CN104894139A - Modified dog alpha1-interferon gene, construction method of expression vector and preparation method of protein encoded by modified dog alpha1-interferon gene - Google Patents

Abstract

The invention discloses a modified dog alpha1-interferon gene and a preparation method of dog alpha1-interferon. The modified dog alpha1-interferon gene has the nucleotide sequence as shown in SEQ ID NO.1 and the amino acid sequence coded by the gene is shown in SEQ ID NO.2. The invention concretely relates to matching and modification for the predilection of a dog alpha1-interferon codon to a yeast codon as well as optimal control on a culture method for efficiently producing dog alpha1-interferon, pollution avoiding measures and operation methods of methanol adding amount and time determination, protein expression time control, namely protein harvest time, sterility test, protein content and activity measurement, culture condition expansion and the like. The method disclosed by the invention is simple and easy to realize, relatively low in cost, and capable of realizing stable and efficient expression of the dog alpha1-interferon in pichia pastoris, and the expressed dog alpha1-interferon has a remarkable effect on treating dog viral infectious diseases and can enhance the immune effect of a rabies vaccine.

Description

A kind of improved dog α 1-interferon gene, the structure of expression vector and the preparation method of albumen thereof

Technical field

The invention belongs to biological pharmacy technical field.More specifically, a kind of improved dog α 1-interferon gene, the structure of expression vector and the preparation method of albumen thereof is related to.

Background technology

Interferon, rabbit (IFN) is under specific inductor effect, a kind of glycoprotein with broad anti-viral activity produced by cell, and when it acts on other cells again, that it can be made to obtain immediately is antiviral, many-sided immunizing power such as antitumor.A large amount of in vitro and in vivo test shows, dog α 1-Interferon, rabbit all has defence and restraining effect to the viral disease of tool significant threat in production.Interferon, rabbit, with features such as its effect are extensive, residual little, has more wide application prospect.But because Interferon, rabbit cost value is high and the problem of animal economic worth, be also mainly limited to the treatment of pet disease in the application of veterinary clinic, at the treatment primary limitation of industry animal in experimental stage, and mainly assisting therapy virus disease and parasitosis.Therefore, the high expression of dog α 1-Interferon, rabbit and the reduction of cost significant.

At present, utilize the work of yeast and escherichia coli expression Interferon, rabbit to achieve some achievements, such as number of patent application 200310109820.6 disclose a kind of containing intestinal bacteria partially addicted to the porcine alpha-interferon of codon.The patent application 200810027952.7 in earlier stage of the present inventor seminar discloses a kind of modified pig alpha-interferon genes, the structure of expression vector and the preparation method of albumen thereof, and the preparation for porcine alpha-interferon achieves good achievement.

Although, the work of yeast expression Interferon, rabbit is utilized to achieve some achievements, but for the homologous gene (sequence of gene and character etc. have difference) in different genes, different plant species source, how modified interferon gene, it is caused to mate with the inclined preferendum of yeast, to improve the expression amount of Interferon, rabbit; How to carry out in expression process cultivating, how to ensure to obtain the dissolved oxygen that protein expression needs, the amount how avoiding polluting, adding methanol induction and time, method, protein content and activity to the control of protein expression time and albumen harvest time, steriling test mensuration etc. all have larger difference, there is no that formation one is stable, complete technical scheme, be unfavorable for the suitability for industrialized production developing dog α 1-Interferon, rabbit, fundamentally can not solve the Cost Problems of dog α 1-Interferon, rabbit, affect it and apply.

In order to improve the expression level of dog α 1-Interferon, rabbit in pichia spp, reduce production cost, form commercial production conditions, be necessary to explore a kind of method of carrying out modifying, determine decorating site, modify quantity for dog α 1-interferon gene, be necessary to improve and optimal design-aside the key condition of its abduction delivering.

Summary of the invention

The technical problem to be solved in the present invention is the defect and the deficiency that overcome existing dog α 1-Interferon, rabbit technology of preparing, a kind of modified dog α 1-interferon gene that can mate with pichia spp is provided, set up a kind ofly can to stablize in pichia spp, the technological method of high expression dog α 1-Interferon, rabbit, for dog α 1-Interferon, rabbit is produced in commercial running, enhance productivity, reduce production cost and establish technical foundation.

