CN104788554B - Cat omega interferon mutant and its preparation method and application - Google Patents
Cat omega interferon mutant and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of cat omega interferon mutant and its preparation method and application, belong to the preparation and application field of cat omega interferon.The present invention obtains cat ω 2 or 11 interferon mutant of ω to 2 interferon of cat ω or 11 interferon of ω progress Amino Acid-Induced Site-Directed Mutation by comparing the gene order and amino acid sequence of 13 hypotypes of cat omega interferon.The present invention further discloses the methods for preparing the cat omega interferon mutant, it include: that cat ω gene carries out point mutation, and mutated gene is optimized, the gene cloning of cat ω 2 or 11 interferon mutant of ω will be encoded into baculovirus transfer vector, infected insect host is recombinated with baculoviral, expression alien gene obtains cat omega interferon protein expressioning product.The present invention can make the antiviral activity of 2 interferon of cat ω improve 45% or more by mutation, and optimizing and being mutated to gene can make the antiviral activity of 2 interferon of cat ω improve 96% or more.The method of the present invention simple process, can efficiently, steadily obtain the cat omega interferon that can be used safely, and antiviral activity is in be obviously improved.
Description
Technical field
The present invention relates to cat omega interferon more particularly to cat omega interferon mutant, and it is dry that the invention further relates to the cat ω
The preparation method and its purposes in the drug or reagent of preparation prevention or treatment cat disease viral disease for disturbing plain mutant, belong to
The preparation and application field of cat omega interferon.
Background technique
Interferon (Interferon, IFN) is that have height by one kind that cell generates under the action of specific inducer
The glycoprotein for spending bioactivity has broad anti-viral activity in this zooblast.It is currently known interferon and indirect kills
Sick and wounded poison, but by inducing the cell of host itself to generate a variety of enzymes come the genetic transcription of viral interference or virus protein group
The translation divided.Interferon can be divided into I according to cell origin difference, physicochemical property and the biological activity difference etc. of generation
Type and Type II IFN；I type IFN mainly includes IFN-α, β, ω, δ, κ, ε, ζ and τ etc., and Type II IFN only has IFN-γ one kind (Cann
AJ.Principles of Molecular Virology.Beijing:Science Press, 2006:177-178).
Species someone with omega interferon, cat, horse, sheep, ox, pig, rabbit for being currently known etc..Cat is doted on as a kind of tradition
Object is very big in the cultivation amount of China, infectious disease especially cat distemper, cat infectious bovine rhinotracheitis and feline calicivirus infection etc.
The generation of viral disease is increasingly severe.There is presently no a kind of specific drug for treating viral disease, there is an urgent need to it is high-quality,
Cheap feline interferon launch, but the expression quantity of current feline interferon is not high, and antiviral activity is not strong.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of 2 interferon mutants of cat ω and 11 interferon of cat ω to be mutated
Body, the cat omega interferon mutant have high antiviral active；
Another technical problem to be solved by this invention is to provide a kind of utilization baculovirus expression vector system preparation
The method of 2 interferon mutant of cat ω or 11 interferon mutant of cat ω；
Third technical problem to be solved by this invention is to provide 2 interferon mutant of cat ω or cat ω 11 is interfered
Purposes of the plain mutant in the drug or reagent of preparation prevention or treatment cat disease viral disease.
In order to solve the above technical problems, the technical solution used in the present invention is:
The present invention discloses a kind of 2 interferon mutant of cat ω first, and amino acid sequence is SEQ ID NO.2 or SEQ
Shown in ID NO.10.The gene of 2 interferon mutant of cat ω is encoded, nucleotides sequence is classified as SEQ ID NO.4, SEQ ID
Shown in NO.5, SEQ ID NO.11 or SEQ ID NO.12.
The invention also discloses a kind of 11 interferon mutant of cat ω, amino acid sequence is shown in SEQ ID NO.7.It compiles
The gene of code 11 interferon mutant of cat ω, nucleotides sequence are classified as shown in SEQ ID NO.8 or SEQ ID NO.9.
Amino acid sequence is shown in SEQ ID NO.1 before 2 interferon of cat ω is mutated, and gene order is SEQ ID NO.3
It is shown；Amino acid sequence is shown in SEQ ID NO.6 before 11 interferon of cat ω is mutated.
Gene order and amino acid sequence of the present invention by comparison 13 hypotypes of cat omega interferon, find each hypotype
Amino acid composition has minimum mutation, and if amino acid becomes L from P, G becomes A, and A becomes V etc.；2 type of cat omega interferon and 4 types tool
There is higher antiviral activity, aligned amino acid sequence finds the amino acid sequence of the omega interferon of the two types in 134-
140 position has more 7 amino acid (RATGEGE), illustrates that this 7 amino acid have centainly the antiviral expression of cat omega interferon
Facilitation；11 types and 8 types have lower antiviral activity, and aligned amino acid sequence finds two types at 125
Amino acid sports V by L, illustrates that V is possible to influence the antiviral activity of cat omega interferon.Based on above-mentioned comparison result, in order to
The antiviral activity of cat omega interferon is improved, the present invention carries out variation site identical with ω 11 in 2 type amino acid sequence of ω
Artificial correction, and the mutational site for using for reference ω 3 introduces the rite-directed mutagenesis of 4 amino acid: R82Q, P105L, V148A, G201A are obtained
The mutant nucleotide sequence of 203 amino acid arrived is as shown in SEQ ID NO.2；By the mutant nucleotide sequence of amino acid through DNAWorks reverse translation
At nucleotide sequence, nucleotide sequence is as shown in SEQ ID NO.4.
The present invention further utilizes OptimumGeneTMTechnology optimizes 2 type interferon mutated gene of cat ω, according to
The codon preference of bioreactor silkworm is transformed gene order, to influence genetic transcription efficiency, translation efficiency and
G/C content, CpG dinucleotides content, codon preference, the secondary structure of mRNA, the mRNA free energy of protein folding are stablized
Property, a variety of relevant parameters such as RNA unstability motif, repetitive sequence optimize, be conducive to improve institute's optimization gene and exist
Transcriptional efficiency and translation efficiency in silkworm, and keep the protein sequence finally translated into constant.It is rod-shaped in silkworm in order to improve
Translation initiation efficiency in viral eukaryotic expression system has added Kozak sequence AAC, before gene in order to improve translation termination
Efficiency, terminator codon are changed to TAA.In addition, also removing the restriction enzymes such as the BamH I inside gene order, EcoR I, Sma I
Enzyme site added BamH I in upstream region of gene, added XbaI restriction enzyme site in downstream of gene, the cat ω 2 of acquisition is dry
Disturbing element mutation optimization gene sequence is shown in SEQ ID NO.5.
The present invention also by original 2 type interferon of cat ω carry out multisite mutation, by 2 type amino acid sequence of ω with 11 phase of ω
Same variation site carries out artificial correction, and the fixed point for using for reference 10 amino acid of mutational site introducing of ω 3, ω 4 and ω 13 is prominent
Become: A12V, P27L, P62L, A78T, R82Q, P103L, P105L, P142L, V148A, G201A, 203 obtained amino acid
Multisite mutation sequence is as shown in SEQ ID NO.10；The multisite mutation sequence of amino acid is nucleated through DNAWorks reverse translation
Nucleotide sequence, as shown in SEQ ID NO.11.Utilize OptimumGeneTMTechnology carries out 2 type interferon multimutation gene of cat ω
Optimization, is transformed gene order according to the codon preference of bioreactor silkworm, after multisite mutation and optimization
Nucleotide sequence is as shown in SEQ ID NO.12.
The present invention is mutated 11 type interferon of cat ω according to the same method of 2 interferon mutant of ω, introduces 4 to it
The rite-directed mutagenesis of a amino acid: R82Q, P105L, V141A, G194A, 11 interferon amino acid sequence such as SEQ of cat ω after mutation
Shown in ID NO.7, nucleotide sequence is as shown in SEQ ID NO.8.The present invention is further applied to line software and analyzes mature cat
Rare codon in 11 interferon mutated gene of ω, it is then by baculoviral preferred codons table, mature cat ω 11 is dry
Disturb that the rare codon in plain gene is all synonymous to replace with baculoviral Preference codon, in the same manner in its 5'
BamHI restriction enzyme site and Kozak sequence is added in end, and XbaI enzyme cutting site is added in the end 3', optimizes mutant gene sequence such as SEQ ID
Shown in NO.9.
