CN110331148B - Gene for coding IFN alpha protein, recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application - Google Patents

Gene for coding IFN alpha protein, recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application Download PDF

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CN110331148B
CN110331148B CN201910768543.0A CN201910768543A CN110331148B CN 110331148 B CN110331148 B CN 110331148B CN 201910768543 A CN201910768543 A CN 201910768543A CN 110331148 B CN110331148 B CN 110331148B
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lactobacillus casei
ifn alpha
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CN110331148A (en
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陈正军
杨雪
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Huazhong Agricultural University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2220/00—Biochemical treatment
    • A23C2220/20—Treatment with microorganisms
    • A23C2220/204—Use of bacteria which are encapsulated, entrapped or immobilised; Fermentation with these bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei

Abstract

The invention provides a gene for coding IFN alpha protein, a recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application, belonging to the technical field of modification, development and application of lactobacillus. The invention combines the broad-spectrum antiviral efficacy of alpha interferon (IFN alpha) and the probiotic effect of lactic acid bacteria by a genetic engineering technology, and utilizes the function factorThe in-situ expression of the seed and the mucous membrane delivery of the probiotics realize the synergy mode to replace the traditional intramuscular injection and intravenous injection, and the detection shows that the titer of the rhIFN alpha secreted by the recombinant lactobacillus casei in the MA104 cell against rotavirus reaches 2.12 multiplied by 105U/mg. The technical scheme of the invention provides a novel solution and application direction for preventing and treating the children viral diarrhea, particularly rotavirus diarrhea, and is favorable for large-scale prevention of viral diseases of young livestock and broad masses of infants and farmers.

Description

Gene for coding IFN alpha protein, recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application
Technical Field
The invention relates to the technical field of modification, development and application of lactic acid bacteria, and in particular relates to a gene for coding IFN alpha protein, a recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application thereof.
Background
Rotavirus (RV) infection is the most main reason for clinically treating diarrhea of infants below 5 years old, and no specific treatment medicine exists at present. Aiming at RV gastroenteritis, symptoms such as vomiting and diarrhea caused by organism dehydration and electrolyte loss are only temporarily relieved basically in the form of oral rehydration. Also, rotavirus is prevalent worldwide, with about forty-five million children dying from rotavirus infection every year worldwide, with the highest rate of infection at the age of 6 months to 2 years, according to incomplete statistics (Catherine et al 2011; Shanlian et al 2018). Since rotainfection is not affected by socioeconomic, environmental hygienic conditions, the incidence of disease is essentially poor in developed and developing countries (Parasharet al 2003). Obviously, the RV diarrhea becomes an important public health problem which harms the healthy growth and life safety of infants, and has harm to the economic and social development of all countries.
With regard to rotavirus diarrhea, many efforts and attempts have been made by scientists and medical workers, and at present, the prevention and control of rotavirus diarrhea is mainly dependent on vaccination. Among them, RotaShield, the first RV vaccine approved in the united states, was found to be associated with intussusception 1 year after marketing, and was shut down and withdrawn from the market (Buttery et al 2011). Later on, although both the Rotarix and Rotateq vaccines proved to be highly protective against RV diarrhea, the detection of nucleic acid fragments of other viruses in them in 2010 had to alert us to the potential safety problems of vaccine contamination (Ruiz-Palacios et al 2006; xylol and Weilingan 2018). In addition, the RV vaccine LLR developed by China has a certain protective effect on the rotavirus diarrhea, but the safety and effectiveness evaluation is limited to the local market. It is worth mentioning that the Chinese application patent CN201711395821.X discloses a lactobacillus genetic engineering subunit vaccine strain capable of stably expressing porcine rotavirus VP4 protein in 2018, 5 months and 4 days, and a preparation method thereof. The invention is a lactobacillus casei gene engineering vaccine with stable expression obtained by inserting porcine rotavirus VP4 gene through homologous recombination on the basis of UPP gene-deleted lactobacillus casei. The specificity of antigen immunity is ensured, and the vaccine strain and the probiotics are skillfully linked together. However, most of the RV vaccines which are on the market or newly invented at present are developed aiming at epidemic strains in a certain time period in a certain area, and the protective effect and the effectiveness in different areas are obviously different, so that the RV vaccines are not beneficial to large-scale prevention and treatment of viral diseases of infants and livestock by the nation and farmers. Therefore, the discovery of new ways to prevent rotavirus diarrhea should be paid more extensive attention by the scholars, and the development of novel broad-spectrum antiviral agents is imminent.
Disclosure of Invention
The invention aims to provide a gene for coding IFN alpha protein, a recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application thereof, wherein the recombinant lactobacillus casei can secrete human IFN alpha optimized protein, and the secreted protein has good rotavirus resisting activity in vitro MA104 (monkey fetal kidney cell) cells.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a gene for coding IFN alpha protein, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.
