CN110205323A - Express the expressing gene, recombinant expression carrier, recombinant lactobacilli and its preparation method and application of 3 type interferon of pig - Google Patents
Express the expressing gene, recombinant expression carrier, recombinant lactobacilli and its preparation method and application of 3 type interferon of pig Download PDFInfo
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Abstract
The present invention relates to a kind of expressing genes for expressing 3 type interferon of pig, recombinant expression carrier, recombinant lactobacilli and its preparation method and application.The present invention carries out MOLECULE DESIGN according to the distinctive codon bias of LP18, pig type iii interferon I is subjected to codon optimization, pass through MOLECULE DESIGN, utilize gene magnification, molecules cloning approach and the PCR such as fusion amplification, agarose gel electrophoresis, the technologies such as glue recycling optimize 1 sequence of pig type iii interferon λ, synthesize containing anchor type secretion peptide 1320 and optimize the sequence carrier of type iii interferon gene, successfully construct 1 recombinant expression carrier of pig type iii interferon λ of optimization, and electrotransformation is to lactobacillus plantarum LP18, obtain the recombinant plant lactobacillus that can stablize expression 1 albumen of pig source IFN- λ, it lays a good foundation for the further development and application research of pig source IFN- λ 1.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of expressing gene for expressing 3 type interferon of pig, recombination table
Up to carrier, recombinant lactobacilli and its preparation method and application.
Background technique
Interferon (interferon, IFN) is the cell factor that a kind of viral interference duplication and infection generate.According to biology
3 classes can be divided by learning activity, and I type is the α generated by virus induction leucocyte and fibroblast and beta interferon；II type is by disease
The interferon that malicious induction of lymphocyte generates；Type iii interferon (also referred to as IFN- λ s or IL-28/29) is to find for 2003
New type, structure with it is genetically similar with IL-10 family member, but show I type IFN sample activity, mainly pass through JAK-
STAT signal path plays antiviral and antitumor and immunoregulation effect.IFN- λ s expression of receptor is mainly in epithelial cell, packet
Include epidermis, respiratory tract and gastrointestinal tract epithelial cell.Relative to I type interferon, type iii interferon is due to its receptor (IFN- λ s)
Targeting advantage, caused side effect are relatively small.In addition, IFN- λ s is mucous membrane, antiviral response is important in epithelial tissue
Medium, and it is most important to the protection of GI epithelium, it may be possible to the potential antiviral therapy agent of mucosa infection is targeted, while also being existed
It plays a significant role in mucosa-immune.The discovery such as Lan F IFN- λ 1 is conducive to the clear of staphylococcus aureus in healthy schneiderian membrane
It removes, and enhances the antibacterial functions of macrophage by IFN- λ 1-IL-28R-ROS-JAK-STAT signal path.Source of people III at present
Type interferon has IFN- λ 1, IFN- λ 2, IFN- λ 3 and IFN- λ 4, and source of mouse type iii interferon includes IFN- λ 2 and IFN- λ 3, but is closed
It is relatively fewer in the research of pig IFN- λ 1, and there has been no related drugs appearances, therefore are expanded for 1 gene of pig type III IFN- λ
Increase optimization, study its active function, can be established for the further immune function of further investigation pig type III IFN- λ 1 and clinical application
Basis.
Summary of the invention
Based on this, it is necessary to provide a kind of expressing gene of expression 3 type interferon of pig, recombinant expression carrier, recombinate newborn bar
Bacterium and its preparation method and application.
A kind of expressing gene for expressing 3 type interferon of pig, including the nucleotide sequence as shown in SEQ ID NO.1.
A kind of recombinant expression carrier for expressing 3 type interferon of pig, including empty carrier and the mesh being inserted in the empty carrier
Gene expression segment, the nucleotide sequence as shown in SEQ ID NO.1, the sky are contained in the destination gene expression segment
Carrier is pSIP411 carrier.
A kind of construction method of above-mentioned recombinant expression carrier includes the following steps: to provide and contains the destination gene expression
The plasmid of segment carries out PCR amplification to the destination gene expression segment using amplimer, the purpose that amplification is obtained
Gene expression segment, which is inserted into the empty carrier, obtains the recombinant expression carrier.