The present invention further discloses a kind of 2 interferon mutant of cat ω or 11 interferon mutants of cat ω of preparing
Method, comprising the following steps:
(1) gene or coding 11 interferon mutant of cat ω of 2 interferon mutant of cat ω will be encoded respectively
Gene cloning into baculovirus transfer vector, building obtain recombinant transfer vector；
(2) by recombinant transfer vector and baculovirus DNA cotransfection insect cell, recombinant baculovirus is obtained；
(3) by recombinate shape virus infection insect cell or insect host, infected insect cell or insect place are cultivated
The corresponding albumen of main expression, purifying to get.
Wherein, the baculovirus transfer vector be selected from AcRP23-lacZ, AcRP6-SC, AcUWl-lacZ,
BacPAK6、Bac to Pac、Bacmid、BlucBacII(pETL)、p2Bac、p2Blue、p89B310、pAc360、pAc373、
pAcAB3、pAcAB 4、PAcAS3、pAcC129、pAcC4、DZI、pAcGP67、pAcIEl、pAcJPl、pAcMLF2、pAcMLF
7、pAcMLF 8、pAcMPl、pAcMP2、pAcRP23、pAcRP25、pAcRW4、pAcsMAG、pAcUWl、pAcUW21、
pAcUW2A、pAcUW2B、pAcUW3、pAcUW31、pAcUW41、pAcUW42、pAcUW43、pAcUW51、pAcVC2、pAcVC
3、pAcYMl、pAcJcC5、pBacl、pBac2、pBlueBacIII、pBlueBacHis、pEV55、mXIV、pIEINeo、
pJVETL、pJVNhel、pJVP10、pJVrsMAG、pMBac、pP10、pPAKl、pPBac、pSHONEX 1.1、pSYN XIV VI
+、pSYNVI+wp、pSYNXIV VI-、pVL1391、pVL 1392、pVL 1393、pVL941、pVL 945、pVL 985、
PVTBac, pBM030 or pUAC-5；Preferably pVL1393；
The baculoviral be selected from silkworm baculovirus parent plant BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV,
HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV；Preferably silkworm baculovirus parent plant
BmBacmid；
The insect host is selected from silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm
(Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia
Pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea
Polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm
(Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn armyworm (Spodoptera
Frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm
(Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), tobacco
Noctuid (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria
dispar)；Preferably silkworm (Bombyx mori)；
It is described to infect the insect larvae or pupa for referring to recombinant baculovirus by eating or infecting through epidermis 1-5 age
Body；Preferably, it by recombinant Bombyx mori baculovirus infected silkworm cell or the silkworm larva or pupa in percutaneous puncture-inoculation 1-5 age, is infecting
Body fluid or the tissue homogenate of silkworm larva or pupa containing various Cat IFNωs are collected after 3-6 days；Wherein, the pupa is most
Excellent is 1-2 days early stage tender pupas.
The invention also discloses interfere containing the gene or the coding cat ω 11 for encoding 2 interferon mutant of cat ω
The expression vector or host cell of the gene of plain mutant.
Transfer vector constructed by the present invention includes: former containing 2,9,11 type interferon gene of cat ω and 2 mutated gene of ω
Carrier pVL1393-FeIFN- ω 2, pVL1393-FeIFN- ω 9, the pVL1393-FeIFN- ω 11, VL1393- of beginning sequence
FeIFN-ω11-M,pVL1393-FeIFN-ω2-M,pVL1393-FeIFN-ω2-MM；Contain 2,9,11 type interferon of cat ω
Carrier pVL1393-FeIFN- ω 2-O, the VL1393-FeIFN- ω 11- of sequence after the 2 mutated gene codon optimization of gene and ω
M-O、pVL1393-FeIFN-ω2-M-O、VL1393-FeIFN-ω2-MM-O。
Present invention recombinant baculovirus obtained includes: recombinant bombyx mori nuclear polyhedrosis virus rBmBacmid (FeIFN-
ω2、FeIFN-ω9、FeIFN-ω11、FeIFN-ω11-M、FeIFN-ω2-M、FeIFN-ω2-MM)、rBmBacmid
(FeIFN-ω2-O、FeIFN-ω11-M-O、FeIFN-ω2-M-O、FeIFN-ω2-MM-O)。
The present invention is by original feline interferon ω 2 (shown in SEQ ID NO.3), 2 mutant gene sequence (ω of 9,11 genes and ω
Shown in 2-M, SEQ ID NO.4) it is expressed in silkworm biological reactor, inhibit method in CRFK/ using few cells lesion
The cat ω type antiviral activity of interferon of silkworm larva expression is detected in VSV*GFP system.The result shows that addition recombination cat ω interference
The cell of fibroin has the ability for resisting virus infection；The FeIFN- ω 2 expressed in silkworm larva body has more significant anti-
Virus activity, potency is up to 4.7 × 105The antiviral activity of U/mL, FeIFN- ω 11 only has 2.3 × 105U/mL；FeIFN-ω2-M
Antiviral activity be apparently higher than FeIFN- ω 2, be 6.8 × 105U/mL illustrates to improve FeIFN- ω by point mutation
Antiviral activity is feasible and effective.
The present invention is further by 2 interferon gene of cat ω (shown in ω 2-O, SEQ ID NO.13) and cat ω 2 after optimization
Interferon mutation optimization gene (shown in ω 2-M-O, SEQ ID NO.5) gene is expressed in silkworm biological reactor, Western
Blotting after recombinant virus infection the result shows that can be detected the special of 19kDa size in the supernatant of silkworm hemolymph sample
Property band.Assay of Antiviral Activity the result shows that, 2 interferon gene of cat ω and mutated gene of optimization are expressed in silkworm larva body
FeIFN- ω 2-O and FeIFN- ω 2-M-O have a more significant antiviral activity, potency is up to respectively reaching 6.5 × 105U/mL
With 9.2 × 105U/mL, and the antiviral activity of FeIFN- ω 2 only has 4.7 × 105U/mL can make FeIFN- ω's 2 by mutation
Antiviral activity improves 45% or more；The antiviral activity of FeIFN- ω 2 can be made to improve 38% or more by optimizing；By to prominent
Becoming gene optimization can make the antiviral activity of FeIFN- ω 2-M improve 35% or more；It can be made by mutation and codon optimization
The antiviral activity of FeIFN- ω 2 improves 96% or more, illustrates that the method for being mutated and optimizing is effective, practical, to raising FeIFN-
The antiviral activity of ω 2 has biggish effect.
The present invention is also by 2 interferon gene multisite mutation gene of cat ω (shown in ω 2-MM, SEQ ID NO.11) and more
Site mutation simultaneously express in silkworm biological reactor, expression product by optimization gene (shown in ω 2-MM-O, SEQ ID NO.12)
Assay of Antiviral Activity the result shows that, the FeIFN- ω 2-MM and FeIFN- ω 2-MM-O expressed in silkworm larva body has more
Apparent antiviral activity, potency reach 6.3 × 105U/mL and 8.2 × 105U/mL, but the raising of antiviral activity does not have
FeIFN- ω 2-M's is more, illustrates that the antiviral activity of FeIFN- ω 2 can not be greatly improved in the mutation of multidigit point, only
Select suitable site mutation that could improve the antiviral activity of FeIFN- ω 2.
The present invention will be after the mutation of 11 interferon gene of cat ω (shown in ω 11-M, SEQ ID NO.8) of mutation and optimization
11 interferon gene of cat ω (shown in ω 11-M-O, SEQ ID NO.9) is expressed in silkworm biological reactor, using few cells
Lesion inhibits method to detect that cat recombination 11 type interferon of ω has disease-resistant in silkworm hemolymph in CRFK cell/VSV*GFP system
Cytotoxic activity, the potency of FeIFN- ω 11-M are 3.7 × 105The potency of U/mL, FeIFN- ω 11-M-O are 4.5 × 105U/mL, than
FeIFN- ω 11 improves a lot, but high not as good as FeIFN- ω 2, illustrates that carrying out identical mutation to 11 interferon gene of cat ω also can
Play the role of improving antiviral activity.
2 interferon mutant of cat ω or 11 interferon mutant of cat ω of the present invention can be applied to preparation prevention or control
Treat the drug or reagent of cat disease viral disease.Wherein, the cat disease viral disease includes: cat distemper, cat infectious bovine rhinotracheitis, cat
Calicivirus infection, the infection of feline leukemia poison, feline immunodeficiency virus infection or vesicular stomatitis virus infection.