The invention also provides a recombinant vector pELSH-IFN alpha, the recombinant vector pELSH-IFN alpha takes plasmid pELSH as an original vector and carries the gene for coding the IFN alpha protein in the scheme;
the nucleotide sequence of the plasmid pELSH is shown as SEQ ID NO: 2, respectively.
Preferably, the site of the gene encoding IFN α protein inserted into plasmid pELSH is NcoI and KpnI.
The invention also provides a recombinant lactobacillus casei containing the recombinant vector pELSH-IFN alpha in the scheme.
The invention also provides application of the recombinant lactobacillus casei in the scheme in preparing a medicament for preventing and treating rotavirus.
The invention also provides application of the recombinant lactobacillus casei in the scheme in preparation of vaccines for preventing and treating rotavirus diarrhea.
The invention also provides application of the recombinant lactobacillus casei in the scheme in preparation of fermented food, health-care products or feed.
Preferably, the fermented food product comprises yoghurt and/or a milk-containing beverage.
Preferably, the nutraceutical comprises a functional dietary supplement.
The invention has the beneficial effects that: the invention provides a gene for coding IFN alpha protein, a recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application. The invention provides a gene for coding IFN alpha protein, which is obtained by performing codon optimization on a mature hIFN alpha polypeptide coding sequence according to the preference of Lactobacillus casei (Lactobacillus casei) to the use of degenerate codons, and compared with other genes for coding IFN alpha protein, the gene of the invention has higher use frequency of Lactobacillus casei (host bacteria); the invention also provides a recombinant vector pELSH-IFN alpha and a recombinant lactobacillus casei containing the recombinant vector pELSH-IFN alpha. The invention combines the broad-spectrum antiviral efficacy of alpha interferon (IFN alpha) with the probiotic effect of lactic acid bacteria through a genetic engineering technology, realizes synergy by utilizing the in-situ expression of functional factors and the mucosal delivery of probiotics to replace the traditional intramuscular injection and intravenous injection, and through detection, the titer of the rhIFN alpha secreted by the recombinant lactobacillus casei in the MA104 cell against rotavirus reaches 2.12 alpha 0105U/mg. The technical scheme of the invention provides a novel solution and application direction for preventing and treating the children viral diarrhea, particularly rotavirus diarrhea, and is favorable for large-scale prevention of viral diseases of young livestock and broad masses of infants and farmers. In addition, the lactobacillus casei is fermented by utilizing the unique flavor substances of the lactobacillus casei, and can be developed into functional yogurt drinks, dietary supplements or feed additives, and the functional yogurt drinks, the dietary supplements or the feed additives are detected and utilizedThe quality guarantee period of the yoghourt obtained by fermenting the recombinant lactobacillus casei can be prolonged.
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FIG. 1 is a mass spectrum of recombinant vector pELSH-IFN alpha;
FIG. 2 shows the PCR verification of JM109-pELSH-hIFN α recon bacterial liquid of example 2, wherein M is Trans 2KPlus DNAmarker, and 1-10 are 10 transformants picked from the plate;
FIG. 3 shows the results of the double restriction enzyme digestion verification of the recombinant vector pELSH-IFN α in example 2, wherein M is DNAmarker, the left 1-10 are electrophoresis charts of EcoRI-Pslpa-F/BamHI-T-R double restriction enzyme products, and the right 1-10 are electrophoresis charts of NcoI/KpnI double restriction enzyme products;
FIG. 4 shows the results of the identification of the secretion and expression of hIFN α recombinant protein in Lactobacillus casei in example 3;
FIG. 5 shows the results of measurement of the optimal secretion expression amount of the protein in example 4;
FIG. 6 shows the qualitative microscopic examination result of anti-rotavirus activity of the recombinant target protein in example 6, in which FIG. a shows normal cells treated neither by loading induction nor by challenge, i.e., a blank control dish; panel b is a diseased cell not loaded induced but challenge treated, i.e. virus control dish; FIG. c shows experimental cells treated by RV challenge after induction of rhIFN alpha recombinant protein sample, i.e. sample dish; FIG. d shows the cells treated by RV challenge after induction with rhIFN alpha protein standard, i.e. the standard dish;
FIG. 7 shows the change of the viable lactic acid bacteria content of the yogurt of the present invention in example 7 during storage.
