CN114426942B - Recombinant lactococcus lactis, microcapsule and application thereof - Google Patents
Recombinant lactococcus lactis, microcapsule and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant lactococcus lactis, a microcapsule and application thereof, and belongs to the technical field of genetic engineering. The invention takes PNZ8149 as a basic vector, a first fusion gene or a second fusion gene is inserted into the PNZ8149, the first fusion gene comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDK and human alcohol dehydrogenase which are sequentially connected in series, and the second fusion gene comprises a signal peptide SPusp45, probiotics LEISS, enterokinase recognition site DDDDK and human aldehyde dehydrogenase which are sequentially connected in series and are respectively fused. The target enzyme is subjected to enzyme cleavage of the signal peptide sequences SPusp and LEISS from the fusion protein under the action of endogenous enterokinase in the intestinal tract, and free alcohol dehydrogenase or acetaldehyde dehydrogenase is released, and the released alcohol dehydrogenase or acetaldehyde dehydrogenase can decompose alcohol.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant lactococcus lactis, a recombinant lactococcus lactis microcapsule and application thereof.
Background
Wine is a common drink in daily life. The drinking brings joy to people and also brings certain harm to the physical health and social stability of people. After the people drink excessive wine, accumulated ethanol can be metabolized in the body to generate a plurality of harmful substances and deposit a plurality of free radicals, so that the people are injured to different degrees, and especially the liver is easy to be diseased. Reducing alcohol intake or absorption is a direct route to mitigating the hazard.
At present, most of the products sold at home and abroad for dispelling effects of alcohol and protecting liver are diuretics chemical synthetic drugs, liver-protecting and stomach-nourishing traditional Chinese medicines, sobering peptide beverages or drugs for improving the activity of alcohol metabolism related enzymes. The efficacy is exerted based on that the product can influence ethanol metabolism or inhibit ethanol absorption after being taken by a human body, thereby reducing the damage of the product to other organs such as liver and the like. Although many anti-hangover and liver-protecting medicines have certain anti-hangover and anti-hangover effects, different side effects are associated, so that the anti-hangover and liver-protecting medicines are unfavorable to the health of human bodies, and particularly, the reactions of different anti-hangover medicines are different.
Disclosure of Invention
In view of the above, the present invention aims to provide a recombinant lactococcus lactis, a microcapsule and an application thereof, which can decompose alcohol by endocrine hADH B enzyme and ALDH2 enzyme in the intestinal tract after oral administration of the recombinant lactococcus lactis of the present invention.
The invention provides a recombinant lactococcus lactis comprising a first recombinant lactococcus lactis and/or a second recombinant lactococcus lactis;
The first recombinant lactococcus lactis comprises a first recombinant expression vector; the second recombinant lactococcus lactis comprises a second recombinant expression vector;
The skeleton plasmids for constructing the first recombinant expression vector and the second recombinant expression vector are PNZ8149 respectively;
A first fusion gene is inserted into the first recombinant expression vector, and comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDK and human-derived alcohol dehydrogenase which are sequentially connected in series;
the second recombinant expression vector is inserted with a second fusion gene, and the second fusion gene comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDDDK and human acetaldehyde dehydrogenase which are sequentially connected in series.
Preferably, the nucleotide sequence of the first fusion gene is shown in SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the second fusion gene is shown as SEQ ID NO: 2.
Preferably, the first and second fusion genes are inserted between the SphI and XbaI restriction sites of PNZ8149, respectively.
Preferably, when the recombinant lactococcus lactis includes a first recombinant lactococcus lactis and a second recombinant lactococcus lactis, the ratio of the effective viable count of the first recombinant lactococcus lactis to the second recombinant lactococcus lactis is (1-3): (1-3).
The invention also provides a microcapsule containing the recombinant lactococcus lactis.