In view of the degenerate of codon and yeast are to the inclined preferendum of amino acid codes, the 14 kind crux codons of the present invention to dog α 1-interferon gene are modified, the inclined preferendum of yeast to amino acid codes can be met, but not do not change the amino acid of interferon protein and amino acid whosely to put in order, thus greatly increasing the expression water of this gene in pichia spp.The present invention is optimized the expression condition of dog α 1-to Interferon, rabbit, comprising the control of dissolved oxygen amount, the measure of decreasing pollution, the interpolation time of methyl alcohol and addition, the temperature of abduction delivering and the time of results albumen, the measuring method of protein content, the mensuration enlarged culturing condition etc. of protein-active.

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

A dog α 1-interferon gene for modified transformation, its nucleotide sequence is as shown in SEQ ID NO.1, and aminoacid sequence is as shown in SEQ ID NO.2.

Particularly, the dog α 1-interferon gene of above-mentioned modified transformation is that in the nucleotide sequence to dog α 1-interferon gene, 14 crucial codons obtain after having carried out modifying, and described 14 crucial codons are the codon that Cys, Pro, Leu, Thr, Gly, Val, Gln, Ala, Ser, Asn, Phe, Asp, Lys and Arg are corresponding.

More specifically, described 14 crucial codons are the codon UGU for Cys, the codon CCG of Pro, the codon GGU of the codon ACG of the codon CUC of Leu, Thr, Gly, the codon GCC of the codon CAG of the codon GUG of Val, Gln, Ala, the codon AGC of Ser, the codon GAC of the codon UUC of the codon AAU of Asn, Phe, Asp, the codon CGU of the codon AAA of Lys, Arg.

A kind of dog α 1-interferon expression carrier, it builds the expression vector pPICZ α C employing secretor type, described expression vector contains EcoR I and Xba I restriction enzyme site, a strong promoter AOX1 promotor, a Zeocin resistance selects site, and strong secretion signal alpha factor signal peptide is nucleotide sequence as shown in SEQ ID NO.1.

Wherein, described alpha factor signal peptide comprises 83 amino acid whose leading peptides and spacer peptide, described spacer peptide is made up of lys-Arg-Glu-Ala-Glu-Ala aminoacid sequence, lys-ArgC holds by the identification of embrane-associated protein enzyme KEX2 gene product, by the identification of STE13 gene product outside-Glu-Ala-.

A preparation method for the protein (i.e. dog α 1-Interferon, rabbit) of the dog α 1-interferon gene coding of above-mentioned modified transformation, comprises the following steps:

S1. according to the nucleotide sequence design and synthesis pair of primers of dog α 1-Interferon, rabbit, synthesized dog alpha 1-interferon gene; Described primer sequence is as follows:

Upstream primer P1:5'-CCGGAATTCCATGTGTCA-3';

Downstream primer P2:5'-GCTCTAGATTACTTTCTTCTTCTGA-3';

Upstream primer P1 adds at its 3' end ecoRi restriction endonuclease sites, downstream primer P2 adds at its 5' end xbai restriction endonuclease sites; And ecoRthe upstream of I restriction enzyme site increases a bases G;

After the order-checking of dog α 1-interferon gene is correct, modification transformation is carried out to 14 crucial codons in the nucleotide sequence of dog α 1-interferon gene, transformation site mainly: be the codon UGU of Cys, the codon CCG of Pro, the codon CUC of Leu, the codon GUG of the codon GGU of the codon ACG of Thr, Gly, Val, the codon CAG of Gln, the codon AAU of the codon AGC of the codon GCC of Ala, Ser, Asn, the codon UUC of Phe, the codon CGU of the codon AAA of the codon GAC of Asp, Lys, Arg;

The present invention removes the dog α 1-Interferon, rabbit codon after signal peptide with reference to preference of the yeast codon transformation, first guarantee that the amino acid of proteins encoded can not change, select the codon of yeast expression system preference on this basis, former rare codon is replaced, consider A+T content and G+C content simultaneously, both even cause protein expression at the too high or too low protein translation efficiency that can reduce, and the sequence of premature termination can not be there is, consider each side factor and reasonably modification is successfully made to protogene;

S2. modified dog α 1-interferon gene is cloned in Yeast expression carrier pPICZ α C, uses Zeocin+YPDS plate screening, activate and cultivate, express through methanol induction, and carry out steriling test;

S3. gather in the crops albumen, in results process, carry out albumen steriling test;

S4. content and the activity of albumen is measured.