Detailed Description
The invention provides a gene for coding IFN alpha protein, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1, specifically:
TGTGATTTGCCAGAAACCCATAGTTTGGATAATCGCCGCACCTTGATGTTGTTGGCCCAAATGAGTCGCATTAGTCCAAGTAGTTGTTTGATGGATCGCCATGATTTTGGCTTTCCACAAGAAGAATTTGATGGCAATCAATTTCAAAAAGCCCCAGCCATTAGTGTTTTGCATGAATTGATTCAACAAATTTTTAATTTGTTTACCACCAAAGATAGTAGTGCCGCCTGGGATGAAGATTTGTTGGATAAATTTTGTACCGAATTGTATCAACAATTGAATGATTTGGAAGCCTGTGTTATGCAAGAAGAACGCGTTGGCGAAACCCCATTGATGAATGCCGATAGTATTTTGGCCGTTAAAAAATATTTTCGCCGCATTACCTTGTATTTGACCGAAAAAAAATATAGTCCATGTGCCTGGGAAGTTGTTCGCGCCGAAATTATGCGCAGTTTGAGTTTGAGTACCAATTTGCAAGAACGCTTGCGCCGCAAAGAA。
in the present invention, the optimized IFN α protein is derived from human (i.e., human IFN α); in the specific implementation process of the invention, based on the protein sequence [ Refseq: NP-076918.1 ] of human interferon alpha submitted on NCBI, the amino acid sequence is shown as SEQ ID NO: 3, specifically: CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE, under the precondition of not changing the amino acid, the coding sequence of hIFN alpha mature polypeptide is optimized according to the preference of Lactobacillus casei (Lactobacillus casei) to the degenerate codon (a sequence with high usage frequency of host bacteria is selected from the degenerate codons for coding the same amino acid, U → T), and the Wuhan Dynasty scientific creative biotechnology Limited company is sent to complete gene synthesis to obtain the target gene (the gene for coding the IFN alpha protein). In the present invention, the gene encoding IFN α protein is used more frequently than other genes encoding IFN α protein by host bacteria.
The invention also provides a recombinant vector pELSH-IFN alpha, the recombinant vector pELSH-IFN alpha takes plasmid pELSH as an original vector and carries the gene for coding the IFN alpha protein in the scheme; the nucleotide sequence of the plasmid pELSH is shown as SEQ ID NO: 2 is shown in the specification; the sites of the gene coding for the IFN alpha protein inserted into the plasmid pELSH are NcoI and KpnI; the mass spectrum of the recombinant vector pELSH-IFN alpha is shown in figure 1.
In the present invention, the plasmid pELSH carries a signal peptide sequence; the amino acid sequence coded by the signal peptide sequence is shown as SEQ ID NO: 4, specifically: MKKKIISAILMSTVILSAAAPLSGVYADTN are provided. In the invention, the signal peptide sequence is derived from lactococcus lactis and is synthesized by Wuhan engine creative biotechnology limited company.
In the invention, the vector pELSH is obtained by transforming and constructing the plasmid pMC11 separated from Lactobacillus casei, and the plasmid itself is separated from Lactobacillus casei MCJ (the Lactobacillus casei MCJ delta 1 is the host bacterium without the plasmid, and the difference between the Lactobacillus casei MCJ delta 1 and the Lactobacillus casei MCJ is that the latter lacks a pMC11 plasmid as the former, and the Lactobacillus casei MCJ delta 1 and the Lactobacillus casei MCJ are stored in the national emphasis laboratory A408 of agriculture university in China), so the plasmid can stably exist and express in the Lactobacillus casei. And the expression host range of the essence particle expression system is narrow, and the shuttle expression can be only carried out in escherichia coli and lactobacillus casei (or lactobacillus paracasei), so that the directionality and the safety of target gene expression are further ensured.
The preparation method of the recombinant vector pELSH-IFN alpha preferably comprises the following steps of carrying out double enzyme digestion on a gene for coding IFN alpha protein and a plasmid pELSH, recovering an enzyme digestion product, and carrying out enzyme ligation on the enzyme digestion product to obtain the recombinant vector pELSH-IFN alpha.
In the invention, the gene for coding IFN alpha protein and a plasmid pELSH double enzyme digestion system are shown in a table 1; the enzyme cutting temperature is preferably 35-40 ℃, and more preferably 37 ℃; the enzyme cutting time is preferably 1.5-3 h, and more preferably 2 h.
TABLE 1 Gene encoding IFN alpha protein and plasmid pELSH double digestion System
Figure BDA0002172778110000051
In the invention, the kit for recovering the enzyme digestion product is preferably a PCR clean recovery kit purchased from OMEGA company; preferably, the method further comprises removing substances such as enzymes in the reaction system after the enzyme digestion product is recovered, and the method is continued after the agarose gel electrophoresis is successfully verified.
In the invention, the enzyme-linked system of the enzyme digestion products (the target fragment and the vector fragment) is shown in table 2; the temperature of the enzyme is preferably 15-18 ℃, and more preferably 16 ℃; the enzyme-linked time is preferably 0.5-2 h, and more preferably 1-1.5 h.