The invention also provides an application of the recombinant lactococcus lactis or the microcapsule in preparing medicines for preventing drunk; the recombinant lactococcus lactis comprises a first recombinant lactococcus lactis, or the recombinant lactococcus lactis comprises a first recombinant lactococcus lactis and a second recombinant lactococcus lactis.
Preferably, the preventing drunkenness comprises reducing alcohol absorption and/or extending alcohol tolerance time.
The invention also provides an application of the recombinant lactococcus lactis or the microcapsule in preparation of anti-hangover medicines.
Preferably, the anti-hangover includes reducing recovery time after drinking and/or reducing acute damage to the liver and/or intestine caused by excessive drinking and/or alleviating vomiting and headache after drinking.
Preferably, the dosage form of the medicament comprises an oral formulation.
The invention provides a recombinant lactococcus lactis, which takes PNZ8149 (lactobacillus food-grade expression vector) as a basic vector, wherein a first fusion gene or a second fusion gene is inserted into PNZ8149, the first fusion gene comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDDDK and human alcohol dehydrogenase which are sequentially connected in series, and the second fusion gene comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDK and human aldehyde dehydrogenase which are sequentially connected in series and are respectively fused. The target enzyme is subjected to enzyme cleavage of the signal peptide sequences SPusp and LEISS from the fusion protein under the action of endogenous enterokinase in the intestinal tract, and free alcohol dehydrogenase or acetaldehyde dehydrogenase is released, and the released alcohol dehydrogenase or acetaldehyde dehydrogenase can decompose alcohol. The invention provides a high-activity human ADH1B (hADH B and/or acetaldehyde dehydrogenase (ALDH 2) expressed probiotics, hADH B enzyme and ALDH2 enzyme are secreted after oral administration to decompose alcohol, and the result shows that the oral administration of the probiotics expressed hADH B reduces alcohol absorption and prolongs alcohol tolerance time, the oral administration of the probiotics expressed hADH B and/or ALDH2 shortens recovery time after alcohol consumption, and more importantly, liver and intestinal tract are protected from acute injury caused by excessive alcohol consumption.
Drawings
FIG. 1 is a schematic diagram showing the construction and action of recombinant lactococcus lactis of the present invention;
FIG. 2 shows the results of detection of recombinant lactococcus lactis for expression of ADH enzyme using anti-hadh antibodies;
FIG. 3 shows the results of detecting expression of ALDH enzyme by recombinant lactococcus lactis using hALDH antibody;
FIG. 4 is a view showing the result of detecting the number of viable bacteria by the culture plate count method;
FIG. 5 shows the case of oral administration of recombinant lactococcus lactis for prolonged alcohol tolerance;
FIG. 6 is a graph showing the time to intoxicate mice;
FIG. 7 is a graph showing exercise count statistics of drunk mice after sobering;
Fig. 8 is a graph showing the case of oral administration of recombinant lactococcus lactis to alleviate liver-intestinal damage caused by acute alcohol intake, wherein a is serum alcohol residue, B is blood triglyceride concentration, C is intestinal mucosal lesion, and D is lipid level in liver of mice.
Detailed Description
The invention provides a recombinant lactococcus lactis comprising a first recombinant lactococcus lactis and/or a second recombinant lactococcus lactis;
The first recombinant lactococcus lactis comprises a first recombinant expression vector; the second recombinant lactococcus lactis comprises a second recombinant expression vector;
The skeleton plasmids for constructing the first recombinant expression vector and the second recombinant expression vector are PNZ8149 respectively;
A first fusion gene is inserted into the first recombinant expression vector, and comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDK and human-derived alcohol dehydrogenase which are sequentially connected in series;
the second recombinant expression vector is inserted with a second fusion gene, and the second fusion gene comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDDDK and human acetaldehyde dehydrogenase which are sequentially connected in series.