Technical solution of the present invention compared with prior art, has the advantages that
The present invention passes through the gene order and amino acid sequence of comparison 13 hypotypes of cat omega interferon, to 2 interferon of cat ω
The rite-directed mutagenesis of 4 amino acid is introduced, 2 interferon mutant of cat ω is obtained.Further cat ω 2 described to coding is interfered the present invention
The nucleotide sequence of plain mutant optimizes, and expresses 2 interferon of cat ω using baculovirus expression vector system and dash forward
The antiviral activity of variant, expressed 2 interferon mutant of cat ω increases substantially.The present invention carries out 11 interferon of cat ω
Identical Amino Acid-Induced Site-Directed Mutation also functions to the effect for improving antiviral activity.The present invention further introduces 2 interferon of cat ω
The rite-directed mutagenesis of 10 amino acid simultaneously optimizes mutant gene sequence, expresses in silkworm biological reactor, obtained
2 interferon mutant of cat ω has more apparent antiviral activity, but the raising of antiviral activity does not introduce 4 amino acid cuts
Point mutation improves more, illustrates that the antiviral work of 2 interferon of cat ω can be increased substantially by only selecting suitable site mutation
Property.
The term definition involved in the present invention arrived
Unless otherwise defined, otherwise all technologies used herein and scientific term all have with it is of the art
Those of ordinary skill usually understands identical meaning.
Term " polynucleotides " or " nucleotide " mean the deoxyribonucleotide of sub-thread or bifilar form, deoxyribose core
Glycosides, ribonucleotide or ribonucleotide and its polymer.Except nonspecific limitation, otherwise the term is covered containing natural nucleotide
Known analog nucleic acid, the analog have similar to reference nucleic acid binding characteristic and be similar to it is naturally-produced
The mode of nucleotide is metabolized.Unless in addition specific limitation, otherwise the term also means oligonucleotide analogs comprising
PNA (peptide nucleic acid), the DNA analog used in antisense technology (thiophosphate, phosphamide acid esters etc.).Unless in addition referring to
Fixed, otherwise specific nucleic acid sequence also impliedly covers variant of its conservative modification (including but not limited to degenerate codon takes
Generation) and complementary series and clearly specified sequence.Particularly, can by generate one of them or more than one selected by (or
It is all) the 3rd sequence replaced through mixing base and/or deoxyinosine residue of codon realize that degenerate codon replaces
(Batzer et al., Nucleic Acid Res.19:5081 (1991)；Ohtsuka et al., J.Biol.Chem.260:2605-
2608(1985)；With Cassol et al., (1992)；Rossolini et al., Mol Cell.Probes 8:91-98 (1994)).
Term " host cell " or " recombinant host cell " mean the cell comprising polynucleotides of the present invention, but regardless of using
Which kind of method is inserted into generate recombinant host cell.
Term " transfection " refers to that eukaryocyte obtains the process of new genetic marker due to exogenous DNA incorporation.
Detailed description of the invention
The double digestion that Fig. 1 is recombinant plasmid pVL-FeIFN- ω x is identified；Wherein, M:DNA molecular mass standard；ω 2: weight
Group 2 double enzyme digestion product of plasmid pVL-FeIFN- ω；9 double enzyme digestion product of ω 9: recombinant plasmid pVL-FeIFN- ω；ω 2-M: recombination
Plasmid pVL-FeIFN- ω 2-M double enzyme digestion product；11 double enzyme digestion product of ω 11: recombinant plasmid pVL-FeIFN- ω；It is negative right
According to；
Fig. 2 is that cell the case where various ratios of fluorescence occurs；Wherein, A: the fluorescence that interferon inhibits VSV virus to show；
The fluorescence that B:VSV virus-infected controls group is shown；C: the fluorescence that part cell infection VSV virus is shown；
The double digestion that Fig. 3 is recombinant plasmid pVL-FeIFN- ω 11-M and pVL-FeIFN- ω 11-M-O is identified；Wherein, M:
DNA molecular quality standard；1: recombinant plasmid pVL-FeIFN- ω 11-M double enzyme digestion product；2: recombinant plasmid pVL-FeIFN- ω
11-M-O double enzyme digestion product；
The double digestion that Fig. 4 is recombinant plasmid pVL-FeIFN- ω 2-O and pVL-FeIFN- ω 2-M-O is identified；Wherein, M:
DNA molecular quality standard；1: recombinant plasmid pVL-FeIFN- ω 2-O double enzyme digestion product；2: recombinant plasmid pVL-FeIFN- ω 2-
M-O double enzyme digestion product；
Fig. 5 is the result for recombinating 2 interferon SDS-PAGE of cat ω；Wherein, M:DNA molecular mass standard；1: recombinant virus
RBmBacmid (FeIFN- ω 2) expression product；2: recombinant virus rBmBacmid (FeIFN- ω 2-O) expression product；3: recombination
Viral rBmBacmid (FeIFN- ω 2-M-O) expression product；4: negative control；
Fig. 6 is the corresponding fluorogram of cytopathy ratio；Wherein, A, "-": cell-free lesion；B, " ± ": several cytopathies
Become；C, "+": 20%~30% cytopathy；D, " ++ ": 50%~60% cytopathy.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1, test material and reagent
Cultivated silkworm breed variety JY1 provides for sericulture research institute of Jiangsu University of Science and Technology, and parental virus Bm-Bacmid DNA is according to document
Method building disclosed in (number of patent application: 201110142492.4, application publication number CN 102286534A)；VSV-GFP
Virus, transfer vector pVL1393, E.coli bacterial strain TOP10, BmN cell, CRFK cell, by Chinese Academy of Agricultural Sciences's biology
Technical research institute saves and provides.
Restriction enzyme, T4DNA ligase is purchased from Promega company, and liposome is purchased from Invitrogen company,
DMEM cell culture medium, fetal calf serum are GIBCO Products.
In relation to the configuration method of solution and culture medium, referring to related tool book, (Joseph et al., Molecular Cloning: A Laboratory refer to
The southern third edition, 2002；Ao Sibai, et al., fine works Molecular Biology, 1998)；Unless otherwise stated, otherwise percentage and
Number is calculated by weight.
Expression and inspection of 12,9,11 and ω of feline interferon ω of embodiment, 2 mutant gene sequence in silkworm biological reactor
It surveys
1, experimental method
The building of acquisition, the synthesis and plasmid of 1.1 cat ω type interferon genes
1.1.1 the acquisition of gene
It is searched from NCBI and obtains FeIFN- ω 2 (GenBank accession number: DQ420221), 9 (GenBank of FeIFN- ω
Accession number: DQ420228), the gene order and amino acid sequence of FeIFN- ω 11 (GenBank accession number: DQ420230.1).
Wherein, 2 interferon amino acid sequence of cat ω is shown in SEQ ID NO.1, and gene order is SEQ ID NO.3 institute
Show；11 interferon amino acid sequence of cat ω is shown in SEQ ID NO.6.
By comparing the gene order and amino acid sequence of 13 hypotypes of cat omega interferon, the amino acid of each hypotype is found
Composition has minimum mutation, and if amino acid becomes L from P, G becomes A；2 type of cat omega interferon and the antiviral work with higher of 4 types
Property, aligned amino acid sequence finds that the amino acid sequence of the omega interferon of the two types has more 7 in the position of 134-140
Amino acid (RATGEGE) illustrates that this 7 amino acid have certain facilitation to the antiviral activity of cat omega interferon；11 types
There is lower antiviral activity with 8 types, aligned amino acid sequence finds that two types are sported in 125 amino acid by L
V illustrates that V is possible to influence the antiviral activity of cat omega interferon.Based on above-mentioned comparison result, specially by 2 type amino acid sequence of ω
Variation site identical with ω 11 carries out artificial correction in column, and the fixed point for using for reference 4 amino acid of mutational site introducing of ω 3 is prominent
Become: R82Q, P105L, V148A, G201A, the mutant nucleotide sequence of 203 obtained amino acid, as shown in SEQ ID NO.2, label
For FeIFN- ω 2-M, gene order is shown in SEQ ID NO.4.
1.1.2 the synthesis of gene order and plasmid construction
Restriction enzyme enzyme site is analyzed with DNAman software, result and transfer vector are analyzed according to restriction enzyme site
The restriction enzyme being not present in objective gene sequence is added at target gene both ends for multiple cloning sites on pVL1393 and pUC57
Enzyme site.BamHI restriction enzyme site and Kozak sequence is added at its end 5' according to analysis result, XbaI enzyme cutting site is added in the end 3',
The gene order determined transfers to Nanjing Genscript Biotechnology Co., Ltd. to synthesize, and is inserted into pUC57 carrier, forms plasmid
PUC57-FeIFN- ω 2, pUC57-FeIFN- ω 9, pUC57-FeIFN- ω 11 and pUC57-FeIFN- ω 2-M.
