CN110331148A - A kind of gene, recombinant vector pELSH-IFN α, recombinant lactobacillus casei and application encoding IFN α albumen - Google Patents
A kind of gene, recombinant vector pELSH-IFN α, recombinant lactobacillus casei and application encoding IFN α albumen Download PDFInfo
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- CN110331148A CN110331148A CN201910768543.0A CN201910768543A CN110331148A CN 110331148 A CN110331148 A CN 110331148A CN 201910768543 A CN201910768543 A CN 201910768543A CN 110331148 A CN110331148 A CN 110331148A
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Classifications
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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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- A23C2220/00—Biochemical treatment
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
The present invention provides a kind of gene, recombinant vector pELSH-IFN α, recombinant lactobacillus casei and applications for encoding IFN α albumen, belong to the transformation and its development and application technical field of lactic acid bacteria.The prebiotic effect of broad-spectrum antiviral effect of alpha interferon (IFN α) and lactic acid bacteria is combined together by the present invention by technique for gene engineering, by the expression in situ of function factor and probiotics can mucosal delivery realize synergy in the way of to substitute traditional intramuscular injection, intravenous, through detecting, the rhIFN α that the recombinant lactobacillus casei is secreted reaches 2.12 × 10 in the potency of MA104 cell anti-rotavirus5U/mg.The technical scheme is that the prevention and treatment of children virus diarrhea especially rotavirus diarrhea provides a kind of novel resolving ideas and application direction, be conducive to prevent a wide range of scale of young age poultry viral disease numerous infants, raiser.
Description
Technical field
The present invention relates to the transformation of lactic acid bacteria and its development and application technical field more particularly to a kind of coding IFN α albumen
Gene, recombinant vector pELSH-IFN α, recombinant lactobacillus casei and application.
Background technique
Rotavirus (Rotavirus, RV) infection is the main reason that clinically 5 years old Infants Below diarrhea is medical,
It there is no specific treatment medicine at present.For RV gastroenteritis, it is basic only in the form of oral rehydration respite because of body dehydration, electricity
Solve the symptoms such as vomiting and diarrhea caused by mass flow is lost.Moreover, rotavirus is worldwide popular, according to incompletely statistics, entirely
Ball has about four or five ten ten thousand children to die of rotavirus infection every year, wherein 6 months to 2 years old age bracket infection rate highest (Catherine
et al 2011；Diao Liandong etc. is 2018).Since rotavirus infection is not influenced by social economy, environmental sanitation condition, so morbidity
Rate is in the basic indifference of developed country and developing country (Parasharet al 2003).Obviously, RV diarrhea, which has become, endangers baby
Child grows up healthy and sound and the important public hygiene problem of life security, has evils to the socio-economic development of various countries.
About rotavirus diarrhea, successively there are scientist and medical worker to make many effort and trial, mesh for it
Before rely primarily on vaccine inoculation it prevented and is controlled.Wherein, the RV vaccine as the first approval in the U.S., RotaShield
It is found to exist with entembole after listing 1 year and be associated with, stopped production withdraw from the market (Buttery et al 2011).Though having later
Both vaccines of Rotarix and RotaTeq are proved to have higher prevention & protection to RV diarrhea, but 2010 detect wherein
Nucleic acid fragment to other viruses has to that us is enabled to worry the existing security hidden trouble (Ruiz-Palacios of vaccine pollution
et al 2006；Take charge of fine jade and Wei Zhidong 2018).In addition, although developing the RV vaccine LLR to produce by China proves to colyliform diarrhea
There is certain protecting effect, but its safety and efficiency evaluation are only limitted to domestic markets.It is worth mentioning, China applies for a patent
CN201711395821.X discloses a kind of lactic acid bacteria base that can stablize expression porcine rotavirus VP4 albumen on May 4th, 2018
Because of the strain of engineering subunit vaccine and its preparation method.The invention is led on the basis of the Lactobacillus casei of UPP gene delection
It crosses homologous recombination insertion porcine rotavirus VP4 gene and obtains its lactic acid bacteria recombinant vaccine for stablizing expression.This not only guarantees
The immune specificity of antigen, even more dexterously links together vaccine strain with probiotics.However, with regard to having listed at present or newly
For the RV vaccine of invention, developed for the epidemic strain of somewhere period, different regions protecting effect and
There are significant differences for validity, and it is a wide range of to young age poultry viral disease to numerous infants, raiser to be unfavorable for country
Scale prevention and treatment.Therefore, the discovery for preventing rotavirus diarrhea new way should obtain scholar and more widely pay close attention to, New-type wide-spectrum
The research and development of antivirotic are extremely urgent.