TABLE 2 fragment and vector fragment enzyme linked systems of interest
Figure BDA0002172778110000061
The invention also provides a recombinant lactobacillus casei containing the recombinant vector pELSH-IFN alpha in the scheme; the preparation method of the recombinant lactobacillus casei is preferably to electrically transform a recombinant vector pELSH-IFN alpha into lactobacillus casei competent cells.
The invention combines the broad-spectrum antiviral efficacy of IFN alpha and the probiotic function of lactobacillus by a genetic engineering technology, and experiments are specifically implemented and verified by taking lactobacillus casei as an example. The host bacteria used in the invention is Lactobacillus casei MCJ delta 1, but not limited to the bacteria, and other probiotics can express the target protein.
The invention also provides an application direction of the recombinant lactobacillus casei in the scheme in preparing a medicament for preventing and treating rotavirus. The recombinant protein secreted and expressed by the recombinant lactobacillus casei has rotavirus resisting activity, so the recombinant lactobacillus casei capable of secreting the target protein also has corresponding antiviral activity.
The invention also provides an application direction of the recombinant lactobacillus casei in the scheme in preparing a vaccine for preventing and treating rotavirus diarrhea. In the invention, the recombinant protein secreted and expressed by the recombinant lactobacillus casei has the effect of preventing and treating rotavirus diarrhea, so the recombinant lactobacillus casei also has the corresponding effect of preventing and treating rotavirus diarrhea.
The invention also provides the application direction of the recombinant lactobacillus casei in the scheme in preparing fermented food, health products or feed; the fermented food preferably comprises yoghurt and/or milk-containing beverage; the nutraceutical preferably comprises a functional dietary supplement.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 construction of recombinant vector pELSH-IFN alpha
1. Double digestion of target fragment and plasmid pELSH
See scheme table 1 above for the double cleavage system. The enzyme was cleaved at 37 ℃ for 2 h. And (3) recovering the enzyme digestion product by using the PCR clean recovery kit, removing substances such as enzyme and the like in the reaction system, and waiting for connection after agarose gel electrophoresis verification is successful.
2. Enzymatic ligation of a fragment of interest to a vector fragment
See scheme table 2 above for enzyme linked systems. Enzyme-linked reaction is carried out for 1h at 16 ℃ to obtain the recombinant vector pELSH-IFN alpha.
Example 2 construction of recombinant Lactobacillus casei
Electrically transforming the recombinant vector pELSH-IFN alpha obtained in the example 1 into Lactobacillus casei competent cells, standing at 37 ℃ for resuscitation, coating an MRS plate containing erythromycin, carrying out inversion culture at the constant temperature of 37 ℃ for 2d, and screening, verifying and obtaining a positive recon. The verification results are shown in FIG. 2 and FIG. 3, wherein FIG. 2 is the PCR verification of JM109-pELSH-hIFN alpha recon bacterial liquid, M is Trans 2KPlus DNA Marker, and 1-10 are 10 transformants picked from the plate. The size of pELSH no-load is 640bp, and the size of PCR product of pELSH-hIFN alpha recombinant plasmid is 1105 bp. As can be seen from the agarose gel electrophoresis results, the band is single and bright, and the sizes of the PCR product fragments in 10 lanes are consistent and consistent with the expected size. FIG. 3 shows the results of double restriction enzyme digestion verification of the recombinant vector pELSH-IFN α, where M is DNA Marker, left 1-10 are EcoRI-Pslpa-F/BamHI-T-R double restriction enzyme products electrophoresis diagrams, and right 1-10 are NcoI/KpnI double restriction enzyme products electrophoresis diagrams. The expected sizes of the products of the recombinant plasmid after EcoRI-Pslpa-F/BamHI-T-R double digestion are 1096bp and 5623bp respectively, and the expected sizes of the products after NcoI/KpnI double digestion are 498bp and 6221bp respectively. As can be seen from the agarose gel nucleic acid electrophoresis result chart, the double cutting results of all plasmids are consistent with the expectation, and the sizes of the bands are consistent, further explaining that the correct pELSH-hIFN alpha positive clone is obtained preliminarily.