In the invention, the nucleotide sequence of the first fusion gene (Noc 1-Usp45-LESS-EK-ADH 1B) is shown in SEQ ID NO:1, specifically:
ccatggtcatgaaaaaaaagattatctcagctattttaatgtctacagtgatactttctgctgcagccccgttgtcaggtgtttacgctgatactaattctgatttggaaatatcgtcgacttgtgatgctgacgatgacgataagagcacagcaggaaaagtaatcaaatgcaaagcagctgtgctatgggaggtaaagaaacccttttccattgaggatgtggaggttgcacctcctaaggcttatgaagttcgcattaagatggtggctgtaggaatctgtcacacagatgaccacgtggttagtggcaacctggtgaccccccttcctgtgattttaggccatgaggcagccggcatcgtggagagtgttggagaaggggtgactacagtcaaaccaggtgataaagtcatcccgctctttactcctcagtgtggaaaatgcagagtttgtaaaaacccggagagcaactactgcttgaaaaatgatctaggcaatcctcgggggaccctgcaggatggcaccaggaggttcacctgcagggggaagcccattcaccacttccttggcaccagcaccttctcccagtacacggtggtggatgagaatgcagtggccaaaattgatgcagcctcgcccctggagaaagtctgcctcattggctgtggattctcgactggttatgggtctgcagttaacgttgccaaggtcaccccaggctctacctgtgctgtgtttggcctgggaggggtcggcctatctgctgttatgggctgtaaagcagctggagcagccagaatcattgcggtggacatcaacaaggacaaatttgcaaaggccaaagagttgggtgccactgaatgcatcaaccctcaagactacaagaaacccattcaggaagtgctaaaggaaatgactgatggaggtgtggatttttcgtttgaagtcatcggtcggcttgacaccatgatggcttccctgttatgttgtcatgaggcatgtggcacaagcgtcatcgtaggggtacctcctgcttcccagaacctctcaataaaccctatgctgctactgactggacgcacctggaagggggctgtttatggtggctttaagagtaaagaaggtatcccaaaacttgtggctgattttatggctaagaagttttcactggatgcgttaataacccatgttttaccttttgaaaaaataaatgaaggatttgacctgcttcactctgggaaaagtatccgtaccgtcctgacgttttgatctaga.
In the invention, the nucleotide sequence of the second fusion gene (Noc 1-Usp45-LESS-EK-ALDH 2) is shown in SEQ ID NO:2, specifically:
ccatggtcatgaaaaaaaagattatctcagctattttaatgtctacagtgatactttctgctgcagccccgttgtcaggtgtttacgctgatactaattctgatttggaaatatcgtcgacttgtgatgctgacgatgacgataagtcagccgccgccacccaggccgtgcctgcccccaaccagcagcccgaggtcttctgcaaccagattttcataaacaatgaatggcacgatgccgtcagcaggaaaacattccccaccgtcaatccgtccactggagaggtcatctgtcaggtagctgaaggggacaaggaagatgtggacaaggcagtgaaggccgcccgggccgccttccagctgggctcaccttggcgccgcatggacgcatcacacaggggccggctgctgaaccgcctggccgatctgatcgagcgggaccggacctacctggcggccttggagaccctggacaatggcaagccctatgtcatctcctacctggtggatttggacatggtcctcaaatgtctccggtattatgccggctgggctgataagtaccacgggaaaaccatccccattgacggagacttcttcagctacacacgccatgaacctgtgggggtgtgcgggcagatcattccgtggaatttcccgctcctgatgcaagcatggaagctgggcccagccttggcaactggaaacgtggttgtgatgaaggtagctgagcagacacccctcaccgccctctatgtggccaacctgatcaaggaggctggctttccccctggtgtggtcaacattgtgcctggatttggccccacggctggggccgccattgcctcccatgaggatgtggacaaagtggcattcacaggctccactgagattggccgcgtaatccaggttgctgctgggagcagcaacctcaagagagtgaccttggagctgggggggaagagccccaacatcatcatgtcagatgccgatatggattgggccgtggaacaggcccacttcgccctgttcttcaaccagggccagtgctgctgtgccggctcccggaccttcgtgcaggaggacatctatgatgagtttgtggagcggagcgttgcccgggccaagtctcgggtggtcgggaacccctttgatagcaagaccgagcaggggccgcaggtggatgaaactcagtttaagaagatcctcggctacatcaacacggggaagcaagagggggcgaagctgctgtgtggtgggggcattgctgctgaccgtggttacttcatccagcccactgtgtttggagatgtgcaggatggcatgaccatcgccaaggaggagatcttcgggccagtgatgcagatcctgaagttcaagaccatagaggaggttgttgggagagccaacaattccacgtacgggctggccgcagctgtcttcacaaaggatttggacaaggccaattacctgtcccaggccctccaggcgggcactgtgtgggtcaactgctatgatgtgtttggagcccagtcaccctttggtggctacaagatgtcggggagtggccgggagttgggcgagtacgggctgcaggcatacactgaagtgaaaactgtcacagtcaaagtgcctcagaagaactcataatctaga.