Summary of the invention
The purpose of the present invention is to provide a kind of gene for encoding IFN α albumen, recombinant vector pELSH-IFN α, recombination are dry
Lactobacillus paracasei and application, the recombinant lactobacillus casei can secrete people's IFN α optimization albumen, and secretory protein MA104 in vitro
(monkey Foetus renal cells) cell has good anti-rotavirus activity.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of gene for encoding IFN α albumen, the nucleotide sequence of the gene such as SEQ ID NO:1
It is shown.
The present invention also provides a kind of recombinant vector pELSH-IFN α, the recombinant vector pELSH-IFN α is with plasmid
PELSH is initial carrier, carries the gene that IFN α albumen is encoded described in above scheme；
The nucleotide sequence of the plasmid pELSH is as shown in SEQ ID NO:2.
Preferably, the site in the insertion plasmid pELSH of the gene of the coding IFN α albumen is NcoI and KpnI.
The present invention also provides a kind of recombinant lactobacillus caseis comprising recombinant vector pELSH-IFN α described in above scheme.
The present invention also provides recombinant lactobacillus caseis described in above scheme in the drug of preparation prevention and treatment rotavirus
Using.
The present invention also provides recombinant lactobacillus caseis described in above scheme in the vaccine for preparing prevention and treatment rotavirus diarrhea
In application.
The present invention also provides the recombinant lactobacillus caseis described in above scheme to prepare fermented food, health care product or feeding
Application in material.
Preferably, the fermented food includes Yoghourt and/or milk-contained drink.
Preferably, the health care product includes functional dietary supplements.
Beneficial effects of the present invention: the present invention provides a kind of gene, recombinant vector pELSH-IFN for encoding IFN α albumen
α, recombinant lactobacillus casei and application.The present invention provides a kind of genes for encoding IFN α albumen, and the gene is according to cheese cream bar
Bacterium (Lactobacillus casei) is to the close to the progress of hIFN α mature polypeptide encoded sequence using inclined preferendum of degenerate codon
Numeral optimizes to obtain, and Lactobacillus casei (host strain) uses gene of the invention compared with the gene of other coding IFN α albumen
Frequency is higher；The present invention also provides a kind of recombinant vector pELSH-IFN α and a kind of include recombinant vector pELSH-IFN α's
Recombinant lactobacillus casei.The present invention passes through technique for gene engineering for broad-spectrum antiviral effect of alpha interferon (IFN α) and lactic acid bacteria
Prebiotic effect be combined together, by the expression in situ of function factor and probiotics can mucosal delivery realize synergy in the way of
To substitute traditional intramuscular injection, intravenous, through detecting, the rhIFN α that the recombinant lactobacillus casei is secreted is anti-in MA104 cell
The potency of rotavirus reaches 2.12 × 105U/mg.The technical scheme is that children virus diarrhea especially colyliform disease
The prevention and treatment of malicious diarrhea provides a kind of novel resolving ideas and application direction, is conducive to numerous infants, raiser to children
The a wide range of scale of age poultry viral disease prevents.Also, the present invention will using the unique flavor substance of Lactobacillus casei
It ferments, and can be developed into functional boruga, dietary supplements or feed addictive, through detecting, utilizes weight of the invention
The Shelf-Life of Yogurt that group Lactobacillus casei fermentation obtains is able to extend.