Example 3 characterization of the secretory expression of hIFN α recombinant proteins in Lactobacillus casei
The positive clone of recombinant Lactobacillus casei (L.casei-pELSH-hIFN. alpha.) from example 2 was streaked and picked, transferred to 5mL of MRS medium containing Em, and incubated overnight at 37 ℃. And taking L.casei MCJ delta 1 and a recombinant strain L.casei-pELSH carrying no-load pELSH as negative controls, and culturing the strain to the same OD. Collecting 1mL of bacterial liquid of each bacterium, centrifuging for 1min at 10000 Xg, and taking supernatant as a sample to be detected to carry out Westernblot identification. The identification results are shown in FIG. 4, wherein M is protein Marker, and lanes 1-3 are recipient bacterium (L.casei MCJ. DELTA.1), plasmid vector transformant (L.casei MCJ. DELTA.1-pELSH) and recombinant bacterium (L.casei MCJ. DELTA.1-pELSH-hIFN. alpha.), respectively.
According to WB results, no protein is detected in empty bacteria and no load, and the size of the protein detected by only recombinant bacteria is consistent with the expected 21kDa, which indicates that the recombinant target protein can be successfully secreted and expressed in Lactobacillus casei L.casei MCJ delta 1.
Example 4 measurement of the amount of protein expressed by optimal secretion
The recombinant lactobacillus casei in example 2 is activated, 1% (volume ratio, for example, 100mL of culture medium is inoculated with 1mL of bacteria liquid) is inoculated to MRS culture medium, culture supernatant of different growth periods (for example, OD600 ═ 1.0, 2.0, 3.0 and the like) is collected, namely total protein of the culture supernatant of the recombinant lactobacillus casei in different growth periods is collected, 15 μ L of sample is taken to be subjected to Westernblot analysis so as to collect bacteria in the optimal growth period, separate and purify protein, and the activity of the recombinant lactobacillus casei in resisting rotavirus is detected by cell experiments. Referring to fig. 5, M is protein Marker (the same protein Marker was used in the experiment, Thermo cat No. 26616), and lanes 1-9 represent the pet lh-hifna-containing recombinant supernatant samples with OD600 ═ 0.942, 1.42, 2.09, 2.87, 3.44, 5.04, 5.67, 5.79 and 5.95, respectively.
As a result of the analysis, the amount of hIFN α protein in the supernatant increased with the growth of the cells when the cell concentration OD600 was 2.87 or less, and reached the highest value when OD600 was 2.87. Then, the amount of hIFN alpha protein in the supernatant gradually decreased with the increase of cell concentration.
EXAMPLE 5 isolation and purification of recombinant protein of interest
The recombinant lactobacillus casei in example 2 is activated and cultured until the protein secretion is maximum, the bacterium liquid is collected, the bacterium liquid is centrifuged at 7500r/min for 30min at 4 ℃, and the supernatant is filtered by a 0.22 mu m filter membrane to remove impurities and bacteria, and then the nickel column affinity chromatography is carried out. Before loading, the nickel column is washed by 20% ethanol and ultrapure water, and then the nickel column is balanced by BufferA. After the column equilibration, the centrifuged and filtered sample was loaded onto a Ni column at a flow rate of 1mL/min by a constant flow pump. After the column is hung, sequentially using Buffer A/Buffer B mixed liquor containing imidazole with different concentrations to start gradient elution of the target product. And (3) according to the real-time peak condition in the elution process, collecting the effluent when the ultraviolet absorbance of the effluent starts to obviously rise, and stopping collecting the liquid when the ultraviolet absorbance of the effluent is reduced to the minimum value. The collected tube samples were analyzed by 12% SDS-PAGE detection. According to the electrophoresis result, the eluted protein in the corresponding collection tube is selected to be dialyzed at the environment of 4 ℃ to remove imidazole, and the concentration is determined by protein standard curve.
Example 6 detection of anti-Rotavirus Activity of recombinant protein of interest obtained in example 5
1. Qualitative microscopic examination is carried out by the following method:
(1)37℃、5％CO2culturing MA104 cells in the environment until a monolayer grows;
(2) discarding the culture solution, and washing with PBS for several times;
(3) sample treatment: diluting the protein sample and the standard to the same concentration, adding into corresponding dishes, respectively, and adding 5% CO at 37 deg.C2The incubator is subjected to induction incubation for 20 h. Simultaneously performing other conditions for equal treatment without adding sample, virus, and RV control vessel with virus only 20 hr later, 37 deg.C, and 5% CO2Culturing;
(4) counteracting toxic substances: activating virus with pancreatin for 30min, removing the original culture solution, adding into RV dish, sample dish and standard dish, and adsorbing in a 37 deg.C carbon dioxide incubator for 1 h. Discarding virus solution, adding maintenance solution containing pancreatin, and placing at 37 deg.C under CO2The environment was incubated for 24h, lesions were observed daily, and the degree of protection of RV infection by recombinant secreted proteins in vitro was recorded. The results are shown in FIG. 6. From FIG. 6, it can be seen that the virus b dish cells have obvious typical lesions compared with the blank dish normal cells: cells are in round clusters, the gaps among the clusters are obvious, and empty pits are formed. Compared with RV control dish lesion cells, the sample dish and the sample dish cells do not observe typical significant rotavirus CPE or are very little, and the cell state is similar to that of normal cells. This fully demonstrates that the hIFN alpha protein secreted and expressed by Lactobacillus casei does have a certain anti-rotavirus activity on MA104 cells.