The amplimer includes upstream primer and downstream primer in one of the embodiments, the upstream primer
Sequence is as shown in SEQ ID NO.2, and the sequence of the downstream primer is as shown in SEQ ID NO.3.
A kind of recombinant lactobacilli for expressing 3 type interferon of pig, the recombinant lactobacilli contain above-mentioned recombinant expression carrier.
A kind of preparation method of recombinant lactobacilli that expressing 3 type interferon of pig, comprising the following steps: prepare the cream of competence
Then bacillus converts above-mentioned recombinant expression carrier into the lactobacillus of the competence, collect positive colony transformant and obtain
The recombinant lactobacilli.
The lactobacillus is lactobacillus plantarum LP18 in one of the embodiments,.
The preparation method of one boar, 3 type interferon, comprising the following steps: above-mentioned recombinant lactobacilli is inoculated in Liquid Culture
In base, when OD value is 0.3~0.5, induction peptide is added, continues culture 8~18 hours, thalline were collected by centrifugation, collects after grinding
Protein liquid obtains the 3 type interferon of pig.
One boar, 3 type interferon is obtained using the preparation method of above-mentioned 3 type interferon of pig.
3 type interferon of pig as claimed in claim 9 is preparing the application in antiviral therapy drug.
The present invention carries out MOLECULE DESIGN according to the distinctive codon bias of LP18, and pig type iii interferon I is carried out password
Son optimization, by MOLECULE DESIGN, optimizes 1 sequence of pig type iii interferon λ, utilizes the molecules such as gene magnification, fusion amplification
The technologies such as cloning approach and PCR, agarose gel electrophoresis, glue recycling, synthesis contain anchor type secretion peptide 1320 and optimization type III
The sequence carrier of interferon gene successfully constructs 1 recombinant expression carrier of pig type iii interferon λ of optimization, and electrotransformation is extremely planted
Object lactobacillus LP18, obtaining can stablize the recombinant plant lactobacillus for expressing pig source IFN- λ 1 albumen, be pig source IFN- λ 1 into one
Step development and application research is laid a good foundation.
Detailed description of the invention
Fig. 1 is 1% Ago-Gel that the pcr amplification product of PCR amplification is carried out by template of 1 plasmid of pcDNA-pIFN- λ
Electrophoretic identification, wherein swimming lane M is DNA molecular quality standard Marker, and swimming lane 1 is pcr amplification product；
Fig. 2 is the structural schematic diagram of recombinant plasmid Lp18:pSIP-pIFN- λ 1；
Fig. 3 is the electrophoresis proof diagram for recombinating lactobacillus plantarum Lp18:pSIP-pIFN- λ 1, wherein swimming lane M is DNA molecular
Quality standard Marker, swimming lane 1~3 are the monoclonal bacterium of electrotransformation lactobacillus plantarum Lp18, and swimming lane 4 is lactobacillus plantarum
Lp18, swimming lane 5 are blank control；
Fig. 4 is the Western Blot figure for detecting 1 albumen of pIFN- λ inducing expression in recombinant plant lactobacillus, wherein
Swimming lane 1 is protein standard Marker, and swimming lane 2 is not plus the lactobacillus plantarum Lp18 of inducer, swimming lane 3 are plus lure
The lactobacillus plantarum Lp18 of agent is led, swimming lane 4 is not plus the recombinant plant lactobacillus of inducer, swimming lane 5 are the recombination for adding inducer
Lactobacillus plantarum；
Fig. 5 is the Western Blot figure for detecting 2~18 hours recombinant protein pIFN- λ 1 and expressing；
Fig. 6 is the immunofluorescence for identifying recombinant bacterium surface expression, and wherein A group is lactobacillus plantarum Lp18, and B group is recombination
Lactobacillus plantarum Lp18:pSIP-pIFN- λ 1；
Fig. 7 is the flow analysis chart of recombinant bacterium expression, and wherein Data.003 is lactobacillus plantarum Lp18, and Data.004 attaches most importance to
Group lactobacillus plantarum Lp18:pSIP-pIFN- λ 1.
Specific embodiment
It is further described below in conjunction with attached drawing and example, unless otherwise indicated, this field routine can be used in the present invention
Technology.Unless otherwise explained, whole technical and scientific term used herein have with by the common skill of disclosure fields
The usually clear identical meaning of art personnel.