1.2 building recombinant baculovirus transfer vectors
Plasmid pUC57-FeIFN- ω 2, the pUC57-FeIFN- ω 9, pUC57- synthesized with BamHI and XbaI double digestion
FeIFN- ω 11 and pUC57-FeIFN- ω 2-M, the mesh segment recycled glass milk method with T4DNA ligase with by BamHI
It is connected with the inactivation baculovirus transfer vector pVL1393 of XbaI double digestion, 16 DEG C of reactions 8 hours or more.Connection product conversion
Competent escherichia coli cell TOP10, choosing colony culture, upgrading grain identify positive colony with BamHI and XbaI double digestion,
It will identify that correct recombinant plasmid send Beijing Qing Ke Bioisystech Co., Ltd to be sequenced, correct plasmid be sequenced and is named as pVL-
FeIFN- ω 2, pVL-FeIFN- ω 9, pVL-FeIFN- ω 11 and pVL-FeIFN- ω 2-M.
The cotransfection of 1.3 recombinant plasmids and parental virus
Recovery, passage and the screening of recombinant virus for carrying out BmN cell according to having method reported in the literature.Work as BmN
When cell culture is to cell monolayer up to 80% or so, old culture medium is outwelled, is washed three times with serum-free TC-100 culture medium, then plus nothing
Blood serum medium.By recombinant plasmid pVL-FeIFN- ω 2, pVL-FeIFN- ω 9, pVL-FeIFN- ω 11 and pVL-FeIFN- ω
The BmN cell that 2-M and parental virus Bm-Bacmid DNA have been recovered with cotransfection after liposome embedded 20min carries out intracellular
Homologous recombination.27 DEG C of culture 5d or so collect cell culture fluid until cell detachment floats, and obtain the recombination containing target gene
Viral Bm-Bacmid (FeIFN- ω 2), Bm-Bacmid (FeIFN- ω 9), Bm-Bacmid (FeIFN- ω 11) and Bm-
Bacmid (FeIFN- ω 2-M), and the purifying and amplification that carry out virus are as seed virus.
1.4 cat ω type interferon are expressed in silkworm body
Recombinant virus culture solution is pressed 105PFU/ dosage injects silkworm from 5 ages, in 27 DEG C, 70%~80% humidity
Under the conditions of cultivate, silkworm larva grows advanced stage, and FeIFN- ω obtains high efficient expression under the action of polyhedrosis gene promoter.It connects
Kind infection 3.5d~4d or so, it can be observed that the symptoms such as the swelling of silkworm larva body segment, abnormal behavior, loss of appetite, work as observation
When being obviously reduced to larva volume, hemolymph is collected, -20 DEG C save backup.
The detection of 1.5 cat ω type interferon protein antiviral activities
Method is inhibited to detect cat ω type interferon in silkworm hemolymph in CRFK/VSV*GFP system using few cells lesion
Antiviral activity.By CRFK cell in good condition with 3.0 × 105The density of a/ml is inoculated in 96 well culture plates.It will
The DMEM culture solution of silkworm hemolymph fetal calf serum containing 70mL/L of ultrasonication and filtration sterilization is configured to different dilutions
The sample diluted is inoculated in the culture hole for being paved with CRFK cell by solution by 100 holes μ L/, each dilution and right
4 multiple holes are at least set according to silkworm blood, while setting the disease of the cell controls group that silkworm hemolymph and VSV*GFP is not added and addition VSV*GFP
Malicious control group, in 37 DEG C, 5%CO2Under the conditions of culture 18~for 24 hours.100TCID will be diluted to50VSV*GFP virus, by 100 μ
The hole L/ is added to have inhaled and abandon in the culture hole of supernatant, is placed in 37 DEG C, 5%CO2Under the conditions of cultivate.Under inverted fluorescence microscope
Observation, when there is fluorescence in cells a large amount of in each hole of virus control group, and the still complete well-grown of the cell in cell controls group, nothing
When fluorescence occurs, then shows that contradistinction system is completely qualified, complete observation can be made.
2, experimental result
The identification of 2.1 recombinant transfer vectors
Recombinant transfer vector pVL-FeIFN- ω 2, pVL-FeIFN- ω 9, pVL-FeIFN- ω 11 and pVL-FeIFN- ω
2-M isolates 2 segments through BamHI and XbaI double digestion, in 1% agarose gel electrophoresis, small fragment respectively 612bp,
591bp, 591bp and 612bp, large fragment are consistent with pVL1393 size, are 9607bp.Electrophoresis result is as shown in Figure 1.By enzyme
Cutting the correct plasmid of identification send Beijing Qing Kexin industry Bioisystech Co., Ltd to carry out nucleic acid sequencing, is compared and is tied with MegaAlign
Fruit shows that sequence is consistent with the sequence originally designed, shows that ω type interferon gene 2,9,11 and ω of the ω 2-M of cat is successfully inserted
Enter into pVL1393 transfer vector between BamHI and XbaI.
The acquisition of 2.2 feline interferon recombinant viruses and the detection of recombinant products
It is interfered using the cat ω type that few cells lesion inhibits method to detect silkworm larva expression in CRFK/VSV*GFP system
Plain antiviral activity.It is observed under inverted fluorescence microscope, the cell growth state in cell controls group is good, and unstressed configuration goes out
It is existing；Lesion occurs for the cell in virus-infected controls group, and almost fluorescence, addition recombination cat omega interferon egg occurs in 100% cell
White cell has the ability (Fig. 2) for resisting virus infection.Protective effect according to cat ω type interferon to CRFK cell, observation
The lesion degree of cell, green cells appearance to be had, this hole cell are just denoted as "+", according to Reed-Muench method meter
Calculate the potency of interferon.Testing result is listed in table 1, and Assay of Antiviral Activity is the result shows that the FeIFN- expressed in silkworm larva body
ω 2 has a more significant antiviral activity, and potency is up to 4.7 × 105The antiviral activity of U/mL, FeIFN- ω 11 only has 2.3 ×
105The antiviral activity of U/mL, FeIFN- ω 9 falls between, and is 3.4 × 105U/mL, as a result trend and reported knot
Fruit is identical；The antiviral activity of FeIFN- ω 2-M is apparently higher than FeIFN- ω 2, is 6.8 × 105U/mL has reached expected effect
Fruit illustrates that it is feasible and effective that FeIFN- ω antiviral activity is improved by point mutation.
The testing result of the recombination cat omega interferon antiviral activity of table 1
Expression and detection of 11 gene of feline interferon ω of the mutation of embodiment 2 and optimization in silkworm biological reactor
Based on the comparison result of embodiment 1,2 interferon gene of cat ω is mutated the raising for being conducive to antiviral activity, for this purpose,
11 type interferon gene of cat ω is mutated according to the same method of ω 2-M.
1, experimental method
Mutation, optimization and the synthesis of 1.1 cat ω, 11 type interferon gene
(GenBank accession number: the DQ420230.1) gene order of FeIFN- ω 11 and amino acid sequence are obtained from NCBI,
According to 1 method of embodiment, the rite-directed mutagenesis of 4 amino acid: R82Q, P105L, V141A, G194A is introduced to it, 196 obtained
The mutant nucleotide sequence of a amino acid derives mutant gene sequence as shown in SEQ ID NO.8 as shown in SEQ ID NO.7.Mutation
Gene order analyzes restriction enzyme enzyme site with DNAman software, analyzes result and transfer vector according to restriction enzyme site
The restriction enzyme being not present in objective gene sequence is added at target gene both ends for multiple cloning sites on pVL1393 and pUC57
Enzyme site.BamHI restriction enzyme site and Kozak sequence is added at its end 5' according to analysis result, XbaI enzyme cutting site is added in the end 3',
Gene order is synthesized by Nanjing Genscript Biotechnology Co., Ltd., and is inserted into pUC57 carrier, forms plasmid pUC57-FeIFN-
ω11-M。
It is analyzed in mature 11 interferon mutated gene of cat ω using online software http://gcua.schoedl.de/
Rare codon, then by baculoviral preferred codons table, by the rare codon in mature 11 interferon gene of cat ω
It is all synonymous to replace with baculoviral Preference codon, through synthesizing, cat ω 11-M interferon gene after must optimizing, according to phase
With method be added BamHI restriction enzyme site and Kozak sequence at its end 5', the end 3' is added XbaI enzyme cutting site, gene order by
Nanjing Genscript Biotechnology Co., Ltd.'s synthesis, and it is inserted into pUC57 carrier, form plasmid pUC57-FeIFN- ω 11-M-O.
Cat ω 11 optimizes mutant gene sequence as shown in SEQ ID NO.9.