Wherein, step S2 comprises the following steps:

S21. single bacterium colony with Zeocin resistance of YPDS grow on plates is chosen with sterilizing toothpick, choose and carry out activation cultivation in the BMGY liquid nutrient medium of 1.5 ml, culture temperature is 28 ~ 30 DEG C, 200 turns/min shaken overnight, to OD600=2 ~ 6, cell is in logarithmic phase;

S22. by cell centrifugal 5 minutes collecting precipitations under 3000 turns/min and room temperature condition that step S21 obtains, be resuspended in the BMMY substratum of 1.5 ml, control dissolved oxygen amount and preventing pollution, cultivate at the test tube relaying persistent oscillation of 15 ml; Described control dissolved oxygen amount and preventing pollution are with four layers of additional two-layer newspaper wrapping BMMY of clean gauze, adopt and carry out alcohol spraying disinfection with ∕ or the volume that controls substratum account for 5 ~ 20% of culture vessel volume;

S23. adding 100% methyl alcohol to final concentration at interval of 24 h is 1%, and add-on is that every milliliter of BMMY substratum adds 10 microlitres, carries out inducing culture.

Steriling test described in step S2 is in protein expression process, observes the color of bacterium liquid, ensures that bacterium liquid is oyster white; Or in culturing process, get bacterium liquid carry out microscopy, observe saccharomycetic form, described microscopy is direct smear microscopy or uses azaleine dyeing microscopic examination.

Gather in the crops described in step S3 albumen be after cultivation 96 ~ 120h under room temperature 1500 ~ 3000 revs/min centrifugal 5 minutes collect supernatants, abandon thalline; Supernatant of getting is made SDS-PAGE immediately and is analyzed or be placed in-70 DEG C of preservations; Steriling test described in step S3 adopts to be heard yeast-leavened taste and observes bacterium liquid color; Or utilize SDS-PAGE to detect.

The protein of being encoded by the dog α 1-interferon gene stating the modified transformation that preparation method prepares, i.e. dog α 1-Interferon, rabbit, also within protection scope of the present invention.

The control of the present invention to dissolved oxygen amount and the measure of decreasing pollution, in experiment expression process, under constantly groping how can keep the prerequisite of the ventilation of yeast better in the not contaminated situation of guarantee yeast, by adopting four layers of additional two-layer newspaper parcel shaking flask mouth of clean gauze or test tube mouth, both ensure that air permeability in turn ensure that yeast is not contaminated.Also need often to carry out alcohol spraying disinfection to ensure the aseptic of external environment as far as possible in shaking table use procedure.Meanwhile, the volume of substratum can only account for 5 ~ 20% of culture vessel, the highest can not more than 20%.

The improvement of the present invention to the method for steriling test comprises: in protein expression process, and observe the color of bacterium liquid, oyster white is the normal color of yeast; In culturing process, get bacterium liquid carry out microscopy, observe saccharomycetic form, can direct smear microscopy, also can use azaleine dyeing microscopic examination.Under the microscope, do not have the yeast dyeed to be white round thalline, the yeast of dyeing is red round thalline, does not have the thalline of other form to occur, this illustrates that the yeast cultivated does not pollute.Hear yeast-leavened taste in results process, sweet is does not pollute, and if there is very smelly taste, and bacterium liquid color is black dull, then illustrate to pollute intestinal bacteria in culturing process; Detect with SDS-PAGE, interferon protein only has the band of 1 about 20KD, also to there will be other protein band a lot of if polluted except object band.

The present invention is add a methyl alcohol at interval of 24h to the interpolation time of methyl alcohol and the control of addition, and adding 100% methyl alcohol to final concentration is 1%, and namely every milliliter adds 10 μ l.

The present invention is to the improvement of the temperature of abduction delivering with the time of results albumen, and be that 28 ~ 30 DEG C of concussion cultivation 96 ~ 120h just can gather in the crops albumen, the method for albumen results is that under room temperature, the centrifugal 5min of 1500 ~ 3000 turns/min collects supernatant, abandons thalline.Which reduce incubation time and reduce production cost accordingly; The present invention to the measuring method of protein content is, adopt spectrophotometer to calculate according to light absorption value, calculation formula is:

Protein concn (mg/mL)=(1.55 × A280nm-0.76 × A260nm) × extension rate.

The present invention adopts cytopathic-effect inhibition assay to measure to the mensuration of protein-active, carries out with reference to prior art.