Detailed description of the invention
Fig. 1 is the mass spectrogram of recombinant vector pELSH-IFN α；
Fig. 2 is JM109-pELSH-hIFN α recon bacterium solution PCR verifying in embodiment 2, and wherein M is Trans 2KPlus
DNAMarker, 1-10 are respectively 10 transformants of the picking from plate；
Fig. 3 is the double digestion verification result of recombinant vector pELSH-IFN α in embodiment 2, and wherein M is DNAMarker, left
1-10 is respectively EcoRI-PslpA-F/BamHI-T-R double enzyme digestion product electrophoretogram, and right 1-10 is NcoI/KpnI double enzyme digestion product
Electrophoretogram；
Fig. 4 be embodiment 3 in hIFN α recombinant protein Lactobacillus casei secreting, expressing qualification result；
Fig. 5 is the best secreting, expressing amount measurement result of albumen in embodiment 4；
Fig. 6 is the qualitative microscopic examination result of anti-rotavirus activity of restructuring destination protein in embodiment 6, wherein figure a is neither
The normal cell of poison processing, i.e. blank control ware are not attacked in sample-adding induction yet；Figure b be not loaded induction but attack poison handle lesion it is thin
Born of the same parents, i.e. virus control ware；Figure c is the experimental cell that RV attacks poison processing after adding the induction of rhIFN α recombinant protein sample, i.e. planchet；
Figure d is the cell that RV attacks poison processing after adding the induction of rhIFN α albumen mark product, i.e. mark product ware；
Fig. 7 is Yoghourt live lactobacillus number changes of contents situation in storage of the present invention in embodiment 7.
Specific embodiment
The present invention provides a kind of gene for encoding IFN α albumen, the nucleotide sequence of the gene such as SEQ ID NO:1
It is shown, specifically:
TGTGATTTGCCAGAAACCCATAGTTTGGATAATCGCCGCACCTTGATGTTGTTGGCCCAAATGAGTCG
CATTAGTCCAAGTAGTTGTTTGATGGATCGCCATGATTTTGGCTTTCCACAAGAAGAATTTGATGGCAATCAATTT
CAAAAAGCCCCAGCCATTAGTGTTTTGCATGAATTGATTCAACAAATTTTTAATTTGTTTACCACCAAAGATAGTA
GTGCCGCCTGGGATGAAGATTTGTTGGATAAATTTTGTACCGAATTGTATCAACAATTGAATGATTTGGAAGCCTG
TGTTATGCAAGAAGAACGCGTTGGCGAAACCCCATTGATGAATGCCGATAGTATTTTGGCCGTTAAAAAATATTTT
CGCCGCATTACCTTGTATTTGACCGAAAAAAAATATAGTCCATGTGCCTGGGAAGTTGTTCGCGCCGAAATTATGC
GCAGTTTGAGTTTGAGTACCAATTTGCAAGAACGCTTGCGCCGCAAAGAA。
In the present invention, the IFN α albumen source of the optimization is in people (i.e. human IFN α)；The present invention was embodied
Protein sequence [(Refseq:NP_076918.1), amino acid sequence such as SEQ based on the human alpha interferon submitted on NCBI in journey
Shown in ID NO:3, specifically: CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISV L
HELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTE
KKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE], under the premise of not changing its amino acid, according to Lactobacillus casei
(Lactobacillus casei) carries out password to hIFN α mature polypeptide encoded sequence using inclined preferendum to degenerate codon
Son optimization (the high sequence of one host strain frequency of use of selection, U → T from the degenerate codon for encoding same amino acid),
It send Wuhan to hold up the synthesis of Kechuang neoformation Technology Co., Ltd.'s full genome and obtains target gene (gene of coding IFN α albumen).This hair
In bright, the gene of coding IFN α albumen host strain frequency of use compared with the gene of other coding IFN α albumen is higher.
The present invention also provides a kind of recombinant vector pELSH-IFN α, the recombinant vector pELSH-IFN α is with plasmid
PELSH is initial carrier, carries the gene that IFN α albumen is encoded described in above scheme；The nucleotides sequence of the plasmid pELSH
Column are as shown in SEQ ID NO:2；It is described coding IFN α albumen gene insertion plasmid pELSH in site be NcoI and
KpnI；The mass spectrogram of the recombinant vector pELSH-IFN α is referring to Fig. 1.