1.2 building recombinant baculovirus transfer vectors
The plasmid pUC57-FeIFN- ω 11-M and pUC57-FeIFN- ω 11-M- synthesized with BamHI and XbaI double digestion
O, the target fragment for being recycled glass milk method with T4DNA ligase and the baculoviral by the inactivation of BamHI and XbaI double digestion
Transfer vector pVL1393 connection, 16 DEG C of reactions 8 hours or more.Connection product converts competent escherichia coli cell TOP10, chooses
Bacterium colony culture is selected, upgrading grain identifies positive colony with BamHI and XbaI double digestion, will identify that correct recombinant plasmid send Beijing
The sequencing of Qing Ke Bioisystech Co., Ltd, is sequenced correct plasmid and is named as pVL-FeIFN- ω 11-M and pVL-FeIFN- ω
11-M-O。
The cotransfection of 1.3 recombinant plasmids and parental virus
Method according to the literature carries out recovery, passage and the screening of recombinant virus of BmN cell.When BmN cell
When culture is to cell monolayer up to 80% or so, old culture medium is outwelled, is washed three times with serum-free TC-100 culture medium, then plus serum-free
Culture medium.By recombinant plasmid pVL-FeIFN- ω 11-M and pVL-FeIFN- ω 11-M-O and parental virus Bm-Bacmid DNA
The BmN cell recovered with cotransfection after liposome embedded 20min, carries out intracellular homologous recombination.27 DEG C of culture 5d or so, until
Cell detachment floats, and collects cell culture fluid, obtains the recombinant virus containing target gene, and carries out the purifying and amplification of virus
As seed virus.
1.4 cat ω type interferon are expressed in silkworm body
Recombinant virus culture solution is pressed 105PFU/ dosage injects silkworm from 5 ages, 27 DEG C, it is 70%~80% relatively wet
It is raised under conditions of degree, silkworm larva grows advanced stage, Bm-Bacmid (FeIFN- ω 11-M) and Bm-Bacmid (FeIFN- ω
11-M-O) high efficient expression is obtained under the action of polyhedrosis gene promoter.Inoculation 3.5d~4d or so, it can be observed that
The symptoms such as the swelling of silkworm larva body segment, abnormal behavior, loss of appetite collect blood strangury when observing that larva volume is obviously reduced
Bar, -20 DEG C save backup.
The detection of 1.5 cat ω type interferon protein antiviral activities
Method is inhibited to detect cat ω type interferon in silkworm hemolymph in CRFK/VSV*GFP system using few cells lesion
Antiviral activity, operating method is the same as embodiment 1.
2, experimental result
The identification of 2.1 recombinant transfer vectors
Recombinant transfer vector pVL-FeIFN- ω 11-M and pVL-FeIFN- ω 11-M-O reflects through BamHI and XbaI double digestion
Fixed, isolating small fragment through 1% agarose gel electrophoresis is all 591bp, and large fragment is in 9000bp or more, with pVL1393
(9607bp) size is consistent.Electrophoresis result is as shown in Figure 3.Digestion identifies that correct plasmid carries out nucleic acid sequencing, uses MegaAlign
Comparison result shows that sequence is consistent with the sequence originally designed, shows that the ω 11-M type interferon gene of cat and optimization gene have become
Function is inserted into pVL1393 transfer vector between BamHI and XbaI.Digestion detects correct Plasmid DNA, and Beijing is sent to hold up the new industry of section
Bioisystech Co., Ltd's sequencing, as a result as shown in SEQ ID NO.8 and SEQ ID NO.9, obtained recombinant plasmid is named as
Recombinant transfer vector pVL-FeIFN- ω 11-M and pVL-FeIFN- ω 11-M-O.
The acquisition of 2.2 feline interferon recombinant viruses and the expression of recombinant products
Inoculation about 1 × 106Cell is in 15cm2In culture bottle, after cell is adherent, removes and cultivated containing fetal calf serum (FBS)
Base is washed three times with the culture medium without FBS, adds 1.5ml without FBS culture medium.It is rod-shaped that 1 μ g silkworm is sequentially added into a sterile tube
Viral parent plant BmBacmid DNA, 2 μ g recombinant transfer plasmid pVL-FeIFN- ω 11-M, pVL-FeIFN- ω 11-M-O and 5 μ
L liposome is supplied volume to 60 μ l with aseptic double-distilled water, is mixed gently, stand 15min after, be added dropwise in culture bottle into
Row cotransfection.1.5ml serum free medium and 300 μ l FBS are added after 27 DEG C of culture 4h.27 DEG C constant temperature incubation 4~5 days, collect
Supernatant is used for the screening of recombinant virus rBmBacmid (FeIFN- ω 11-M, FeIFN- ω 11-M-O).It is inoculated with appropriate cell
(about 70~80%) after cell is adherent, suck culture medium in the small plate of 35mm, and it is dilute that cotransfection supernatant is carried out various concentration
It releases, takes 1ml corotation dye liquor to be added in attached cell, be evenly distributed.After 27 DEG C of infection 1h, infection liquid is sucked, by 2% low melting point fine jade
Sepharose melts in 60 DEG C of water-baths, is cooled to 40 DEG C and mixes with 40 DEG C of 2 × TC-100 culture mediums preheated (containing 20%FBS)
Even, every plate adds 4ml glue, is sealed after to be solidified with Parafilm, and 3~5d, micro- sem observation are cultivated in 27 DEG C of inversions.It will not contain
Polyhedrosis plaque is picked out, and above step is repeated, and obtains pure recombinant Bombyx mori baculovirus by the purifying of 2~3 wheels
rBmBacmid(FeIFN-ω11-M、FeIFN-ω11-M-O)。
Recombinant Bombyx mori baculovirus rBmBacmid (FeIFN- ω 11-M, FeIFN- ω 11-M-O) is infected into normal growth
BmN cell, culture collects supernatant after 3 days, a large amount of recombinant virus rBmBacmid (FeIFN- ω 11- contained in supernatant
M、FeIFN-ω11-M-O)。
The detection of 2.3 expression products
It is interfered using the cat ω type that few cells lesion inhibits method to detect silkworm larva expression in CRFK/VSV*GFP system
Plain antiviral activity.It is observed under inverted fluorescence microscope, the cell growth state in cell controls group is good, and unstressed configuration goes out
Existing, lesion occurs for the cell in virus-infected controls group, and almost fluorescence, addition recombination cat omega interferon egg occurs in 100% cell
White cell has the ability for resisting virus infection.Cell is observed in protective effect according to cat ω type interferon to CRFK cell
Lesion degree, green cells appearance to be had, this hole cell is just denoted as "+", calculates according to Reed-Muench method dry
The potency of element is disturbed, the results are shown in Table 2.The result shows that the FeIFN- ω 11-M and FeIFN- ω 11- expressed in silkworm larva body
M-O has more significant antiviral activity, and potency, which reaches, respectively reaches 3.7 × 105U/mL and 4.5 × 105U/mL, FeIFN- ω 11
Antiviral activity there was only 2.3 × 105U/mL, the antiviral activity that gene mutation can be such that FeIFN- ω 11 expresses improve 61%,
The product antiviral activity that optimizing to mutated gene can be such that mutated gene expresses improves 21%, and mutation and optimization can make to express
The activity of interferon improves 96% or more than FeIFN- ω 11, illustrates that the effect that mutation generates is higher than optimization, mutation and optimization
Combine the antiviral activity that can greatly improve FeIFN- ω 11.
The testing result of the recombination cat omega interferon antiviral activity of table 2
The mutation and optimization of 3 feline interferon ω of embodiment, 2 gene and expression and detection in silkworm biological reactor
It is based on Examples 1 and 2 as a result, Cat IFNω mutation and optimization are conducive to the raising of antiviral activity,
2 type interferon gene of cat ω and mutated gene carry out ω 2 and ω 2-M gene according to the optimization method of embodiment 2 thus excellent
Change, the specific method is as follows:
1, experimental method
The building of 1.1 cat ω, 2 type interferon mutated gene and the optimization and conjunction of 2 interferon gene of ω and mutant gene sequence
At
1.1.1 the building of 2 type interferon mutated gene of cat ω
The present invention is based on embodiments 1 to the gene order of 13 hypotypes of cat omega interferon and the comparison knot of amino acid sequence
Fruit, in order to improve expression quantity of the cat omega interferon in baculovirus expression system, the present invention by 2 type amino acid sequence of ω with
The identical variation site ω 11 carries out artificial correction, and the mutational site for using for reference ω 3 introduces the rite-directed mutagenesis of 4 amino acid:
R82Q, P105L, V148A, G201A, the mutant nucleotide sequence of 203 obtained amino acid is as shown in SEQ ID NO.2.By amino acid
Mutant nucleotide sequence through DNAWorks reverse translation at nucleotide sequence, nucleotide sequence is as shown in SEQ ID NO.4.
1.1.2 the optimization and synthesis of cat ω 2 type interferon gene and mutant gene sequence
Utilize OptimumGeneTMTechnology optimizes 2 type interferon gene of cat ω and mutated gene, according to biological anti-
It answers the codon preference of device silkworm to be transformed gene order, is rolled over to genetic transcription efficiency, translation efficiency and albumen is influenced
Folded G/C content, CpG dinucleotides content, codon preference, the secondary structure of mRNA, the free stabilizability of mRNA, RNA are not
A variety of relevant parameters such as stability motif, repetitive sequence optimize, and are conducive to improve institute's optimization gene in silkworm
Transfer efficient record and translation efficiency, and keep the protein sequence finally translated into constant.