The present invention improves enlarged culturing condition, selects suitable culture vessel, and according to the dilution proportion of 1:50 ~ 1:100, the addition of corresponding expansion methyl alcohol, improves the speed that concussion is cultivated.Concrete improvement is as follows:

Abduction delivering is carried out according to the ratio of 1:50 ~ 1:100 in the process that enlarged culturing is expressed, namely choose 5 ~ 6 bacterium colonies to activate in 55 ~ 65 ml BMGY, 30 DEG C of 200 turns/min shake cultivation 16 ~ 18 h, then directly join in BMMY and add methyl alcohol by activating the thalline cultivated and BMGY substratum and carry out abduction delivering, a small amount of glycerine can not affect the expression of albumen, and reduce once centrifugal, the probability of decreasing pollution.In order to ensure that enough dissolved oxygen amounts will control the addition of methyl alcohol, after dilution, the addition of methyl alcohol is still according to the addition of a small amount of abduction delivering, namely adds a methyl alcohol at interval of 24h, and adding 100% methyl alcohol to final concentration is 0.5 ~ 1%, and namely every milliliter adds 5 ~ 10 μ l; The selection of container in Induction Process, the triangular flask that capacity is excessive can not be used, if more than 2L, strengthen the difficulty of operation and increase the probability polluted, therefore, enlarged culturing uses the triangular flask of 1L, volume of culture is about 500 ml, and 30 DEG C of 300 turns/min shake cultivation 96 ~ 120h and gather in the crops albumen.Although yeast culture matrix amasss into container cubic capacity 50%, because the ratio of inoculation is 1:50 ~ 1:100, so still can meet the dissolved oxygen amount of expressing needs, the protein-active of results can reach 10 ⁶u/ml.

The present invention has following beneficial effect:

(1) the present invention is directed to dog α 1-interferon gene, solve and how reasonably transformation is modified to its sequence, relate generally to dog α 1-Interferon, rabbit codon to the coupling of the inclined preferendum of yeast codons, transformation, achieve the pichia spp high expression of α 1-interferon gene sequence.

(2) the invention solves the measure of avoiding polluting, the time of adding methyl alcohol and add the amount of methyl alcohol, cultural method, i.e. albumen harvest time to the control of protein expression time, High-efficient Production dog α 1-Interferon, rabbit, steriling test, protein content and the gordian technique such as determination of activity, enlarged culturing condition, thus making dog α 1-Interferon, rabbit in pichia spp, obtain the expression of stability and high efficiency, the antiviral activity of Interferon, rabbit improves 300 times.

(3) the dog α 1-Interferon, rabbit that the present invention expresses has significant therapeutic effect to canine viral transmissible disease.

(4) the inventive method is simple, and cost is lower, has possessed widely used basis aborning, has had wide market outlook.

Accompanying drawing explanation

Fig. 1 is the multiple clone site schematic diagram of expression vector in the invention process, with reference to the pichia spp operational manual (www.invitrogen.com) of Invitrogen.

Fig. 2 is the physical map of expression vector in the invention process, with reference to the pichia spp operational manual (www.invitrogen.com) of Invitrogen.

Fig. 3 is the SDS-PAGE result of dog α 1-interferon gene Pichia anomala expression in the invention process.

Fig. 4 is that embodiment of the present invention dog α 1-is to the Western-blotting analytical results of interferon gene Pichia anomala expression product.

Embodiment

Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment does not limit in any form to the present invention.Unless stated otherwise, the present invention adopts reagent, method and apparatus are the art conventional reagent, method and apparatus.

Unless stated otherwise, following examples agents useful for same and material are commercial.

embodiment 1

1, according to the nucleotide sequence design and synthesis pair of primers of dog α 1-Interferon, rabbit, synthesized dog alpha 1-interferon gene;

Reference literature of the present invention (Gurkan C, 2005; Zhao Xiang, 2000) analyze pichia pastoris phaff expression system to the inclined preferendum of codon, the complete genome sequence of (A33693) dog α 1 Interferon, rabbit announced in Genbank is carried out inclined preferendum transformation, adds respectively in upstream and downstream simultaneously ecoRi and xbai restriction enzyme site (underscore part) and protection base, and ecoRthe upstream of I restriction enzyme site increases a bases G.Modifying gene is synthesized by Shanghai biotechnology company limited full genome, and improved dog α 1-interferon gene is inserted the multiple clone site of plasmid pUC57, recombinant plasmid called after pUC57-CaIFN-α 1.According to the nucleotide sequence of dog α 1 Interferon, rabbit after transformation, design pair of primers also synthesizes:

Described primer sequence is as follows:

Upstream primer P1:5'-CCGGAATTCCATGTGTCA-3';

Downstream primer P2:5'-GCTCTAGATTACTTTCTTCTTCTGA-3'.