in the present invention, the first fusion gene and the second fusion gene are preferably obtained by full-sequence chemical synthesis.
In the present invention, the first fusion gene and the second fusion gene are inserted between SphI and XbaI restriction sites of PNZ8149, respectively. The method of inserting the first fusion gene and the second fusion gene into PNZ8149 is not particularly limited, and conventional methods in the art may be employed.
In the present invention, the original strain of the first recombinant lactococcus lactis and the second recombinant lactococcus lactis is preferably lactococcus lactis NZ3900.
In the present invention, the mode of transferring the first recombinant expression vector and the second recombinant expression vector into lactococcus lactis NZ3900, respectively, is preferably electrotransfer; the conditions for the electrical transfer are preferably: 2000V, 25 μf and 200Ω.
The invention takes PNZ8149 (lactobacillus food-grade expression vector) as a basic vector, a first fusion gene or a second fusion gene is inserted into the PNZ8149, the first fusion gene comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDK and human alcohol dehydrogenase which are sequentially connected in series, and the second fusion gene comprises a signal peptide SPusp45, probiotics LEISS, enterokinase recognition site DDDDK and human aldehyde dehydrogenase which are sequentially connected in series and are respectively fused. The target enzyme cleaves the signal peptide sequences SPusp, LEISS from the fusion protein in the gut under the action of endogenous enterokinase in the gut, releasing the free alcohol dehydrogenase or acetaldehyde dehydrogenase.
In the invention, when the recombinant lactococcus lactis comprises a first recombinant lactococcus lactis and a second recombinant lactococcus lactis, the ratio of the effective viable count of the first recombinant lactococcus lactis to the second recombinant lactococcus lactis is (1-3): (1-3), preferably 1:1.
The invention also provides a microcapsule containing the recombinant lactococcus lactis.
The method for preparing the microcapsule is not particularly limited in the present invention, and conventional methods in the art may be employed. The first recombinant lactococcus lactis or the second recombinant lactococcus lactis are respectively embedded; the effective viable count of the recombinant lactococcus lactis in each microcapsule is preferably 1-5 x 109cfu.
The invention also provides an application of the recombinant lactococcus lactis or the microcapsule in preparing medicines for preventing drunk; the recombinant lactococcus lactis comprises a first recombinant lactococcus lactis, or the recombinant lactococcus lactis comprises a first recombinant lactococcus lactis and a second recombinant lactococcus lactis.
In the present invention, the prevention of alcohol consumption includes reducing alcohol absorption and/or extending alcohol tolerance time.
The invention also provides an application of the recombinant lactococcus lactis or the microcapsule in preparation of anti-hangover medicines.
In the present invention, the anti-alcohol includes reducing recovery time after drinking and/or reducing acute damage to the liver and/or intestines caused by excessive drinking and/or alleviating vomiting and headache after drinking.