In order to improve the translation initiation efficiency in silkworm baculovirus eukaryotic expression system, add before gene
Kozak sequence AAC, in order to improve translation termination efficiency, terminator codon is changed to TAA.In addition, also removing inside gene order
BamH I, EcoR I, the restriction enzyme sites such as Sma I, added BamH I in upstream region of gene, added XbaI in downstream of gene
Restriction enzyme site is cloned into eukaryon transfer vector pVL1393 so as to subsequent.Gene order is artificial by biotechnology company
Synthesis, 2 interferon of cat ω are mutated optimization gene sequence, i.e. for the gene order of ω 2-M-O as shown in SEQ ID NO.5,2 type of ω is dry
The sequence after plain gene optimizes is disturbed, i.e., the gene order of ω 2-O is as shown in SEQ ID NO.13, and is inserted into pUC57 respectively
Carrier forms plasmid pUC57-FeIFN- ω, is named as pUCS-FeIFN- ω 2-O and pUCS-FeIFN- ω 2-M-O.
1.2 building recombinant baculovirus transfer vectors
Plasmid pUC57-FeIFN- ω 2-O and pUC57-FeIFN- the ω 2-M-O synthesized with BamHI and XbaI double digestion,
The target fragment for being recycled glass milk method with T4DNA ligase and the inactivation baculoviral turn by BamHI and XbaI double digestion
The pVL1393 connection of transfer body, 16 DEG C of reactions 8 hours or more.Connection product converts competent escherichia coli cell TOP10, selects
Bacterium colony culture, upgrading grain identify positive colony with BamHI and XbaI double digestion, will identify that correct recombinant plasmid send Beijing to hold up
The sequencing of Bioisystech Co., Ltd of section, is sequenced correct plasmid and is named as pVL-FeIFN- ω 2-O and pVL-FeIFN- ω 2-M-
O。
The cotransfection of 1.3 recombinant plasmids and parental virus
Method according to the literature carries out recovery, passage and the screening of recombinant virus of BmN cell.When BmN cell
When culture is to cell monolayer up to 80% or so, old culture medium is outwelled, is washed three times with serum-free TC-100 culture medium, then plus serum-free
Culture medium.Recombinant plasmid pVL-FeIFN- ω 2-O and pVL-FeIFN- ω 2-M-O and parental virus Bm-Bacmid DNA is used
The BmN cell that cotransfection has been recovered after liposome embedded 20min, carries out intracellular homologous recombination.27 DEG C of culture 5d or so, until thin
Born of the same parents, which fall off, to be floated, collect cell culture fluid, obtain containing target gene recombinant virus Bm-Bacmid (FeIFN- ω 2-O,
FeIFN- ω 2-M-O), and the purifying and amplification that carry out virus are as seed virus.
1.4 cat ω type interferon are expressed in silkworm body
Recombinant virus culture solution is pressed 105PFU/ dosage injects silkworm from 5 ages, 27 DEG C, it is 70%~80% relatively wet
It is raised under conditions of degree, silkworm larva grows advanced stage, Bm-Bacmid (FeIFN- ω 2-O) and Bm-Bacmid (FeIFN- ω 2-
M-O) high efficient expression is obtained under the action of polyhedrosis gene promoter.Inoculation 3.5d~4d or so, it can be observed that family
The symptoms such as the swelling of silkworm larva body segment, abnormal behavior, loss of appetite collect blood strangury when observing that larva volume is obviously reduced
Bar, -20 DEG C save backup.
The detection of 1.5 cat ω type interferon protein antiviral activities
Method is inhibited to detect cat ω type interferon in silkworm hemolymph in CRFK/VSV*GFP system using few cells lesion
Antiviral activity.Operating method is the same as embodiment 1.
1.6 Western blotting detection
After the silkworm hemolymph of recombinant virus infection is diluted 10 times of ultrasonic disruptions with PBS (pH 7.4), SDS- is carried out
PAGE gel electrophoresis, concentration glue are 5%, resolving gel concentration 15%, then with half-dried robin, by protein delivery to gathering inclined difluoro second
On alkene (PVDF) film, 3%BSA closing is prepared with PBST, the serum after the 2 protein immunization mouse of His-FeIFN- ω of prokaryotic expression
For primary antibody (1:1000 dilution), the goat anti-mouse IgG of HRP label is secondary antibody (1:5000 dilution), finally uses DAB (diamino
Benzidine) colour developing, after terminated with deionized water, testing result.
2 experimental results
The identification of 2.1 recombinant transfer vectors
Recombinant transfer vector pVL-FeIFN- ω 2-O and pVL-FeIFN- ω 2-M-O reflects through BamHI and XbaI double digestion
Fixed, isolating small fragment through 1% agarose gel electrophoresis is all 591bp, and large fragment is in 9000bp or more, with pVL1393
(9607bp) size is consistent.Electrophoresis result is as shown in Figure 4.Digestion identifies that correct plasmid carries out nucleic acid sequencing, uses MegaAlign
Comparison result shows that sequence is consistent with the sequence originally designed, shows 2 type interferon gene of ω and the mutation optimization of the optimization of cat
Gene has been successfully plugged into pVL1393 transfer vector between BamHI and XbaI.
The building and acquisition of 2.2 recombinant Bombyx mori baculovirus rBmBacmid (FeIFN- ω 2-O, FeIFN- ω 2-M-O)
Inoculation about 1 × 106Cell is in 15cm2In culture bottle, after cell is adherent, removes and cultivated containing fetal calf serum (FBS)
Base is washed three times with the culture medium without FBS, adds 1.5ml without FBS culture medium.It is rod-shaped that 1 μ g silkworm is sequentially added into a sterile tube
Viral parent plant BmBacmid DNA, 2 μ g recombinant transfer plasmid pVL-FeIFN- ω 2-O, pVL-FeIFN- ω 2-M-O and 5 μ l
Liposome is supplied volume to 60 μ l with aseptic double-distilled water, is mixed gently, and after standing 15min, is added dropwise in culture bottle and carries out
Cotransfection.1.5ml serum free medium and 300 μ l FBS are added after 27 DEG C of culture 4h.27 DEG C constant temperature incubation 4~5 days, in collection
Clear liquid is used for the screening of recombinant virus rBmBacmid (FeIFN- ω 2-O, FeIFN- ω 2-M-O).It is inoculated with appropriate cell (about 70
~80%) in the small plate of 35mm, after cell is adherent, culture medium is sucked, cotransfection supernatant is subjected to various concentration dilution, is taken
1ml corotation dye liquor is added in attached cell, is evenly distributed.After 27 DEG C of infection 1h, infection liquid is sucked, by 2% low fusion agarose
Gel melts in 60 DEG C of water-baths, is cooled to 40 DEG C and is uniformly mixed with 40 DEG C of 2 × TC-100 culture mediums preheated (containing 20%FBS),
Every plate adds 4ml glue, is sealed after to be solidified with Parafilm, and 3~5d, micro- sem observation are cultivated in 27 DEG C of inversions.It will not contain more
The plaque of angle body is picked out, and above step is repeated, and obtains pure recombinant Bombyx mori baculovirus by the purifying of 2~3 wheels
rBmBacmid(FeIFN-ω2-O、FeIFN-ω2-M-O)。
Amplification of the 2.3 recombinant virus rBmBacmid (FeIFN- ω 2-O, FeIFN- ω 2-M-O) in bombyx mori cell
By recombinant Bombyx mori baculovirus rBmBacmid (FeIFN- ω 2-O, FeIFN- ω 2-M-O) infection normal growth
BmN cell, culture collected supernatant after 3 days, in supernatant i.e. containing a large amount of recombinant virus rBmBacmid (FeIFN- ω 2-O,
FeIFN-ω2-M-O)。
2.4 Western blotting testing results
Western blotting result (Fig. 5) shows after recombinant virus infection in the supernatant of silkworm hemolymph sample
The specific band of 19kDa size can be detected.