In the present invention, the plasmid pELSH carries signal peptide sequence；The amino acid sequence of the signal peptide sequence encodes
As shown in SEQ ID NO:4, specifically: MKKKIISAILMSTVILSAAAPLSGVYADTN.In the present invention, the signal peptide sequence
Column derive from Lactococcus lactis, hold up Kechuang neoformation Technology Co., Ltd. by Wuhan and synthesize.
In the present invention, carrier pELSH separation quality grain pMC11 transformation from Lactobacillus casei is constructed and is obtained, due to matter
Grain is isolated from Lactobacillus casei MCJ in itself, and (Lactobacillus casei MCJ △ 1 is without the plasmid
The difference of host strain of the present invention, Lactobacillus casei MCJ △ 1 and Lactobacillus casei MCJ is: after
It is the former that person, which has lacked a pMC11 plasmid,；The Lactobacillus casei MCJ △ 1 and Lactobacillus
Casei MCJ is preserved in state Key Laboratory of Agricultural Microbiology A408), therefore it is in Lactobacillus casei
It can be stabilized and express.And this plasmid expression system expressive host narrow range, only can Escherichia coli and Lactobacillus casei (or
Lactobacillus paracasei) in shuttling expressing, further ensure target gene expression directionality and safety.
In the present invention, the preparation method of the recombinant vector pELSH-IFN α is preferred the following steps are included: coding IFN α
The gene and plasmid pELSH double digestion of albumen recycle digestion products, and digestion products enzyme connects, and obtain recombinant vector pELSH-IFN α.
In the present invention, the gene and plasmid pELSH double digestion system of the coding IFN α albumen are referring to table 1；The digestion
Temperature be preferably 35~40 DEG C, more preferably 37 DEG C；The time of the digestion is preferably 1.5~3h, more preferably 2h.
The gene and plasmid pELSH double digestion system of the coding IFN α albumen of table 1
In the present invention, the kit of the recycling digestion products is preferably PCR cleaning QIAquick Gel Extraction Kit, is purchased from OMEGA
Company；It preferably further include that substances, the agarose gel electrophoresis such as the enzyme in removing reaction system are tested after the recycling digestion products
To even after demonstrate,proving successfully.
In the present invention, the system of digestion products (the target fragment and carrier segments) enzyme even is referring to table 2；What the enzyme connected
Temperature is preferably 15~18 DEG C, and more preferably 16 DEG C；The time of the enzyme even is preferably 0.5~2h, more preferably 1~1.5h.
2 target fragment of table and carrier segments enzyme disjunctor system
The present invention also provides a kind of recombinant lactobacillus caseis comprising recombinant vector pELSH-IFN α described in above scheme；
The preparation method of heretofore described recombinant lactobacillus casei is preferably by recombinant vector pELSH-IFN α electrotransformation cheese cream bar
Bacterium competence cell.
The present invention by broad-spectrum antiviral effect of IFN α and the prebiotic function of lactic acid bacteria by technique for gene engineering in conjunction with rise
Come, experiment is embodied and is verified by taking Lactobacillus casei as an example.The host strain that the present invention uses is Lactobacillus casei
Lactobacillus casei MCJ △ 1, but it is not limited only to the bacterium, as long as other probiotics can express the equal of target protein
It can.
The present invention also provides recombinant lactobacillus caseis described in above scheme in the drug of preparation prevention and treatment rotavirus
Application direction.The recombinant protein of heretofore described recombinant lactobacillus casei secreting, expressing is active with anti-rotavirus, therefore energy
The recombinant lactobacillus casei for secreting this destination protein also has corresponding antiviral activity.
The present invention also provides recombinant lactobacillus caseis described in above scheme in the vaccine for preparing prevention and treatment rotavirus diarrhea
In application direction.In the present invention, the recombinant protein of the recombinant lactobacillus casei secreting, expressing has prevention and treatment rotavirus abdomen
The effect of rushing down, therefore the recombinant lactobacillus casei also accordingly has effects that prevent and treat rotavirus diarrhea.