In the present invention, the dosage form of the drug preferably includes an oral preparation; the oral formulation preferably comprises microcapsules.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.
Examples
Cloning and expression of recombinant adh and ALDH genes
Studies have shown that mutation of amino acid 143 of ADH1 from glycine to alanine greatly increases alcohol-decomposing activity (Lee, S.L., Chau, G.Y., Yao, C.T., Wu, C.W., and Yin, S.J.Functional assessment of human alcohol dehydrogenase family in ethanol metabolism: significance of first-pass metabolism[J]. Alcohol Clin Exp Res, 2006, 30 (7): 1132-1142.). of alcohol dehydrogenase, and thus we synthesized the mutant gene.
Plasmid pNZ8149 (Cat#VS-ELV 00300-01) carrying a high efficiency constitutive promoter (GapA promoter, patent CN 111518801A) was selected as a vector (FIG. 1).
The human ADH1B gene and the human ALDH2 gene are cloned into a plurality of cloning sites, and the N end is fused with a Usp45-LESS-EK sequence.
The invention takes PNZ8149 (lactobacillus food-grade expression vector) as a basic vector, and a fusion gene is inserted into the PNZ8149, wherein the fusion gene comprises a signal peptide SPusp, a probiotic LEISS and an enterokinase recognition site DDDDDDK which are sequentially connected in series and is respectively fused with human alcohol dehydrogenase or acetaldehyde dehydrogenase. The full sequence is SphI-SPusp-LEISS-DDDDK-ADH 1B-XbaI, and SphI-SPusp-ALDH 2-DDDDK-ADH1B-XbaI. The target enzyme cleaves the signal peptide sequences SPusp, LEISS from the fusion protein in the gut under the action of endogenous enterokinase in the gut, releasing the free alcohol dehydrogenase or acetaldehyde dehydrogenase.
The expression construct was transformed into lactococcus cremoris NZ3900 (Cat#VS-ELS 03900-01) by electrotransformation and screened for recombinant bacteria using Elliker agar plates. The electrotransformation conditions were 2000V, 25. Mu.F and 200Ω. Recombinant bacteria were PCR identified and sequenced using forward (5'-catgccatggtcatgaaaaaaaagattatcagct-3', shown as SEQ ID NO: 3) and reverse (5'-gctctagatcaaaacgtcagacggtacg-3', shown as SEQ ID NO: 4) primers.
Recombinant lactococcus lactis was resuspended in M17 liquid medium (1%), incubated at 30℃for 8 h, centrifuged at 4℃ (4000 g 10 min) and the supernatant collected. The supernatant was then precipitated by low temperature ethanol precipitation. Equal amounts of protein samples were separated by electrophoresis on a 10% sds-polyacrylamide gel, proteins transferred to PVDF membrane, incubated with blocking buffer (5% skim milk) for 1h at room temperature, and blotted with anti-hadh antibody and hALDH antibody (SantaCruz). The results of specific antibody detection showed that our recombinant bacteria were able to successfully express ADH enzyme (FIG. 2) and ALDH (FIG. 3).
Production of recombinant bacteria
2 Recombinant bacteria are respectively cultured for 6-8 hours and then are collected by centrifugation (4000 g, 4 ℃ and 10 min). Then, the thalli are subjected to microencapsulation: the recombinant bacteria were suspended in 3% (w/v) sodium alginate solution. The suspension was dropped into soybean oil containing 0.2% tween-80 at a ratio of 1:5, and then stirred with a magnetic stirrer at 600 rpm for 10 minutes. Then 0.05M CaCl2 solution was slowly added and stirred (600 rpm,10 min). Microcapsules were collected after centrifugation for 10 minutes (350 g at 4 ℃). The microcapsules were added to lyoprotectants (19.5% maltodextrin and 2.5% skim milk powder, by mass, respectively), resuspended, transferred to petri dishes, stored at-80 ℃ until completely frozen, then transferred to a vacuum freeze dryer, and the 2 bacteria lyophilized the recombinant lactococcus lactis microcapsules, respectively.