One, material and method
1.1 reagents, bacterial strain
Plasmid DNA Mini Kit, Bioflux gel reclaims kit are purchased from the limited public affairs of the healthy and free from worry Life Science in Suzhou
Department；MRS, M17 culture medium are purchased from Hai Bo biotech firm；His monoclonal antibody, HRP mark anti-mouse secondary antibody and FITC to mark anti-mouse secondary antibody purchase
In protein company；Clone Express Multis One Step Cloning Kit and Phanta Max Super-
Fidelity DNA Polymerase is purchased from Vazyme company；Induction peptide SppIP is closed in Nanjing Jin Sirui biotechnology company
At；Lactobacillus plantarum Lp18, Lactococcus lactis NZ3900 are provided by this laboratory, are also commercially available；Contain pig source pIFN- λ 1
(pIFN- λ 1) gene and plasmid pcDNA-pIFN- λ 1 Jing Guo sequence optimisation is provided by this laboratory, can also be according to sequence voluntarily
Synthesis；PSIP411 carrier is granted by Shandong University, is also commercially available.
1.2 design of primers and synthesis
According to gene order in GenBank (FJ455508.1), using Primer5.0 software Design primers for expanding base
Because of sequence, primer pIFN- λ 1-F and pIFN- λ 1-R is for expanding 1 sequence of IFN- λ, fragment length 650bp；Universal primer SF
With SR for screening positive bacterium colony；Primer is synthesized by Jilin provincial treasury U.S. Biotechnology Co., Ltd, and primer information is shown in Table 1.
Table 1
1.3 the amplification of 1 gene of pIFN- λ
MOLECULE DESIGN is carried out according to the distinctive codon bias of Lp18, three type interferon I of pig is subjected to codon optimization,
By MOLECULE DESIGN, the sequence carrier of type iii interferon gene is synthesized containing anchor type secretion peptide 1320 and optimized.Developed by molecule
Frame are as follows: secretion peptide (1320)-IFN λ 1-His, expression characteristic are anchor type expression, may there is a small amount of presence in supernatant.
Using 1 plasmid of pcDNA-pIFN- λ containing above-mentioned developed by molecule frame as template, PCR amplification is carried out.Reaction system:
KOD Plus 0.5uL, 1 μ L of template, each 1 μ L of primer pIFN- λ 1-F and pIFN- λ 1-R, 2.5 μ L of 10x buffer, dNTPs
2.5μL、MgSO4 1μL、ddH2O supplies 25 μ L.Reaction condition: 95 DEG C of 2min, 58 DEG C of 30s, 72 DEG C of 1min, totally 30 recycle.Expand
Volume increase object is identified through 1% agarose gel electrophoresis, and carries out recovery product according to plastic recovery kit.
1 segment of pcr amplification product pIFN- λ is identified through 1% agarose gel electrophoresis, obtains the special of 1 treaty 650bp
Band is consistent, as shown in Figure 1 with expected size.
The building and identification of 1.4 recombinant bacterium expression plasmids
Extract plasmid pSIP411, NcoI/XhoI double digestion obtain linearisation skeleton carrier sip, by skeleton carrier sip with
Pcr amplification product mixing, carries out seamless clone, reaction system is as follows:
Seamless cloning experimentation system
In 30 DEG C of incubators, NZ3900 is expanded using GM17 fluid nutrient medium, 12h is incubated overnight and obtains plateau
NZ3900.The GM17 culture medium containing 2%Gly is inoculated with 1% volume fraction again, when culture to OD value is 0.3~0.5,
4000rpm/min horizontal centrifugal 10min, collecting Gly stimulates culture.Supernatant is abandoned, supernatant is blotted, is added autoclaved
Washing buffer, 1mL/ pipe, is resuspended thallus, and 8000rpm/min is centrifuged 5min, abandons supernatant.Second step is repeated, is eventually adding
Appropriate Washing buffer, is resuspended thallus, is used for electrotransformation with the every pipe packing of 50 μ L volumes.
