 Co-γ irradiation treatment.

[0076] Recombinant protein LlrG high dose group (LlrG-H group): 5 mg / kg recombinant protein LlrG (Example 1) aqueous solution, continuous gavage for 30 days and then 60 Co-γ irradiation treatment.

[0077] 3. Evaluation method

[0078] 3.1 Peripheral blood assay

[0079] 20 μL of blood was collected from the mouse eyeball and placed in a buffer solution, and the number of peripheral blood leukocytes and platelets was detected using a blood cell analyzer.

[0080] The experimental results are as follows Figure 2 , Figure 3 As shown. Among them, Figure 2 The effect of recombinant protein LlrG on peripheral blood leukocytes of irradiated mice. Figure 3 The effect of recombinant protein LlrG on peripheral blood platelets of irradiated mice. ### P<0.001 compared with NC group: * P < 0.05 and ** P<0.01 compared with the IR group (n=10).

[0081] 3.2 Organ index determination

[0082] After the mice were killed by dislocation, their spleens were quickly removed, fat was removed, and they were cleaned with pre-cooled saline. The surface moisture of the organs was absorbed with filter paper and weighed to calculate the organ index of the mice. The tissues were pre-cooled in a liquid nitrogen tank and stored at -80°C.

[0083] Spleen organ index = spleen weight / mouse body weight × 100%

[0084] The experimental results are as follows Figure 4 shown. Figure 4 The effect of recombinant protein LlrG on spleen organ index of irradiated mice. ### P < 0.001 compared with the NC group; *P<0.05 compared with the IR group (n=10).

[0085] 3.3 Determination of bone marrow DNA content

[0086] After the mouse was dissected, the left femur was taken out, and all the bone marrow cells in the femur were flushed out with 5 mL of 5 mmol / L CaCl2 solution, placed at 4°C for 30 min, centrifuged at 2500 rpm for 10 min, the supernatant was discarded, 5 mL of 0.2 mol / L HClO4 was added, and the mixture was heated at 90°C for 15 min. After cooling, the mixture was centrifuged at 2500 rpm for 10 min, the supernatant was taken, the absorbance was measured at a wavelength of 260 nm, and the bone marrow cell DNA content was calculated. The absorbance value was the bone marrow cell DNA content.

[0087] The experimental results are as follows Figure 5 The figure shows the effect of recombinant protein LlrG on the DNA content of bone marrow in irradiated mice. ### P < 0.001 compared with the NC group; ** P < 0.01 and *** P<0.001 compared with the IR group (n=10).

[0088] 3.4 Antioxidant enzyme assay

[0089] (1) Determination of antioxidant enzymes in serum: After blood was collected from the mouse eyeball, the blood sample was centrifuged at 4000 rpm, 4°C, and 10 min to prepare serum. The SOD enzyme activity, GSH-PX enzyme activity, GSH content, and MDA content in serum were detected using Nanjing Jiancheng SOD kit, MDA kit, and GSH kit.

[0090] (2) Determination of antioxidant enzymes in spleen and liver:

[0091] The spleen and liver were taken out from -80°C, and physiological saline (liver weight: physiological saline = 1:9) was added, and the homogenized suspension was quickly homogenized in a high-speed tissue grinder. The homogenized suspension was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The SOD enzyme activity, MDA content and GSH content in the liver and spleen homogenates were determined according to the kit instructions.

[0092] The experimental results are as follows Figure 6 to Figure 14 shown. Figure 6 The effect of recombinant protein LlrG on SOD in the serum of irradiated mice. Figure 7 The effect of recombinant protein LlrG on MDA in the serum of irradiated mice. Figure 8 The effect of recombinant protein LlrG on GSH in the serum of irradiated mice. Fig. 9 The effect of recombinant protein LlrG on SOD in the spleen of irradiated mice. Fig.10The effect of recombinant protein LlrG on MDA in the spleen of irradiated mice. Fig.11 The effect of recombinant protein LlrG on GSH in the spleen of irradiated mice. Fig.12 The effect of recombinant protein LlrG on SOD in the liver of irradiated mice. Fig.13 The effect of recombinant protein LlrG on MDA in the liver of irradiated mice. Fig.14 Effect of recombinant protein LlrG on GSH in liver of irradiated mice ( ### P < 0.001 compared with the NC group; * P<0.05, ** P < 0.01 and *** P<0.001 compared with the IR group (n=10).

[0093] Depend on Figure 6 to Figure 14 It can be seen that radiation can significantly reduce the SOD enzyme activity and GSH content in the serum, spleen and liver of mice, while increasing the MDA content, indicating that radiation can lead to an increase in the level of oxidative stress in mice. Intragastric administration of the recombinant protein LlrG of the embodiment of the present invention can reduce the oxidative stress level in the blood and tissues of mice, increase the activity of antioxidant enzymes, and reduce the content of lipid peroxidation product MDA.

[0094] In summary, the results of animal experiments show that the recombinant protein LlrG (Example 1) can increase the number of white blood cells and platelets in irradiated mice, improve the spleen organ index and bone marrow DNA content in irradiated mice, increase the activity of antioxidant enzymes in the serum, spleen and liver of irradiated mice and reduce the MDA content.

[0095] Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the claims. Any technical personnel in this field may make several possible changes and modifications without departing from the concept of the present application. Therefore, the scope of protection of the present application shall be based on the scope defined by the claims of the present application.

Claims

1. Application of Lactococcus lactis protein LlrG in the preparation of antioxidant and / or anti-radiation products.

2. The use according to claim 1, characterized in that: The amino acid sequence of the protein LlrG is the same as the protein sequence of GenBank No. AAK05845.

1.

3. The use according to claim 1, characterized in that: The product can be used for resisting oxidative damage, preventing oxidative damage, resisting radiation damage and / or preventing radiation damage.

4. The use according to the preceding claim, characterized in that The radiation damage is damage to the hematopoietic system and / or the immune system.

5. The use according to claim 1, characterized in that: The product has at least one of the following effects on mammals exposed to ionizing radiation: (1) Reduce the damage of H2O2 to cells; (2) Increase the number of white blood cells and platelets in peripheral blood after radiation; (3) Increased bone marrow DNA content and spleen organ index after radiation; (4) Reduce the levels of MDA, AST and ALT in the blood after radiation; (5) Reduce the MDA content in the spleen and liver after radiation; (6) Increase the levels of SOD and GSH in the blood after radiation; (7) Increase the levels of SOD and GSH in the spleen and liver after radiation.

6. Use according to the preceding claim, characterized in that The mammals include humans.

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