2. The titer was determined by the following method:
(1)37℃、5％CO2culturing MA104 cells to grow into a monolayer;
(2) purified and measured concentration of hIFN alpha 5 times dilution, according to the amount of 0.1 mL/hole added to the marker corresponding concentration of cell culture plate hole, 37 degrees, 5% CO2Performing induction culture for 20 h;
(3) removing culture solution, adding rotavirus solution at 100 μ L/well for counteracting virus, setting virus-free control and interferon-free control, and controlling at 37 deg.C with 5% CO2Continuing culturing;
(4) CPE was observed day by day for 3 consecutive days. The titer, i.e. the activity of hIFN alpha on MA104 cells for inhibiting rotavirus replication, was calculated by replacing the lesion wells in the titer-determination wells with the lesion-inhibition wells according to the Reed-Muench method. The results of the evaluation are shown in Table 3. The titer of rhIFN alpha against rotavirus in MA104 cells calculated from Table 3 can reach 2.12 × 105U/mg。
TABLE 3 evaluation of the inhibition of rotavirus replication by hIFN α on MA104 cells
Figure BDA0002172778110000101
Figure BDA0002172778110000111
The anti-rotavirus activity of the rhIFN alpha protein on MA104 cells is calculated according to a Reed-Muench method:
Figure BDA0002172778110000112
through measurement and calculation, the titer of the rhIFN alpha against rotavirus in the MA104 cell is 2.12 multiplied by 105U/mg。
Example 7 fermentation experiment and viable count detection of functional yogurt
Dissolving 60g of skim milk powder in 455mL of distilled water to prepare skim milk, uniformly stirring, mixing with 20mL of recombinant lactobacillus casei growth logarithmic phase bacterial liquid and 25mL of oyster mushroom juice to obtain a mixed liquid, fermenting at the constant temperature of 37 ℃ for 20h, when a chromatography phenomenon is observed, namely whey is separated out, shaking to obtain a uniform texture, indicating that the state is good, successfully fermenting, and detecting whether the number of viable lactic acid bacteria reaches the standard or not.
Storing the fermented yoghourt in a refrigerator at 4 ℃, taking out the fermented yoghourt respectively at 3 rd, 6 th, 9 th, 12 th and the like, diluting the yoghourt by 10 times by using sterilized distilled water, coating the diluted yoghourt on an MRS agar plate containing the erythromycin antibiotic, inverting the diluted yoghourt to a constant-temperature incubator at 37 ℃ for about 36 hours, counting, calculating and analyzing, detecting the shelf life of the yoghourt and comparing the yoghourt with the market. The results are shown in FIG. 7. As can be seen from FIG. 7, the viable count of the recombinant lactobacillus casei in the yoghurt fermented by the recombinant lactobacillus casei still reaches 10 after the product is stored for 21 days at 4 DEG C8CFU/mL, two orders of magnitude higher than the national standard, indicates that it has a good application foundation.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> gene for coding IFN alpha protein, recombinant vector pELSH-IFN alpha, recombinant lactobacillus casei and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 498
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgtgatttgc cagaaaccca tagtttggat aatcgccgca ccttgatgtt gttggcccaa 60
atgagtcgca ttagtccaag tagttgtttg atggatcgcc atgattttgg ctttccacaa 120
gaagaatttg atggcaatca atttcaaaaa gccccagcca ttagtgtttt gcatgaattg 180
attcaacaaa tttttaattt gtttaccacc aaagatagta gtgccgcctg ggatgaagat 240
ttgttggata aattttgtac cgaattgtat caacaattga atgatttgga agcctgtgtt 300
atgcaagaag aacgcgttgg cgaaacccca ttgatgaatg ccgatagtat tttggccgtt 360
aaaaaatatt ttcgccgcat taccttgtat ttgaccgaaa aaaaatatag tccatgtgcc 420
tgggaagttg ttcgcgccga aattatgcgc agtttgagtt tgagtaccaa tttgcaagaa 480
cgcttgcgcc gcaaagaa 498
<210> 2
<211> 6254
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctgaaaagtg gttcgaaatg acaatcgtgt cattttggca aagaatcaaa gtctagaagt 60
ctagtagtaa acaccgatgt cgttgggcat cggtgttttt gcttggcaaa aaagcgggga 120
cgatcatgat cggagaaacg ccacaaaaaa ttgtgccgat ttcgataaaa ataactggca 180
gtcaccgcag aattagggca cactaaaaag tgttctcatt gaacgctaat tttttatgga 240
gacaaaaaac ccgtgcagct tgcggactac acgggtttgt gatgttcata aattcattcg 300