The present invention also provides recombinant lactobacillus caseis described in above scheme in preparing fermented food, health care product or feed
Application direction；The fermented food preferably includes Yoghourt and/or milk-contained drink；The health care product preferably includes functionality
Dietary supplements.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1 constructs recombinant vector pELSH-IFN α
1. target fragment and plasmid pELSH double digestion
Double digestion system is referring to above scheme table 1.37 DEG C of digestion 2h.Using PCR cleaning QIAquick Gel Extraction Kit to digestion products
Recycled, remove the substances such as enzyme in reaction system, agarose gel electrophoresis be proved to be successful after to even.
2. the enzyme of target fragment and carrier segments connects
Enzyme disjunctor system is referring to above scheme table 2.Enzyme even reacts 16 DEG C, 1h, obtains recombinant vector pELSH-IFN α.
Embodiment 2 constructs recombinant lactobacillus casei
The recombinant vector pELSH-IFN α electrotransformation Lactobacillus casei competent cell that embodiment 1 is obtained, 37 DEG C of standings
It is coated with the MRS plate containing erythromycin after recovery, screened after 37 DEG C of constant temperature are inverted culture 2d, verifying, obtain positive recombinant.
Referring to figs. 2 and 3, wherein Fig. 2 is JM109-pELSH-hIFN α recon bacterium solution PCR verifying, M Trans to verification result
2KPlus DNA Marker, 1-10 are respectively 10 transformants of the picking from plate.PELSH zero load size is 640bp,
PELSH-hIFN α recombinant plasmid PCR product size is 1105bp.By Ago-Gel nucleic acid electrophoresis result figure it is found that band list
One is bright, and the PCR product clip size of 10 swimming lanes is consistent, is consistent with expection.Fig. 3 indicates recombinant vector pELSH-IFN α's
Double digestion verification result, M are DNA Marker, and left 1-10 is respectively EcoRI-PslpA-F/BamHI-T-R double enzyme digestion product electricity
Swimming figure, right 1-10 are NcoI/KpnI double enzyme digestion product electrophoretogram.Recombinant plasmid is through the bis- enzymes of EcoRI-PslpA-F/BamHI-T-R
Cutting the expected size of rear product is respectively 1096bp and 5623bp, and the expected size of product is respectively after NcoI/KpnI double digestion
498bp and 6221bp.By Ago-Gel nucleic acid electrophoresis result figure it is found that all plasmids are double to cut result and expected consistent, band
Size is consistent, and further explanation tentatively obtains correct pELSH-hIFN α positive colony.
Embodiment 3hIFN α recombinant protein is identified in the secreting, expressing of Lactobacillus casei
It crosses and positive colony recombinant lactobacillus casei (L.casei-pELSH-hIFN α) is forwarded in picking embodiment 2
In MRS culture medium of the 5mL containing Em, 37 DEG C of stationary cultures are stayed overnight.With L.casei MCJ Δ 1 and the recombinant bacterium of carrying zero load pELSH
L.casei-pELSH is negative control, by thallus culture to identical OD.Each bacterium bacterium solution 1mL is collected, 10000 × g is centrifuged 1min,
Its supernatant is taken to carry out Westernblot identification as measuring samples.Referring to fig. 4, wherein M is albumen Marker, swimming to qualification result
Road 1-3 is respectively recipient bacterium (L.casei MCJ Δ 1), plasmid vector transformant (L.casei MCJ Δ 1-pELSH) and recombination
Bacterium (L.casei MCJ Δ 1-pELSH-hIFN α).
By WB result it is found that albumen is not detected in empty bacterium and zero load, only recombinant bacterium detects albumen size and expection
21kDa is consistent, and illustrates that restructuring destination protein successful secretion can be expressed in Lactobacillus casei L.casei MCJ Δ 1.