According to dog α 1-interferon gene sequence (A33693) display that Genbank announces, dog α 1 Interferon, rabbit full genome is 564 bp, 187 amino acid of encoding, and wherein N holds front 23 amino acid to be signal peptide, and maturation protein is containing 164 amino acid.The dog α 1-Interferon, rabbit codon after signal peptide is removed with reference to preference of the yeast codon transformation, first guarantee that the amino acid of proteins encoded can not change, select the codon of yeast expression system preference on this basis, former rare codon is replaced, consider A+T content and G+C content simultaneously, both even cause protein expression at the too high or too low protein translation efficiency that can reduce, and the sequence of premature termination can not be there is, consider each side factor and reasonably modification is successfully made to protogene, wherein relate to the transformation of Cys Pro Leu Thr Gly Val Gln Ala Ser Asn Phe Asp Lys Arg14 seed amino acid codon.

The nucleotide sequence of improved dog α 1-interferon gene is as shown in SEQ ID NO.1, and the sequence of its coded amino acid is as shown in SEQ ID NO.2.

2, improved dog α 1-interferon gene is cloned in Yeast expression carrier pPICZ α C, uses Zeocin+YPDS plate screening, activate and cultivate, express through methanol induction, and carry out steriling test;

Concrete steps are as follows:

A () chooses single bacterium colony with Zeocin resistance of YPDS grow on plates with sterilizing toothpick, choose and carry out activation cultivation in the BMGY liquid nutrient medium of 1.5 ml, 28 ~ 30 DEG C, 200 turns/min shaken overnight, to OD600=2 ~ 6, now cell is in logarithmic phase.

B () 3000 turns/min room temperature centrifugal 5 minutes collecting precipitations, are resuspended in the BMMY of 1.5 ml, continue shaking culture, wrap up, shaking culture in the test tube of 15 ml with four layers of additional two-layer newspaper of clean gauze.

C () adds 100% methyl alcohol to final concentration at interval of 24 h is 1%, carries out inducing culture.

(3) gather in the crops albumen, in results process, carry out albumen steriling test;

C () step is cultivated 96 ~ 120h and is collected sample, centrifugal, gets supernatant and does SDS-PAGE analysis immediately or be placed in-70 DEG C of preservations.

3, the SDS-PAGE result of the present embodiment dog α 1-interferon gene Pichia anomala expression as shown in Figure 3.Wherein M is pre-dyed Marker, and 1 is the recombination yeast that pPICZ α C – IFN transforms, and 2 is that pPICZ α C empty carrier is expressed.

4, content and the activity of albumen is measured

(1) glue and race glue: carry out with reference to " DNA techniques handbook " (Wang Jiazheng etc., 2000).

1) 12% separation gel is prepared, 10.01 milliliters:

30% acrylamide stock solution 4.0 mL

4 × separation gel damping fluid 2.5 mL

10% ammonium persulphate 100 μ L

TEMED 10 μL

Distilled water 3.4 mL.

In mounted glass plate, slowly add separation gel solution and be about 1.5cm to distance sheet glass top, the water layer of one deck 1.0cm is covered gently on separation gel solution, to prevent the oxygen in air to the restraining effect of gel polymerisation, after about 30min gel polymerisation completes, outwell the water layer of covering, rinse with water gel top is blotted on gel top several times afterwards as far as possible water with filter paper.

2) 4% concentrated glue is recorded, 4.008ml:

30% acrylamide stock solution 0.54 mL

4 × concentrated glue damping fluid 1.0 mL

10% ammonium persulphate 80 μ L

TEMED 8 μL

Distilled water 2.38 mL.

Slowly add concentrated sol solution to sheet glass top, quick insertion comb, avoid producing bubble, about 30 min carefully extract comb after concentrating glue polymerization.Add appropriate Tris-glycine running buffer, with electrophoresis wash buffer comb hole before loading.Assemble electrophoresis equipment, protein sample is mixed with 2 × sample buffer (10 μ L+10 μ L) in an Eppendorf pipe, carefully adds in sample well with pipettor.Switch on power, constant voltage electrophoresis: spacer gel stage 120V, separation gel stage 150V, until dye front migrates to bottom gel.