2.5 expression product Assay of Antiviral Activity
Using cytopathic-effect inhibition assay, the antiviral activity of expression product is measured in Vero-VSV*GFP system (see figure
6), operating method and step such as embodiment 1.Protective effect according to cat ω type interferon to CRFK cell, observes the disease of cell
Change degree, green cells appearance to be had, this hole cell are just denoted as "+", calculate interferon according to Reed-Muench method
Potency, the results are shown in Table 3.The result shows that 2 gene of feline interferon ω and mutated gene of optimization were expressed in silkworm larva body
FeIFN- ω 2-O and FeIFN- ω 2-M-O has more significant antiviral activity, and potency respectively reaches 6.5 × 105U/mL and
9.2×105U/mL, and the antiviral activity of FeIFN- ω 2 only has 4.7 × 105U/mL can make resisting for FeIFN- ω 2 by being mutated
Virus activity improves 45% or more；The antiviral activity of FeIFN- ω 2 can be made to improve 38% or more by optimizing；By to mutation
Gene optimization can make the antiviral activity of FeIFN- ω 2-M improve 35% or more；It can be made by mutation and codon optimization
The antiviral activity of FeIFN- ω 2 improves 96% or more, illustrates that the method for being mutated and optimizing is effective, practical, to raising FeIFN-
The antiviral activity of ω 2 has biggish effect.
The testing result of the recombination cat omega interferon antiviral activity of table 3
4 feline interferon ω of embodiment, 2 gene multisite mutation and expression and detection in silkworm biological reactor
It is based on embodiment 1,2 and 3 as a result, optimization can make the raising 20-40% of cat omega interferon antiviral activity, and dash forward
Change can make its antiviral activity improve 40% or more, and mutation and optimization can make antiviral activity improve 90%, thus the present embodiment
2 type interferon gene of cat ω is subjected to multisite mutation, the specific method is as follows:
1, experimental method
The building of 1.1 cat ω, 2 type interferon mutated gene and the optimization and conjunction of 2 interferon gene of ω and mutant gene sequence
At
1.1.1 the building of 2 type interferon multimutation gene of cat ω
The present invention is based on embodiments 1 to the gene order of 13 hypotypes of cat omega interferon and the comparison knot of amino acid sequence
Fruit, in order to improve expression quantity of the cat omega interferon in baculovirus expression system, the present invention by 2 type amino acid sequence of ω with
The identical variation site ω 11 carries out artificial correction, and the mutational site for using for reference ω 3, ω 4 and ω 13 introduces 10 amino acid
Rite-directed mutagenesis: A12V, P27L, P62L, A78T, R82Q, P103L, P105L, P142L, V148A, G201A, 203 obtained ammonia
The multisite mutation sequence of base acid is as shown in SEQ ID NO.10.The multisite mutation sequence of amino acid is turned over through DNAWorks is inverse
It is translated into nucleotide sequence, nucleotide sequence is as shown in SEQ ID NO.11.
1.1.2 the optimization and synthesis of 2 type interferon multimutation gene order of cat ω
Utilize OptimumGeneTMTechnology optimizes 2 type interferon multimutation gene of cat ω, according to bioreactor
The codon preference of silkworm is transformed gene order, to influence genetic transcription efficiency, translation efficiency and protein folding
G/C content, CpG dinucleotides content, codon preference, the secondary structure of mRNA, the free stabilizability of mRNA, RNA are unstable
Property a variety of relevant parameters such as motif, repetitive sequence optimize, be conducive to improve institute's optimization gene and turn effect in silkworm
Rate record and translation efficiency, and keep the protein sequence finally translated into constant.
In order to improve the translation initiation efficiency in silkworm baculovirus eukaryotic expression system, add before gene
Kozak sequence AAC, in order to improve translation termination efficiency, terminator codon is changed to TAA.In addition, also removing inside gene order
BamH I, EcoR I, the restriction enzyme sites such as Sma I, added BamH I in upstream region of gene, added XbaI in downstream of gene
Restriction enzyme site is cloned into eukaryon transfer vector pVL1393 so as to subsequent.Gene order is by artificial synthesized, 2 base of cat ω
Nucleosides because the nucleotide sequence of multisite mutation is as shown in SEQ ID NO.11, after 2 gene multisite mutation of cat ω and optimization
Acid sequence is inserted into pUC57 carrier as shown in SEQ ID NO.12, forms plasmid pUC57-FeIFN- ω, is named as pUCS-
FeIFN- ω 2-MM and pUCS-FeIFN- ω 2-MM-O.
1.2 building recombinant baculovirus transfer vectors
The plasmid pUC57-FeIFN- ω 2-MM and pUC57-FeIFN- ω 2-MM- synthesized with BamHI and XbaI double digestion
O, the target fragment for being recycled glass milk method with T4DNA ligase and the baculoviral by the inactivation of BamHI and XbaI double digestion
Transfer vector pVL1393 connection, 16 DEG C of reactions 8 hours or more.Connection product converts competent escherichia coli cell TOP10, chooses
Bacterium colony culture is selected, upgrading grain identifies positive colony with BamHI and XbaI double digestion, will identify that correct recombinant plasmid send Beijing
The sequencing of Qing Ke Bioisystech Co., Ltd, is sequenced correct plasmid and is named as pVL-FeIFN- ω 2-MM and pVL-FeIFN- ω 2-
MM-O。
The cotransfection of 1.3 recombinant plasmids and parental virus
Method according to the literature carries out recovery, passage and the screening of recombinant virus of BmN cell.When BmN cell
When culture is to cell monolayer up to 80% or so, old culture medium is outwelled, is washed three times with serum-free TC-100 culture medium, then plus serum-free
Culture medium.By recombinant plasmid pVL-FeIFN- ω 2-MM and pVL-FeIFN- ω 2-MM-O and parental virus Bm-Bacmid DNA
The BmN cell recovered with cotransfection after liposome embedded 20min, carries out intracellular homologous recombination.27 DEG C of culture 5d or so, until
Cell detachment floats, collect cell culture fluid, obtain containing target gene recombinant virus Bm-Bacmid (FeIFN- ω 2-MM,
FeIFN- ω 2-MM-O), and the purifying and amplification that carry out virus are as seed virus.
1.4 cat ω type interferon are expressed in silkworm body
Recombinant virus culture solution is pressed 105PFU/ dosage injects silkworm from 5 ages, 27 DEG C, it is 70%~80% relatively wet
It is raised under conditions of degree, silkworm larva grows advanced stage, and FeIFN- ω 2-MM and FeIFN- ω 2-MM-O starts in polyhedrosis gene
High efficient expression is obtained under the action of son.Inoculation 3.5d~4d or so, it can be observed that the swelling of silkworm larva body segment, behavior are different
Often, the symptoms such as loss of appetite collect hemolymph, -20 DEG C save backup when observing that larva volume is obviously reduced.
The detection of 1.5 cat ω type interferon protein antiviral activities
Method is inhibited to detect cat ω type interferon in silkworm hemolymph in CRFK/VSV*GFP system using few cells lesion
Antiviral activity.Operating method is the same as embodiment 1.
1.6 Western blotting detection
After the silkworm hemolymph of recombinant virus infection is diluted 10 times of ultrasonic disruptions with PBS (pH 7.4), SDS- is carried out
PAGE gel electrophoresis, concentration glue are 5%, resolving gel concentration 15%, then with half-dried robin, by protein delivery to gathering inclined difluoro second
On alkene (PVDF) film, 3%BSA closing is prepared with PBST, the serum after the 2 protein immunization mouse of His-FeIFN- ω of prokaryotic expression
For primary antibody (1:1000 dilution), the goat anti-mouse IgG of HRP label is secondary antibody (1:5000 dilution), finally uses DAB (diamino
Benzidine) colour developing, after terminated with deionized water, testing result.
2, experimental result
The identification of 2.1 recombinant transfer vectors
Recombinant transfer vector pVL-FeIFN- ω 2-MM and pVL-FeIFN- ω 2-MM-O reflects through BamHI and XbaI double digestion
Fixed, isolating small fragment through 1% agarose gel electrophoresis is all 591bp, and large fragment is in 9000bp or more, with pVL1393
(9607bp) size is consistent.Digestion identifies that correct plasmid carries out nucleic acid sequencing, with MegaAlign comparison result show sequence with
The sequence originally designed is consistent, shows that the ω 2-MM type interferon gene of cat and optimization gene have been successfully plugged into pVL1393 and have turned
In transfer body between BamHI and XbaI.