2 Bacteria recombinant lactococcus lactis microcapsules are respectively dissolved in artificial gastric acid (0.2% sodium chloride, pH value is 1.2) and are stood for 2 hours. Then, the capsulorhexis solution (19:81 NaH2PO4 0.2 to 0.2 Na2HPO 4) was added and the mixture was broken for 30 minutes at 37℃with a rotator at a speed of 200 rpm, the recombinant lactococcus lactis was released, and the viable bacteria count was measured by the plate count method (FIG. 4).
Determination of drunk regimens
Red start (red star) er pan (Fenwei wine, 56% vol) purchased from supermarkets was selected for the mouse drunk experiments. For establishing a safe and stable acute drunk model, 9-week-old male C57BL/6J mice are randomly divided into 3 groups (n=5 in each group) with weights of 20-22 g, and 4 mg/g BW, 6 mg/g BW and 8 mg/g BW are respectively given. The degree of intoxication was determined by the specular reflection. Briefly, the back of the mouse was placed on the ground with the abdomen and limbs facing upward. If the mouse fails to turn over within 30 seconds, it is considered to lose normal orthostatic reflex. The point in time at which the flip-flop disappears is defined as the drunk point, and the point in time between the first drinking and drunk is defined as the alcohol tolerance time. The alcohol tolerance time was less than 20 minutes for all three groups of mice, and to exclude the influence of individual differences, we define that mice that did not lose the orthostatic reflex within 1h were considered to be free from acute intoxication. In view of the rate of intoxication and safety, the subsequent experiments selected a dose of 6 mg/g BW (Table 1).
TABLE 1 establishment of mouse intoxication model 6 mg/g BW alcohol was selected as therapeutic dose
Detection of alcohol tolerance time in different treatment groups
In the intestinal environment, the expression peak of recombinant lactococcus lactis secreting the foreign protein occurs at about 1-2 hours. The anti-intoxication effect of oral recombinant lactococcus lactis was examined by administration of l. lactis (1X 109 CFU) expressing hADH B (n=8). Lactobacillus lactate containing pNZ structure was used as a control (n=8). After 1 h, all mice were perfused with 6 mg/g BW alcohol and the alcohol tolerance time (time from drinking to loss of positive reflection) was recorded
Motion restoration monitoring
After drinking for 1h, mice were placed in a motion detection system, respectively, and their motion was detected every 15 seconds. When the mice drunk, the machine reading was 0. When the number of movements at four consecutive recording time points was not zero, the mice were considered to have recovered motor ability.
Histological analysis
Hematoxylin eosin (hematoxylin and eosin, H & E) staining paraffin-embedded 4% paraformaldehyde fixed tissues, staining of 5 μm thick sections, and observations under either 10X or 20X objective.
Results
Recombinant oral recombinant lactococcus lactis for prolonging alcohol tolerance time
The 3 groups of mice were perfused with gastric ADH, ALDH recombinant lactococcus lactis and control bacteria (pNZ 8149-transfected lactococcus lactis, replaced with pNZ) respectively, and the results showed that the alcohol tolerance time (i.e., the time from drinking to loss of positive inversion) was significantly prolonged in the recombinant lactococcus lactis mice expressing hadh b, while the ALDH probiotics were not significantly affected (Table 2, FIG. 5).
TABLE 2 alcohol tolerance time (i.e., time to disappearance of the specular reflection) for two groups of mice, 8 per group
Influence of recombinant oral recombinant lactococcus lactis on drunk rate of mice
The 3 groups of mice were perfused with gastric ADH, ALDH recombinant lactococcus lactis and control bacteria (pNZ 8149-null lactococcus lactis, replaced with pNZ) respectively, and the results showed that the drunk rate of recombinant lactococcus lactis mice expressing hadh b was significantly reduced, while the ALDH probiotics were not significantly affected (Table 3).