It is fixed that the best secreting, expressing of 4 albumen of embodiment measures
Recombinant lactobacillus casei in embodiment 2 is activated, 1% (volume ratio, such as 100mL culture medium inoculated 1mL bacterium solution) connects
Kind collects the culture solution supernatant of different growing stage (such as OD600=1.0,2.0,3.0), i.e. recombination cheese to MRS culture medium
Lactobacillus body different growing stage culture solution supernatant total protein takes 15 μ L samples to carry out Westernblot analysis, with best at it
Growth period receives bacterium and isolates and purifies albumen, and cell experiment to be done detects its anti-rotavirus activity.Testing result is referring to Fig. 5, M
Albumen Marker (albumen marker used in experiment is same, Thermo article No. 26616), swimming lane 1-9 is respectively represented
The recon of OD600=0.942,1.42,2.09,2.87,3.44,5.04,5.67,5.79 and 5.95 α containing pELSH-hIFN
Supernatant sample.
Analysis result can obtain, as cell concentration OD600≤2.87, in supernatant hIFN α protein content with cell grow and
Increase, reaches highest in OD600=2.87.Later, hIFN α protein content then increasing gradually with cell concentration in supernatant
It reduces.
5 restructuring destination protein of embodiment isolates and purifies
Bacterium solution, 4 DEG C, 7500r/ are collected in activation culture embodiment 2 when recombinant lactobacillus casei to protein secretion maximum
Min is centrifuged 30min and carries out affinity chromatography after supernatant crosses 0.22 μm of filter membrane removal of impurities degerming.Before loading, first with 20% second
Alcohol, ultrapure water nickel column, then nickel column is balanced with BufferA.After column equilibration, by the sample after centrifugation, filtration treatment with 1mL/
The flow velocity of min is loaded on Ni column by weighing apparatus stream pump.After the completion of hanging column, successively with the Buffer A/ of the imidazoles containing various concentration
Buffer B mixed liquor starts gradient elution purpose product.According to the real-time appearance situation of elution process, when the ultraviolet suction of efflux
Efflux is collected when luminosity starts obviously to rise, until the ultraviolet absorptivity of efflux stops receiving liquid when dropping to minimum.It will
Each pipe sample collected carries out 12%SDS-PAGE detection and analysis.According to electrophoresis result, the elution egg in corresponding collecting pipe is selected
The white dialysis under 4 DEG C of environment removes imidazoles, measures concentration by albumen mark song.
The anti-rotavirus Activity determination for the restructuring destination protein that 6 embodiment 5 of embodiment obtains
1. qualitative microscopy, the method is as follows:
(1) 37 DEG C, 5%CO2Environment culture MA104 cell is to growing up to single layer；
(2) culture solution is discarded, PBS is washed several times；
(3) sample treatment: diluted protein sample and mark product are separately added into corresponding ware, 37 DEG C, 5%CO to same concentrations2
Incubator induction is incubated for 20h.Simultaneously do other condition equivalent processes but be not added sample, be not added poison normal cell blank control and
Sample is not added, only adds the RV of virus to compare ware, 37 DEG C, 5%CO after 20h2Culture；
(4) it attacks poison: first with pancreatin activated virus 30min, then inhaling and abandon original fluid addition RV ware, planchet and mark product
Ware sets 37 DEG C of carbon dioxide incubator absorption 1h.Virus liquid is discarded, the maintaining liquid containing pancreatin is added and puts 37 DEG C, CO2Environment is incubated
It educates for 24 hours, observes lesion, the outer degree of protection infected to RV of record recombinant secretor proteosome daily.As a result referring to Fig. 6.It can by Fig. 6
Know and compared with blank control ware normal cell, obvious typical cytopathic occurs for viral b ware cell: cell is in circle cluster, gap between cluster
Significantly, free nest is formed.And compared with RV control ware sick cell, planchet and mark product ware cell do not observe that typical case is significant
Rotavirus CPE is very little in other words, and cell state is similar with normal cell.This has absolutely proved Cheesecake lactobacillus
The hIFN α albumen of secreting, expressing is on MA104 cell really with certain anti-rotavirus activity.