The building and acquisition of 2.2 recombinant Bombyx mori baculovirus rBmBacmid (FeIFN- ω 2-MM, FeIFN- ω 2-MM-O)
Inoculation about 1 × 106Cell is in 15cm2In culture bottle, after cell is adherent, removes and cultivated containing fetal calf serum (FBS)
Base is washed three times with the culture medium without FBS, adds 1.5ml without FBS culture medium.It is rod-shaped that 1 μ g silkworm is sequentially added into a sterile tube
Viral parent plant BmBacmid DNA, 2 μ g recombinant transfer plasmid PVLFeIFN- ω 2-MM, PVLFeIFN- ω 2-MM-O and 5 μ l
Liposome is supplied volume to 60 μ l with aseptic double-distilled water, is mixed gently, and after standing 15min, is added dropwise in culture bottle and carries out
Cotransfection.1.5ml serum free medium and 300 μ l FBS are added after 27 DEG C of culture 4h.27 DEG C constant temperature incubation 4~5 days, in collection
Clear liquid is used for the screening of recombinant virus rBmBacmid (FeIFN- ω 2-MM, FeIFN- ω 2-MM-O).It is inoculated with appropriate cell (about
70~80%) in the small plate of 35mm, after cell is adherent, culture medium is sucked, cotransfection supernatant is subjected to various concentration dilution, is taken
1ml corotation dye liquor is added in attached cell, is evenly distributed.After 27 DEG C of infection 1h, infection liquid is sucked, by 2% low fusion agarose
Gel melts in 60 DEG C of water-baths, is cooled to 40 DEG C and is uniformly mixed with 40 DEG C of 2 × TC-100 culture mediums preheated (containing 20%FBS),
Every plate adds 4ml glue, is sealed after to be solidified with Parafilm, and 3~5d, micro- sem observation are cultivated in 27 DEG C of inversions.It will not contain more
The plaque of angle body is picked out, and above step is repeated, and obtains pure recombinant Bombyx mori baculovirus by the purifying of 2~3 wheels
rBmBacmid(FeIFN-ω2-MM、FeIFN-ω2-MM-O)。
Amplification of the 2.3 recombinant virus rBmBacmid (FeIFN- ω 2-MM, FeIFN- ω 2-MM-O) in bombyx mori cell
Recombinant Bombyx mori baculovirus rBmBacmid (FeIFN- ω 2-MM, FeIFN- ω 2-MM-O) is infected into normal growth
BmN cell, culture collects supernatant after 3 days, a large amount of recombinant virus rBmBacmid (FeIFN- ω 2- contained in supernatant
MM、FeIFN-ω2-MM-O)。
2.4 Western blotting testing results
Western blotting the result shows that, after recombinant virus infection in the supernatant of silkworm hemolymph sample can detect
To the specific band of 19kDa size.
2.5 expression product Assay of Antiviral Activity
Using cytopathic-effect inhibition assay, the antiviral activity of expression product, operation are measured in Vero-VSV*GFP system
Method and steps such as embodiment 1, the protective effect according to cat ω type interferon to CRFK cell, observes the lesion degree of cell,
Green cells appearance to be had, this hole cell are just denoted as "+", and the potency of interferon is calculated according to Reed-Muench method,
It the results are shown in Table 4.The result shows that the FeIFN- ω 2- that 2 gene of feline interferon ω and mutated gene of optimization are expressed in silkworm larva body
MM and FeIFN- ω 2-MM-O has more apparent antiviral activity, and potency respectively reaches 6.3 × 105U/mL and 8.2 × 105U/
ML, but the no FeIFN- ω 2-M's of raising of antiviral activity is more, illustrates that the mutation of multidigit point can not be greatly improved
The antiviral activity of FeIFN- ω 2 only selects suitable site mutation that could improve the antiviral activity of FeIFN- ω 2.
The testing result of the recombination cat omega interferon antiviral activity of table 4
Claims (5)
1. encoding the gene of 2 interferon mutant of cat ω, which is characterized in that its nucleotides sequence is classified as shown in SEQ ID NO.5.
2. the expression vector containing gene described in claim 1.
3. a kind of method for preparing 2 interferon mutant of cat ω, which comprises the following steps:
(1) by gene cloning described in claim 1 into baculovirus transfer vector, building obtains recombinant transfer vector；
(2) by recombinant transfer vector and baculovirus DNA cotransfection insect cell, recombinant baculovirus is obtained；
(3) by recombinate shape virus infection insect cell or insect host, infected insect cell or insect host table are cultivated
Up to corresponding albumen, purifying to get.
4. according to the method for claim 3, it is characterised in that: the baculovirus transfer vector is selected from AcRP23-
lacZ、AcRP6-SC、AcUWl-lacZ、BacPAK6、Bac to Pac、Bacmid、BlucBacII(pETL)、p2Bac、
p2Blue、p89B310、pAc360、pAc373、pAcAB3、pAcAB 4、PAcAS3、pAcC129、pAcC4、DZI、pAcGP67、
pAcIEl、pAcJPl、pAcMLF2、pAcMLF 7、pAcMLF 8、pAcMPl、pAcMP2、pAcRP23、pAcRP 25、
pAcRW4、pAcsMAG、pAcUWl、pAcUW21、pAcUW2A、pAcUW2B、pAcUW3、pAcUW31、pAcUW41、pAcUW42、
pAcUW43、pAcUW51、pAcVC2、pAcVC 3、pAcYMl、pAcJcC5、pBacl、pBac2、pBlueBacIII、
pBlueBacHis、pEV55、mXIV、pIEINeo、pJVETL、pJVNhel、pJVP10、pJVrsMAG、pMBac、pP10、
pPAKl、pPBac、pSHONEX 1.1、pSYN XIV VI+、pSYNVI+wp、pSYNXIV VI-、pVL1391、pVL 1392、
PVL 1393, pVL941, pVL 945, pVL 985, pVTBac, pBM030 or pUAC-5；
The baculoviral be selected from silkworm baculovirus parent plant BmBacmid, BmNPV, AcMNPV, ApNPV, HaNPV,
HzNPV, LdMNPV, MbMNPV, OpMNPV, SlMNPV, SeMNPV or SpltNPV；
The insect host is selected from silkworm (Bombyx mori), wild silkworm (Bombyx mandarina), castor silkworm
(Philosamia cynthia ricim), wild silkworm (Dictyoploca japanica), Philosamia cynthia (Philosamia cynthia
Pryeri), tussah (Antheraea pernyi), yamama (Antheraea yamamai), wild giant silkworm (Antheraea
Polyphymus), autographa california (Atographa califorica), tea geometrid (Ectropis obliqua), cabbage army worm
(Mamestra brassicae), prodenia litura (Spodoptera littoralis), autumn armyworm (Spodoptera
Frugiperda), cabbage looper (Trichoplusia ni), armyworm (Thaumetopoea wilkinsoni), bollworm
(Heliothis armigera), U.S. bollworm (Heliothis zea), oriental tobacco budworm (Heliothis assulta), tobacco
Noctuid (Heliothis virescens), oriental armyworm (Pseudaletia separata) or gypsymoth (Lymantria
dispar)；
It is described to infect the insect larvae or pupal cell for referring to recombinant baculovirus by eating or infecting through epidermis 1-5 age.
5. according to the method for claim 4, it is characterised in that: the baculovirus transfer vector is pVL1393；It is described
Baculoviral is silkworm baculovirus parent plant BmBacmid；The insect host is silkworm (Bombyx mori).
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| CN108329390B (en) * | 2018-01-29 | 2020-08-21 | 中国农业科学院生物技术研究所 | Chicken lambda 3 interferon, mutant thereof, expression method and application thereof |
| CN108707194B (en) * | 2018-04-12 | 2020-09-29 | 中国农业科学院生物技术研究所 | Pig omega 7 interferon mutant and preparation method and application thereof |
| CN108484750B (en) * | 2018-04-28 | 2020-08-14 | 中国农业科学院生物技术研究所 | Pig lambda 3 interferon mutant and preparation method and application thereof |
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| CN108949787A (en) * | 2018-07-05 | 2018-12-07 | 上海海洋大学 | A kind of goldfish Tgf2 transposase and its preparation and store method |
| CN109206502B (en) * | 2018-09-26 | 2022-03-04 | 东北农业大学 | Cat interferon omega, preparation method thereof and application thereof in antivirus |
| CN109206501B (en) * | 2018-09-26 | 2022-03-04 | 东北农业大学 | Cat interferon omega, coding gene thereof and application of cat interferon omega in antiviral aspect |
| CN110257396A (en) * | 2019-05-13 | 2019-09-20 | 华南农业大学 | A kind of anti-ALV-J chicken Lambda interferon and the preparation method and application thereof |
| CN110256552B (en) * | 2019-06-11 | 2021-02-19 | 中国农业科学院生物技术研究所 | Duck lambda interferon mutant and preparation method and application thereof |
| CN113355286A (en) * | 2020-03-02 | 2021-09-07 | 成都彤琦恩生物科技有限公司 | Mammalian cell culture process for efficiently expressing recombinant feline interferon omega 2 |
| CN113462648A (en) * | 2020-03-31 | 2021-10-01 | 成都彤琦恩生物科技有限公司 | Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant |
| CN112500472B (en) * | 2020-12-04 | 2022-09-23 | 广州源博医药科技有限公司 | Cat omega interferon mutant and preparation method and application thereof |
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