TABLE 3 influence of pNZ and hADH B on intoxication and recombination of hADH B probiotic bacteria to reduce the rate of intoxication in mice
Recombinant oral probiotics shorten exercise recovery time of drunk mice
Drunk mice need 6-10 hours to recover. To accurately record recovery time of mice in different treatment groups, mice were placed in a exercise recorder after 1h of alcohol consumption. The number of movements was recorded every 15s (fig. 6). When the recorder receives non-zero data 4 consecutive times, the mice are considered to have restored their motor ability. Statistically, we found that hADH B treated mice had significantly shorter motor recovery time (5.5±0.41, n=6) than pNZ probiotic treated mice (6.4±0.41, n=7), and that 1/4 of the mice in the probiotic treated group expressing hADH B had no motor loss throughout the process (fig. 6,7 and table 4). These results indicate that probiotics expressing hADH B can shorten recovery time in drunk mice.
Table 4 influence of pNZ (n=8) and hADH B (n=8) on motion recovery time
Recombinant oral probiotics can alleviate liver-intestinal damage caused by acute alcohol intake
Alcohol is mainly absorbed in the intestine and eventually transported to the liver where it breaks down. Thus, the intestinal liver axis plays an important role in regulating ethanol metabolism, and the intestines and liver are also the most directly damaged organs after drinking. To detect acute poisoning in both groups of mice, intestinal mucosal lesions were observed. The cup cells of pnz-treated drunk mice showed more hypertrophy than non-drunk mice, and probiotic treatment expressing hadh b reduced the damage to the gut from acute drinking (C in fig. 8), indicating a reduction in alcohol absorption through the gut. The alcohol content in the blood of the mice reaches the peak value after drinking for 2-3 hours. To test the effect of different probiotics on alcohol absorption, blood alcohol levels at different points were measured after consumption using an ethanol assay kit (BioAssay Systems, ECET-100). In the first h, there was no difference in alcohol content in the blood of the two groups of mice. The serum alcohol residue in the pNZ group continued to increase 2h after drinking, while the serum alcohol residue in the hADH group showed a significant decrease trend, and was significantly lower than that in the pNZ group (a in fig. 8). Further studies have found that hADH B treatment reduced blood triglyceride concentration (B in fig. 8) while also reducing lipid levels in the liver of hADH B treated mice (D in fig. 8). In conclusion, the probiotic treatment expressing hadh b can alleviate intestinal damage caused by acute alcohol intake and reduce fat content in liver and blood.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.
Sequence listing
<110> Animal institute of China academy of sciences
<120> Recombinant lactococcus lactis, microcapsule and use thereof
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ccatggtcat gaaaaaaaag attatctcag ctattttaat gtctacagtg atactttctg 60
ctgcagcccc gttgtcaggt gtttacgctg atactaattc tgatttggaa atatcgtcga 120
cttgtgatgc tgacgatgac gataagagca cagcaggaaa agtaatcaaa tgcaaagcag 180
ctgtgctatg ggaggtaaag aaaccctttt ccattgagga tgtggaggtt gcacctccta 240
aggcttatga agttcgcatt aagatggtgg ctgtaggaat ctgtcacaca gatgaccacg 300
tggttagtgg caacctggtg accccccttc ctgtgatttt aggccatgag gcagccggca 360
tcgtggagag tgttggagaa ggggtgacta cagtcaaacc aggtgataaa gtcatcccgc 420
tctttactcc tcagtgtgga aaatgcagag tttgtaaaaa cccggagagc aactactgct 480
tgaaaaatga tctaggcaat cctcggggga ccctgcagga tggcaccagg aggttcacct 540