After 1.3 glue recovery product is connected in pSIP411 carrier using seamless clone technology, electrotransformation to clone
In host galactococcus NZ3900, the seamless clone of 5 μ L is assembled into product, freshly prepared NZ3900 competent cell is added, incubated on ice
20min is educated, 2mm specification is transferred to and is pre-chilled in electric revolving cup (BTX), 2000kV clicks 5ms after drying surface moisture, is rapidly added 30 DEG C
In the M17 culture medium of preheating.Conversion bacterium after 30 DEG C are cultivated 3h carries out gentle centrifugation, collects all bacterium solutions, is coated on red
Mycin concentration is 30 DEG C of overnight incubations on the M17 solid plate of 25mg/L, it is seen that clear bacterium colony appears in plate, as doubts
Like positive colony body.Picking 10 or so positive colonies carry out clone PCR in 8 μ L solution, as template, and system is such as
Shown in following table.
Clone PCR experimental system
Regular-PCR, PCR reactant are carried out again with universal primer SF and SR to positive colony after agarose gel electrophoresis verifying
System is as shown in the table, and PCR product is sent to company and is sequenced.
PCR reaction system
Building, expression and the identification of 1.5 recombination 1 lactobacillus plantarums of pIFN- λ
The Lp18 of recovery was cultivated to plateau, in MRS culture medium of the 1% volume inoculation containing 2%Gly, 37 DEG C are trained
Supporting to OD value is 0.3~0.5, and 10000 × g/min is centrifuged 10min, collects the culture of Gly stimulation culture, abandons supernatant, is added
Thallus is resuspended in autoclaved washing lotion, and supernatant is abandoned in centrifugation, and second step (cleans three times) altogether twice in repetition, and washing lotion is recycled to be resuspended
Thallus completes the preparation of Lp18 competence, and packing to each tubule, 40 μ L/ pipe or 80 μ L/ pipe are spare.
The structure of the recombinant plasmid Lp18:pSIP-pIFN- λ 1 containing pIFN- λ 1 constructed by seamless clone technology is such as
Shown in Fig. 2, recombinant plasmid is added in the competence of preparation, ice bath 20min is added in the electric revolving cup of pre-cooling, with 1750kV electricity
5ms is hit, in the non-resistant MRS fluid nutrient medium that thallus is incubated in 37 DEG C after transfer electric shock rapidly, cultivates 3h in 37 DEG C of incubators, from
The heart collects bacterium, is all coated in Erythromycinresistant plate, continues 24~48h of culture, observes positive colony transformant.Containing
There are the multiple monoclonals of picking on the plate of Erythromycinresistant, extracts positive plasmid and send company's sequencing identification.It is correct through company's sequencing
Afterwards, electricity is transferred in expressive host bacterium lactobacillus plantarum Lp18 recombinant plasmid again, and 3 monoclonals of picking carry out bacterium solution PCR identification.
The visible 1552bp purpose band of 1% agarose gel electrophoresis is consistent, as shown in Figure 3 with expection.Show recombinant plasmid success electricity
It is transferred in lactobacillus plantarum Lp18, verifies correct recombinant plant lactobacillus and be named as Lp18:pSIP-pIFN- λ 1.
The inducing expression of 1.6 recombinant plant lactobacillus
1% inoculation of picking positive restructuring lactobacillus plantarum Lp18:pSIP411-pIFN- λ 1 and lactobacillus plantarum Lp18 difference
In MRS fluid nutrient medium (erythromycin concentration 25mg/L), when OD value is 0.3~0.5, (concentration is addition induction peptide SppIP
50 μ g/L), continue to cultivate 10h.Thalline were collected by centrifugation by 4000rpm, is cleaned with PBS to remove MRS culture medium, then heavy with PBS
Outstanding thallus.Bead is added to be ground using grinder, collects protein liquid, -80 DEG C save backup.
The Western Blot of 1.7 recombinant plant lactobacillus expression products is identified
It takes the protein liquid of -80 DEG C of preservations appropriate, 5xSDS-PAGE Loading Buffer is added and is uniformly mixed, boiling water
Boil 6~8min.After 12%SDS-PAGE electrophoresis, is transferred by half dry type transferring film and is analyzed on pvdf membrane for Western Blot:
5% skimmed milk room temperature closes 3h, and PBST is washed 3 times；1: the 5000His anti-mouse primary antibody of label carries out 4 DEG C and is incubated overnight, and PBST is washed 3 times；
The anti-mouse secondary antibody of HRP label is incubated for 1h, and PBST is washed 3 times；ECL development is observed and is taken pictures.