(2) dyeing and decolouring:

After electrophoresis, careful stripping running gel, dye with the coomassie brilliant blue R_250 staining fluid of at least 5 times of gel volumes, slow jolting 1 h on decolorization swinging table, take out gel rinsing several in water, again with the 1h that decolours in Xylene Brilliant Cyanine G destainer, in order to decolour completely, need therebetween to change destainer 2 ~ 3 times.After protein band is clear, observations.If only have 1 protein band at corresponding molecular weight place, illustrate that interferon protein is expressed, and do not pollute; If also have other protein band beyond object band, illustrate that interferon protein pollutes, so these albumen just can not employ, and the words of use do not have effect yet, only can increase the stimulation to carcass.

To the Western-blotting analytical results of interferon gene Pichia anomala expression product as shown in Figure 4, wherein M is pre-dyed Marker to the present embodiment dog α 1-, and 1 is the recombination yeast that pPICZ α C – IFN transforms, and 2 is the yeast that pPICZ α C transforms.

(3) mensuration of protein content

Determination of protein concentration: testing protein solution is suitably diluted, uses GeneRAY

UV-Photometer nucleic acid protein spectrophotometer is measured respectively the value at wavelength A260nm and A280nm place, according to following formulae discovery protein concentration:

Protein concn (mg/mL)=(1.55 × A280nm-0.76 × A260nm) × extension rate,

(4) active detection

Concrete steps are as follows:

1) bed board: discard the nutrient solution in Vero Tissue Culture Flask, uses collected by trypsinisation cell, is inoculated in 96 porocyte culture plates, every hole 100 μ L, and 37 DEG C, overnight incubation under 5%CO2 condition, make cell attachment be individual layer.

2) interferon solution is prepared: get and express supernatant after penicillin, paraxin room temperature treatment half an hour, be inoculated in without in any antibiotic LB nutrient solution, make steriling test.Continuous 4 times of gradient dilutions are made after the assay was approved with cell maintenance medium.

3) application of sample: get cultured Vero 96 porocyte culture plate in advance, dilution for difference Interferon, rabbit sample is moved into 96 porocyte culture plates, every hole 100 μ L.Each concentration does 6 repetitions, arranges cell controls and virus control simultaneously, does not add Interferon, rabbit.37 DEG C, overnight incubation under 5%CO2 condition.

4) virus liquid is prepared: attack toxic agent amount 100 μ L TCID50, the VSV cell maintenance medium deposited of going bail for is diluted to working concentration.

5) attack poison: discard Vero96 porocyte culture plate nutrient solution, dry, add the virus liquid of working concentration, every hole 100 μ L.Cell control well does not add virus liquid.37 DEG C, cultivate about 24 h to 36 h under 5%CO2 condition.

6) cytopathy judges: when complete cytopathy appears in virus control wells, judged result, and according to the lesion degree of cell, gives a mark by 5 grading systems, protectiveness during record different concns.

Cytopathic degree of protection divides 5 deciles into:

4: without obvious cytopathy

3: cytopathy is about 25%

2: have an appointment 50% cytopathy

1: cytopathy is about 75%

0: have 100% pathology.

7) result calculates: calculate according to Reed-Muench method.In the extent of dilution of 50% cytopathy protection, contained Interferon, rabbit amount is 1 Interferon, rabbit unit.

(5) enlarged culturing

A () is with 5 single bacterium colonies with Zeocin resistance of sterilizing toothpick tall and slender YPDS grow on plates, choose and carry out activation cultivation in the BMGY liquid nutrient medium of 5ml, 30 DEG C, 200 turns/min shaking culture 16 ~ 18 h, to OD600=2 ~ 6, now cell is in logarithmic phase.

B () aseptically, by without centrifugal 5 milliliters of BMGY liquid nutrient mediums containing thalline, directly joins in 500ml BMMY, bottleneck four layers of clean additional two-layer newspaper of gauze are wrapped up, 300 turns/min shaking culture in the triangular flask of 1L.

C () adds 100% methyl alcohol to final concentration at interval of 24h is 1%, carries out inducing culture, namely adds methyl alcohol 10 ml in 500 ml BMMY.

D () is cultured to 96h and collects albumen, be positioned over 4 DEG C of refrigerators for subsequent use.

(6) the Interferon, rabbit assay after codon modify

After electrophoresis, with thin layer chromatography scanner, interferon protein zone is scanned, be then combined with the method for measuring of Bradford Tot Prot.