gcagggggaa gcccattcac cacttccttg gcaccagcac cttctcccag tacacggtgg 600
tggatgagaa tgcagtggcc aaaattgatg cagcctcgcc cctggagaaa gtctgcctca 660
ttggctgtgg attctcgact ggttatgggt ctgcagttaa cgttgccaag gtcaccccag 720
gctctacctg tgctgtgttt ggcctgggag gggtcggcct atctgctgtt atgggctgta 780
aagcagctgg agcagccaga atcattgcgg tggacatcaa caaggacaaa tttgcaaagg 840
ccaaagagtt gggtgccact gaatgcatca accctcaaga ctacaagaaa cccattcagg 900
aagtgctaaa ggaaatgact gatggaggtg tggatttttc gtttgaagtc atcggtcggc 960
ttgacaccat gatggcttcc ctgttatgtt gtcatgaggc atgtggcaca agcgtcatcg 1020
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tcagcaggaa aacattcccc accgtcaatc cgtccactgg agaggtcatc tgtcaggtag 300
ctgaagggga caaggaagat gtggacaagg cagtgaaggc cgcccgggcc gccttccagc 360
tgggctcacc ttggcgccgc atggacgcat cacacagggg ccggctgctg aaccgcctgg 420
ccgatctgat cgagcgggac cggacctacc tggcggcctt ggagaccctg gacaatggca 480
agccctatgt catctcctac ctggtggatt tggacatggt cctcaaatgt ctccggtatt 540
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ttgcctccca tgaggatgtg gacaaagtgg cattcacagg ctccactgag attggccgcg 900
taatccaggt tgctgctggg agcagcaacc tcaagagagt gaccttggag ctggggggga 960
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ggaacccctt tgatagcaag accgagcagg ggccgcaggt ggatgaaact cagtttaaga 1200
agatcctcgg ctacatcaac acggggaagc aagagggggc gaagctgctg tgtggtgggg 1260
gcattgctgc tgaccgtggt tacttcatcc agcccactgt gtttggagat gtgcaggatg 1320
gcatgaccat cgccaaggag gagatcttcg ggccagtgat gcagatcctg aagttcaaga 1380
ccatagagga ggttgttggg agagccaaca attccacgta cgggctggcc gcagctgtct 1440
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ggagtggccg ggagttgggc gagtacgggc tgcaggcata cactgaagtg aaaactgtca 1620
cagtcaaagt gcctcagaag aactcataat ctaga 1655
<210> 3
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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Claims (8)
1. A recombinant lactococcus lactis, which is a first recombinant lactococcus lactis;
The first recombinant lactococcus lactis comprises a first recombinant expression vector;
the skeleton plasmid for constructing the first recombinant expression vector is PNZ8149;
A first fusion gene is inserted into the first recombinant expression vector, and comprises a signal peptide SPusp, probiotics LEISS, enterokinase recognition site DDDDK and human-derived alcohol dehydrogenase which are sequentially connected in series;
the nucleotide sequence of the first fusion gene is shown as SEQ ID NO: 1.
2. The recombinant lactococcus lactis according to claim 1, wherein said first fusion gene is inserted between SphI and XbaI restriction sites of PNZ 8149.
3. A microcapsule comprising the recombinant lactococcus lactis of claim 1.
4. Use of a recombinant lactococcus lactis according to claim 1 or 2 or a microcapsule according to claim 3 for the preparation of a medicament for the prevention of intoxication.
5. The use according to claim 4, wherein the prevention of alcohol intoxication comprises reducing alcohol absorption and/or extending alcohol tolerance time.
6. Use of a recombinant lactococcus lactis according to claim 1 or 2 or a microcapsule according to claim 3 for the preparation of a medicament for alleviating hangover.
7. The use according to claim 6, wherein the anti-alcohol comprises shortening recovery time after drinking and/or reducing acute damages of the liver and/or intestinal tract caused by excessive drinking.
8. The use according to any one of claims 4 to 7, wherein the dosage form of the medicament comprises an oral formulation.
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