The result shows that protein band plus in the albumen of inducer is not detected in recombinant plant lactobacillus, and recombinate
The lactobacillus plantarum of pIFN- λ 1 adds the specific band of visible 27kDa of inducer or so, as shown in figure 4, showing 1 base of pIFN- λ
Because of the successful expression in lactobacillus plantarum Lp18.
The optimization of 1.8 exogenous gene expression conditions
Optimal 1 albumen of pIFN- λ is expressed to obtain lactobacillus plantarum, carries out recombinant plant lactobacillus difference expression condition
Grope.The optimization for selecting different induction times is prepared at 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h time point
Protein sample carries out Western Blot detection to it.As a result as shown in figure 5, as seen from the figure, recombinant protein is just opened in 4h
Begin to express, and as the extension of recombinant plant lactobacillus incubation time, the expression of recombinant protein increase accordingly, can be held in 18h
Continued reaches, and according to cost and time, selects the albumen of 12h expression to carry out correlative study optimal.
The immunofluorescence analysis of 1.9 exogenous gene expressions
1% recombinant plant lactobacillus is inoculated in liquid MRS culture medium (erythromycin concentration 25mg/L), 10h is cultivated
Thallus is collected afterwards to be washed 2 times with equivalent PBS.Primary antibody uses the source of mouse Anti-His tag antibody containing 1: 50 to be incubated overnight, PBS cleaning 5
Time, the secondary antibody 1: 200 of FITC label is incubated for 2h, and PBS is cleaned 5 times.Carry out film-making, the expression feelings of oily microscopic observation foreign gene
Condition.By immunofluorescence, the foreign protein of expression is identified.Lactobacillus plantarum Lp18 has no fluorescence as shown in Figure 6, and weighs
The visible single thallus fluorescence of group lactobacillus plantarum Lp18:pSIP-pIFN- λ 1 further demonstrates that target gene can be in plant cream
It is expressed in bacillus Lp18, and the foreign gene expressed can be shown to lactobacillus surface.
The flow cytometry of 1.10 exogenous gene expressions
1% recombinant plant lactobacillus is inoculated in liquid MRS culture medium (erythromycin concentration 25mg/L), 10h is cultivated
Thallus is collected afterwards to be washed 2 times with equivalent PBS.Primary antibody uses the source of mouse Anti-His tag antibody containing 1: 50 to be incubated overnight, PBS cleaning 5
Time, the secondary antibody 1: 200 of FITC label is incubated for 2h, and PBS is cleaned 5 times.Take appropriate PBS that thallus is resuspended for flow cytometer.Pass through
Flow cytometry carries out identification and analysis to lactobacillus plantarum Lp18 and recombinant plant lactobacillus Lp18:pSIP-pIFN- λ 1, by Fig. 7
Know that lactobacillus plantarum Lp18 is 4.31%, the positive rate of recombinant plant lactobacillus Lp18:pSIP-pIFN- λ 1 is 22.96%.
Type iii interferon shows the antivirus action of wide spectrum as a kind of mucous membrane cytokines in mucomembranous surface, is
One kind having promising clinical application cell factor very much.Source of people IFN- λ 1 has been applied to antiviral and antitumor and on having at present
City's treatment product, and the effect of the antiviral mucous membrane of pig source IFN- λ 1 is needed to be studied.Lactobacillus plantarum is as gene work
Journey bacterium has inherent advantage: the colonization ability and Adhering capacity of stronger intestinal epithelial cell, has to Intestinal Mucosal Immunity and adjusts
Section effect.The present invention selects the host strain Lp18 of high-adhesiveness, by optimization pSIP411 expression vector and expresses protein gene,
1 albumen of successful expression pig source IFN- λ.Heterologous antigen is expressed in cell surface, fluorescence detection result card by 1320 signal peptides
Its surface display ability is illustrated, this facilitates the immune response for stimulating body.The present invention is by groping the expression feelings of different condition
Condition determines optimal expression quantity, as the time, to increase IFN- λ 1 expressing quantity higher.The recombinant plant lactobacillus table of optimization
Anti- mucosal virus up to protein binding pIFN- λ acts on, and can enhance antiviral effect.The recombinant plant lactobacillus that the present invention constructs