Result shows, after genetic modification, the content of dog α 1 interferon fusion protein is 0.453 mg/mL, and compare with the bacterial strain expression amount 0.08mg/mL not through transforming, the protein content of Interferon, rabbit is significantly improved.

SEQUENCE LISTING

 

<110> Agricultural University Of South China

 

<120> mono-kind improved dog α 1-interferon gene, the structure of expression vector and the preparation method of albumen thereof

 

<130>

 

<160> 2

 

<170> PatentIn version 3.3

 

<210> 1

<211> 495

<212> DNA

The nucleotide sequence of <213> dog α 1 Interferon, rabbit

 

<400> 1

tgccacctgc ccgacaccca cggcctgcgc aactggaggg tcctgacgct cctgggacag 60

 

atgaggagac tctccgccgg ctcttgtgac cactacacca atgactttgc cttccccaag 120

 

gagctgtttg atggccagcg gctccaggag gcgcaggccc tctctgtggt ccacgtgatg 180

 

acccagaagg tcttccacct cttctgcccg gacacgtcct ctgctccttg gaacatgact 240

 

ctcctggagg aactgtgctc ggggctctct gagcagctgg atgacctgga ggcctgtccc 300

 

ctgcaggagg cggggctggc cgagaccccc ctcatgcatg aggactccac cctgaggacc 360

 

tacttccaaa ggatctccct ctacctgcaa gacaggaacc acagcccgtg tgcctgggag 420

 

atggtccgag cagaaatcgg gagatccttc ttctcctcga caatcttgca agaaagaatc 480

 

aggaggagga aatga 495

 

 

<210> 2

<211> 164

<212> PRT

<213> dog α 1 Interferon, rabbit aminoacid sequence

 

<400> 2

 

Cys His Leu Pro Asp Thr His Gly Leu Arg Asn Trp Arg Val Leu Thr

1 5 10 15

 

 

Leu Leu Gly Gln Met Arg Arg Leu Ser Ala Gly Ser Cys Asp His Tyr

20 25 30

 

 

Thr Asn Asp Phe Ala Phe Pro Lys Glu Leu Phe Asp Gly Gln Arg Leu

35 40 45

 

 

Gln Glu Ala Gln Ala Leu Ser Val Val His Val Met Thr Gln Lys Val

50 55 60

 

 

Phe His Leu Phe Cys Pro Asp Thr Ser Ser Ala Pro Trp Asn Met Thr

65 70 75 80

 

 

Leu Leu Glu Glu Leu Cys Ser Gly Leu Ser Glu Gln Leu Asp Asp Leu

85 90 95

 

 

Glu Ala Cys Pro Leu Gln Glu Ala Gly Leu Ala Glu Thr Pro Leu Met

100 105 110

 

 

His Glu Asp Ser Thr Leu Arg Thr Tyr Phe Gln Arg Ile Ser Leu Tyr

115 120 125

 

 

Leu Gln Asp Arg Asn His Ser Pro Cys Ala Trp Glu Met Val Arg Ala

130 135 140

 

 

Glu Ile Gly Arg Ser Phe Phe Ser Ser Thr Ile Leu Gln Glu Arg Ile

145 150 155 160

 

 

Arg Arg Arg Lys

               

Claims (10)

1. a dog α 1-interferon gene for modified transformation, is characterized in that, modify the nucleotide sequence of improved dog α 1-interferon gene as shown in SEQ ID NO.1, encoding amino acid sequence is as shown in SEQ ID NO.2.

2. the dog α 1-interferon gene of modified transformation according to claim 1, it is characterized in that, modify 14 crucial codons in the nucleotide sequence of dog α 1-interferon gene, described 14 crucial codons are the codon that Cys, Pro, Leu, Thr, Gly, Val, Gln, Ala, Ser, Asn, Phe, Asp, Lys and Arg are corresponding.

3. the dog α 1-interferon gene of modified transformation according to claim 1, it is characterized in that, 14 crucial codons in the nucleotide sequence of dog α 1-interferon gene are modified, described 14 crucial codons are the codon UGU of Cys, the codon CCG of Pro, the codon CUC of Leu, the codon ACG of Thr, the codon GGU of Gly, the codon GUG of Val, the codon CAG of Gln, the codon GCC of Ala, the codon AGC of Ser, the codon AAU of Asn, the codon UUC of Phe, the codon GAC of Asp, the codon AAA of Lys, the codon CGU of Arg.