2. titration, the method is as follows:
(1) 37 DEG C, 5%CO2Culture MA104 cell is to growing up to single layer；
(2) the 5 times of successives dilutions of hIFN α progress that will purify and measured concentration, are added to label by the amount in the hole 0.1mL/
In the cell culture plate well of respective concentration, 37 DEG C, 5%CO2Fiber differentiation 20h；
(3) inhale abandon culture solution, by 100 holes μ L/ be added rotavirus liquid carry out attacking poison, while set be not added virus control and
The control of interferon, 37 DEG C, 5%CO are not added2Continue to cultivate；
(4) CPE, continuous 3d are observed day by day.Hole is inhibited to replace in malicious valence measurement hole with lesion according to Reed-Muench method
Lesion hole calculates the activity i.e. potency that hIFN α inhibits rotavirus replication on MA104 cell.Results of measuring is referring to table 3.By
Table 3 be calculated rhIFN α MA104 cell anti-rotavirus potency up to 2.12 × 105U/mg。
Table 3hIFN α inhibits the active results of measuring of rotavirus replication on MA104 cell
RhIFN α albumen anti-rotavirus activity on MA104 cell is calculated according to Reed-Muench method:
Through calculating, rhIFN α is 2.12 × 10 in the potency of MA104 cell anti-rotavirus5U/mg。
7 functional yoghourt fermenting experiment of embodiment and viable count detection
By 60g skimmed milk powder be dissolved in 455mL distilled water be made into separated milk stir evenly, with 20mL recombinate cheese cream bar
Bacterium growth logarithmic phase bacterium solution, 25mL oyster mushroom juice are mixed to get mixed liquor, set 37 DEG C of Environmental Incubators fermentation 20h, when having observed layer
Phenomenon is analysed, that is, when having whey precipitation, rocks rear homogeneous, illustrates in good condition, ferment successfully, detecting its live lactobacillus number is
It is no up to standard.
The Yoghourt fermented is put into 4 DEG C of refrigerator storages, is taken out respectively in storage 3d, 6d, 9d, 12d etc., with sterilizing
Distilled water is coated with the MRS agar plate containing antibiotic erythromycin after doing 10 times of doubling dilutions, is upside down in 37 DEG C of constant incubator trainings
36h or so is supported, counts and calculates analysis, detects the shelf-life of Yoghourt of the present invention and compared with market.As a result see Fig. 7.As shown in Figure 7
The Yoghourt to be fermented using this recombinant lactobacillus casei, after product is stored 21 days under the conditions of 4 DEG C, the work of Lactobacillus casei recombinant bacterium
The bacterium number amount order of magnitude is still up to 108CFU/mL is more than two orders of magnitude of national standard, illustrates it with good application foundation.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Hua Zhong Agriculture University
<120>a kind of gene, recombinant vector pELSH-IFN α, recombinant lactobacillus casei and application for encoding IFN α albumen
<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 (9)
1. a kind of gene for encoding IFN α albumen, the nucleotide sequence of the gene is as shown in SEQ ID NO:1.
2. a kind of recombinant vector pELSH-IFN α, the recombinant vector pELSH-IFN α using plasmid pELSH as initial carrier, are carried
It has the right to require 1 gene for encoding IFN α albumen；
The nucleotide sequence of the plasmid pELSH is as shown in SEQ ID NO:2.
3. recombinant vector pELSH-IFN α according to claim 2, which is characterized in that the gene of the coding IFN α albumen
Insertion plasmid pELSH in site be NcoI and KpnI.
4. a kind of recombinant lactobacillus casei comprising recombinant vector pELSH-IFN α described in Claims 2 or 3.
5. application of the recombinant lactobacillus casei described in claim 4 in the drug of preparation prevention and treatment rotavirus.
6. application of the recombinant lactobacillus casei described in claim 4 in the vaccine of preparation prevention and treatment rotavirus diarrhea.
7. recombinant lactobacillus casei described in claim 4 is preparing the application in fermented food, health care product or feed.
8. application according to claim 7, which is characterized in that the fermented food includes Yoghourt and/or milk-contained drink.
9. application according to claim 7, which is characterized in that the health care product includes functional dietary supplements.
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