attaggttca ttttagcata gattataacc agttgcattt ataaatcttt ggttgaggat 360
ctgcgtgcaa aattattgtg aatttcaact tgaactactt gtcgtcctcg cttggcttga 420
aacaattcca aatccatatc atcaattttc atatttattt cttcgattga tctaattaga 480
acgctttttt ttaaatcctt gtaagcgtag tttttggggg cgcctaacca ctttttaaag 540
tcatccggag cgccttggac tatcgggatt gattgaccat tatctttatc agactcacgc 600
atcaatttat acagcaaaat agcatatttg cttttgagct ggacagtgtc atgtaaaaga 660
tactgagtat agtgaccatt atccttgaga cgtaaaagaa aaggcgcaaa gtcttcatta 720
attttaagtt ctattttacc attttcccaa accttagcac gctcaaacca accagtcatc 780
gtgattgatc gttctacctc gttgtaaatt ctgacactct tagcgcgcat gtcattgaga 840
ttttgagcta attgagaata tgtacgaccg ctacgtttta attcgagtac gttagataat 900
tcgtacatag atgtttgaat gacattgaag ttcttgtcat caggcttaat tttggaaatt 960
acataatcca taattttaag ttcacgtgca ttcaagtcat ggcgagcttt ggtgataagg 1020
tcattgtgtt cagcaaccaa aaaatagcgt tgatcgttta tttcgtcttt gttaaagtca 1080
cggtcatttt tccataagag cttctttaca tctaccatac agaatcacct caagaagtat 1140
tatatattaa aaggagaaca attttgtctc cttttaattt ttacccccct atcgtgtgct 1200
ttttaccccc ctatcgtgtg ctttttaccc ccctatcgtg tgctttttac ccccctatcg 1260
tgtgcttttt acccccctat tagtcgattc aaggccttgg ttgaggcatt cttgcttccc 1320
taagtactta aagtatttaa aagtatttag aagtatatat agcgcgcgag gcttgcgttt 1380
gtgttttgct tgttcataac cacaaaagac cttggggagc ccctctcccc aaatcccata 1440
ccaacccttg gtcgcttcgt tccctaaaaa ggtgtggctt cgccacattt aactccggct 1500
atgcctgcgc gcctttggct tgccctaacg cgcttcgctt gcgtggctca gaagagcctc 1560
agctgaagct tcggcagggc tagcgccatt caccgacaaa agctcgttgc ctaaatttga 1620
tcaatcacta atgcttttca ttggtgccat gaacatatgc tgataaaaac ggcttagaac 1680
gcttatattt gcgttctaag ccgtttttga tgtaaagagc attaattgta cccgtcagag 1740
taagaaacgc tagcacgggc tctttagtgc tttttaaaac tcactgtttt gataaccaat 1800
cctcaacgat atttgatttg gtcactctga catggtactt agccagatcc ggattttgat 1860
tcacttcctg ttgctttttt gcgaccagct gatccaattt tgcaatcgcc tgatcggtca 1920
aggtgaacat gacccggtga gtttcgtctt gtttcgtcat ctcaaaaacc tcctgaatgg 1980
tgatgatgaa cggtcgtctg gcaaaccaac acgacgttaa aattgcgtta cgttgccggg 2040
cgatttttgg caaacagttg caacgaccct ggtcaaaacg tgcatgtttt gaccagggta 2100
aaagatcaaa ataaattgag ttgcgatcca atccatctca aaaatctgtt cggctggatc 2160
gaccactttt tgagactgct tggaacgcgc ctaaaatcaa gcaagagcat aaggtgaaac 2220
tgctatcaga cttgatagaa accgcagtgc ggtttggttt gtcgtgatag cacgttgggg 2280
tcaaaaggtg tagtaaaaca cgattcactc cacgtgaaaa aatttcgagc gtgtagtaaa 2340
acgcgtttta ctacacgtgg attatgcgag ctccattaat gaatcggcca acgcgcgggg 2400
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 2460
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 2520
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 2580
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 2640
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 2700
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 2760
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 2820
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 2880
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 2940
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 3000
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 3060
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 3120
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 3180
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 3240
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 3300
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 3360
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 3420
tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 3480
ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 3540
ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 3600
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 3660
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 3720
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 3780
aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 3840
tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 3900
ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 3960
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 4020
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 4080
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 4140
accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 4200
gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 4260
cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 4320
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 4380
atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtccagc tgacggccgt 4440
cagctggcta ccaagacgaa gaggatgaag aggatgagga ggcagattgc cttgaatata 4500
ttgacaatac tgataagata atatatcttt tatatagaag atcgaccgtg ctataattat 4560
actaatttta taaggaggaa aaaatatggg catttttagt atttttgtaa tcagcacagt 4620
tcattatcaa ccaaacaaaa aataagtggt tataatgaat cgttaataag caaaattcat 4680
ataaccaaat taaagagggt tataatgaac gagaaaaata taaaacacag tcaaaacttt 4740
attacttcaa aacataatat agataaaata atgacaaata taagattaaa tgaacatgat 4800
aatatctttg aaatcggctc aggaaaaggc cattttaccc ttgaattagt aaagaggtgt 4860
aatttcgtaa ctgccattga aatagaccat aaattatgca aaactacaga aaataaactt 4920
gttgatcacg ataatttcca agttttaaac aaggatatat tgcagtttaa atttcctaaa 4980
aaccaatcct ataaaatata tggtaatata ccttataaca taagtacgga tataatacgc 5040
aaaattgttt ttgatagtat agctaatgag atttatttaa tcgtggaata cgggtttgct 5100
aaaagattat taaatacaaa acgctcattg gcattacttt taatggcaga agttgatatt 5160
tctatattaa gtatggttcc aagagaatat tttcatccta aacctaaagt gaatagctca 5220
cttatcagat taagtagaaa aaaatcaaga atatcacaca aagataaaca aaagtataat 5280
tatttcgtta tgaaatgggt taacaaagaa tacaagaaaa tatttacaaa aaatcaattt 5340
aacaattcct taaaacatgc aggaattgac gatttaaaca atattagctt tgaacaattc 5400
ttatctcttt tcaatagcta taaattattt aataagtaag ttaagggatg cataaactgc 5460
atcccttaac ttgtttttcg tgtgcctatt ttttgtgaat cgggtcgatc ggggaagaac 5520
agtatgtcga gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat 5580
attatatttt actggatgaa ttgttttagt acctaagctt gcgaattcaa gcggtaggtg 5640
aaatattaca aatagtattt ttcggtcatt ttaacttgct atttcttgaa gaggttagta 5700
caatatgaat cgtggtaagt aataggacgt gcttcaggcg tgttgcctgt acgcatgctg 5760
attcttcagc aagactacta cctcatgaga gttatagact catggatctt gctttgaagg 5820
gttttgtaca ttataggctc ctatcacatg ctgaacctat ggcctattac atttttttat 5880
atttcaagga ggaaaagacc acatgaaaaa aaagattatc tcagctattt taatgtctac 5940
agtgatactt tctgctgcag ccccgttgtc aggtgtttac gctgacacaa accaccatca 6000
tcatcatcat ctcgagccat ggagatctgt cgacgtgctg caggcatgcc ccgggggtac 6060
ctgataaccg gcgctacgat atttggagtt gaggttcaaa gtcaaatggt actgatgacc 6120
ggtaaaattt aatattttga accttgctta ggcagctgac ttcacattgt tgagatcagc 6180
tgccttttgc ttatagttca ttgagtagaa acggttctgt tgcgaagttt gaaaatcaaa 6240
cgcaagctgg atcc 6254
<210> 3
<211> 166
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45
Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile
50 55 60
Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp
65 70 75 80
Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
85 90 95
Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met
100 105 110
Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr
115 120 125
Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
130 135 140
Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 4
<211> 30
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Lys Lys Lys Ile Ile Ser Ala Ile Leu Met Ser Thr Val Ile Leu
1 5 10 15
Ser Ala Ala Ala Pro Leu Ser Gly Val Tyr Ala Asp Thr Asn
20 25 30

Claims (6)

1. A recombinant lactobacillus casei comprising the recombinant vector pELSH-IFN α;
the recombinant vector pELSH-IFN alpha takes a plasmid pELSH as an original vector and carries a gene for coding IFN alpha protein;
the nucleotide sequence of the gene for coding the IFN alpha protein is shown as SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of the plasmid pELSH is shown as SEQ ID NO: 2 is shown in the specification;
the sites where the gene encoding IFN α protein was inserted into plasmid pELSH were NcoI and KpnI.
2. The use of the recombinant lactobacillus casei of claim 1 in the preparation of medicaments for the control of rotavirus.
3. Use of the recombinant lactobacillus casei of claim 1 for the preparation of a vaccine for the prevention and treatment of rotavirus diarrhea.
4. Use of the recombinant lactobacillus casei of claim 1 for the preparation of fermented food, health care products or feed.
5. Use according to claim 4, wherein the fermented food product comprises yoghurt and/or a milk-containing drink.
6. The use according to claim 4, wherein the nutraceutical comprises a functional dietary supplement.
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