4. a dog α 1-interferon expression carrier, it is characterized in that, it builds the expression vector pPICZ α C employing secretor type, described expression vector contains EcoR I and Xba I restriction enzyme site, a strong promoter AOX1 promotor, a Zeocin resistance selects site, and strong secretion signal alpha factor signal peptide is nucleotide sequence as shown in SEQ ID NO.1.

5. dog α 1-interferon expression carrier according to claim 3, it is characterized in that, described alpha factor signal peptide comprises 83 amino acid whose leading peptides and spacer peptide, described spacer peptide is made up of lys-Arg-Glu-Ala-Glu-Ala aminoacid sequence, lys-ArgC holds by the identification of embrane-associated protein enzyme KEX2 gene product, by the identification of STE13 gene product outside-Glu-Ala-.

6. a preparation method for the protein of the dog α 1-interferon gene coding of modified transformation described in claim 1, is characterized in that, comprise the following steps:

S1. according to the nucleotide sequence design and synthesis pair of primers of dog α 1-Interferon, rabbit, synthesized dog alpha 1-interferon gene; Described primer sequence is as follows:

Upstream primer P1:5'-CCGGAATTCCATGTGTCA-3';

Downstream primer P2:5'-GCTCTAGATTACTTTCTTCTTCTGA-3';

Add at the 3' end of upstream primer P1 ecoRi restriction endonuclease sites, adds at the 5' end of downstream primer P2 xbai restriction endonuclease sites; And ecoRthe upstream of I restriction enzyme site increases a bases G;

After the order-checking of dog α 1-interferon gene is correct, modification transformation is carried out to 14 crucial codons in the nucleotide sequence of dog α 1-interferon gene, transformation site mainly: the codon UGU of Cys, the codon CCG of Pro, the codon CUC of Leu, the codon GUG of the codon GGU of the codon ACG of Thr, Gly, Val, the codon CAG of Gln, the codon AAU of the codon AGC of the codon GCC of Ala, Ser, Asn, the codon UUC of Phe, the codon CGU of the codon AAA of the codon GAC of Asp, Lys, Arg;

S2. modified dog α 1-interferon gene is cloned in Yeast expression carrier pPICZ α C, uses Zeocin+YPDS plate screening, activate and cultivate, express through methanol induction, and carry out steriling test;

S3. gather in the crops albumen, in results process, carry out albumen steriling test;

S4. content and the activity of albumen is measured.

7. preparation method according to claim 6, is characterized in that, step S2 comprises the following steps:

S21. choose single bacterium colony with Zeocin resistance of YPDS grow on plates with sterilizing toothpick, choose and carry out activation cultivation in BMGY liquid nutrient medium, culture temperature is 28 ~ 30 DEG C, 200 turns/min shaken overnight, and to OD600=2 ~ 6, cell is in logarithmic phase;

S22. by cell centrifugal 5 minutes collecting precipitations under 3000 turns/min and room temperature condition of step S21 acquisition, be resuspended in BMMY substratum, control dissolved oxygen amount and preventing pollution, cultivate at test tube relaying persistent oscillation, described control dissolved oxygen amount and preventing pollution are with four layers of clean additional two-layer newspaper wrapping BMMY of gauze and alcohol spraying disinfection is carried out in employing with the volume of ∕ or control substratum accounts for 5 ~ 20% of culture vessel volume;

S23. adding 100% methyl alcohol to final concentration at interval of 24 h is 1%, and add-on is that every milliliter of BMMY substratum adds 10 microlitres, carries out inducing culture.

8. preparation method according to claim 6, is characterized in that, steriling test described in step S2 is in protein expression process, observes the color of bacterium liquid, ensures that bacterium liquid is oyster white; Or in culturing process, get bacterium liquid carry out microscopy, observe saccharomycetic form, described microscopy is direct smear microscopy or uses azaleine dyeing microscopic examination.

9. preparation method according to claim 6, is characterized in that, gather in the crops described in step S3 albumen be after cultivation 96 ~ 120h under room temperature 1500 ~ 3000 revs/min centrifugal 5 minutes collect supernatants, abandon thalline; Supernatant of getting is made SDS-PAGE immediately and is analyzed or be placed in-70 DEG C of preservations; Steriling test described in step S3 adopts to be heard yeast-leavened taste and observes bacterium liquid color; Or utilize SDS-PAGE to detect.

10. the protein of the dog α 1-interferon gene coding of the modified transformation prepared by preparation method described in claim 6.