With potential mucosa-immune application value, provide fundamental basis for the development and application research of pig source IFN- λ 1.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Academy of Military Sciences's military medical research institute military affairs veterinary institute
<120>expressing gene, recombinant expression carrier, recombinant lactobacilli and its preparation method and application of 3 type interferon of pig are expressed
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 771
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atggagattt tagccatgaa tttcaaaaca gctgcaaaag taaccgtcgt tgccggcgca 60
tccgtactat tcttagctgc ttgtggctca agcaagagtt cttcaagttc taagcaaacg 120
gccaattgga ccgaatcagc cgaattacca acgatggaca tttctaagtc caccgatgtg 180
gttagttcta acgcgttgaa taacaccaat gaagggttat accgtctcgg taagaaccca 240
gtcccaactt ttaagccaac gacaacgcgg aagggctgtc acatgggcca atttcaaagt 300
ttatcaccac aagaattaaa gggctttaag aaagccaagg atgctttgga agaatcatta 360
tcattaaaga attggagttg tagcagtcca ttatttccac ggacgcggga tttacggcaa 420
ttacaagtgt gggaacggtt agtggcctta gaagctgaat tggatttgac tttaaaggtc 480
ttgcgggccg cggctgattc aagtttaggg gtcacgttag accaaccact tcggacgtta 540
catcatattc atgtcgaact tcaagcttgt attcgggctc aaccaacggc aggtagtcgg 600
ttacaaggcc ggttaaatca ttggttacac cggttacaag aagccacaaa gaaagaaagt 660
caaggctgcc ttgaagccag tgtgacattt aacttatttc acttattagt tcgggactta 720
cggagtgtta cgagtggtga tttgcatatt caccaccacc accaccatta g 771
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
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tattacaagg agattttagc atg 23
<210> 3
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<212> DNA
<213>artificial sequence (Artificial Sequence)
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ccggggtacc gaattcctcg ac 22
Claims (10)
1. a kind of expressing gene for expressing 3 type interferon of pig, which is characterized in that including the nucleotide as shown in SEQ ID NO.1
Sequence.
2. a kind of recombinant expression carrier for expressing 3 type interferon of pig, which is characterized in that including empty carrier and be inserted in the zero load
Destination gene expression segment in body contains the nucleotides sequence as shown in SEQ ID NO.1 in the destination gene expression segment
Column, the empty carrier are pSIP411 carrier.
3. a kind of construction method of recombinant expression carrier as claimed in claim 2, which comprises the steps of: provide
Plasmid containing the destination gene expression segment carries out PCR amplification to the destination gene expression segment using amplimer,
The destination gene expression segment that amplification obtains is inserted into the empty carrier and obtains the recombinant expression carrier.
4. construction method according to claim 3, which is characterized in that the amplimer includes that upstream primer and downstream are drawn
Object, the sequence of the upstream primer is as shown in SEQ ID NO.2, and the sequence of the downstream primer is as shown in SEQ ID NO.3.
5. a kind of recombinant lactobacilli for expressing 3 type interferon of pig, which is characterized in that the recombinant lactobacilli contains claim 2
The recombinant expression carrier.
6. a kind of preparation method for the recombinant lactobacilli for expressing 3 type interferon of pig, which comprises the following steps: preparation
Then the lactobacillus of competence converts recombinant expression carrier as claimed in claim 2 into the lactobacillus of the competence, receive
Collection positive colony transformant obtains the recombinant lactobacilli.
7. preparation method according to claim 6, which is characterized in that the lactobacillus is lactobacillus plantarum LP18.
8. the preparation method of 3 type interferon of a boar, which comprises the following steps: by recombination described in claim 5
Lactobacillus is inoculated in fluid nutrient medium, when OD value is 0.3~0.5, is added induction peptide, is continued culture 8~18 hours, is centrifuged
Thallus is collected, protein liquid is collected after grinding, obtains the 3 type interferon of pig.
9. 3 type interferon of a boar, which is characterized in that obtained using the preparation method of 3 type interferon of pig as claimed in claim 8
?.
10. 3 type interferon of pig as claimed in claim 9 is preparing the application in antiviral therapy drug.
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| CN114920817A (en) * | 2022-05-12 | 2022-08-19 | 华中农业大学 | Porcine interferon lambda 4 recombinant protein and preparation method and application thereof |
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