CN106337054A - Method for preparing high activity recombinant porcine epidermal growth factor - Google Patents
Method for preparing high activity recombinant porcine epidermal growth factor Download PDFInfo
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- CN106337054A CN106337054A CN201610705741.9A CN201610705741A CN106337054A CN 106337054 A CN106337054 A CN 106337054A CN 201610705741 A CN201610705741 A CN 201610705741A CN 106337054 A CN106337054 A CN 106337054A
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- growth factor
- epidermal growth
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- egf
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- 102000009024 Epidermal Growth Factor Human genes 0.000 title claims abstract description 42
- 229940116977 epidermal growth factor Drugs 0.000 title claims abstract description 42
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/102—Plasmid DNA for yeast
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing a high activity recombinant porcine epidermal growth factor. A nucleotide sequence shown by SEQIDNO.2 and a recombinant vector and recombinant bacteria containing the nucleotide sequence are firstly disclosed. The method can effectively express the recombinant porcine epidermal growth factor, and the prepared recombinant porcine epidermal growth factor has high activity and good industrial application prospect.
Description
Technical field
The present invention relates to genetic engineering field is and in particular to a kind of highly active Recombinant Swine somatomedin preparation method.
Background technology
Epidermal growth factor (epidermal growth factor, egf) is a kind of multi-functional somatomedin, in body
Interior all have strong rush splitting action in vitro to Various Tissues cell.Epidermal growth factor is by the receptor epidermis with cell surface
Growth factor receptorses (egfr) combine, excite in receptor tyrosine kinase activity, thus starting signal transduction cascade
And leading to multiple biochemical changes, intracellular calcium rises, and increases glycolysiss and protein synthesis, increases some genes
The expression of (inclusion EGF-R ELISA), ultimately results in dna synthesis and cell proliferation.Epidermal growth factor content in breast
Abundant, its receptor is widely distributed in digestive tract, therefore has important function in the gastrointestinal development of infant animal.
In modern intensiveization cultivation, application pig's epidermal growth factor can strengthen the health level of young animal, strengthens
Animal entirety production performance, is one of the means of pig fast-growth and the delayed contradiction of allelotaxises that fundamentally solve.Therefore, people
Work production is prepared epidermal growth factor and is particularly important.Jiao Xiaojun, " recombinant yeast pichia pastoris high density fermentation expresses pig epidermis
" the pig epidermises such as somatomedin and its Purification ", Agricultural University Of South China's journal the 1st phase of volume 32 in January, 2011, and vast sea
Expression in Pichia sp. for the somatomedin and identification ", Scientia Agricultura Sinica 2007,40 (11): 2593-2599 all disclose profit
With the method for the recombinant expressed pig's epidermal growth factor of Pichia sp., but the activity of restructuring pig's epidermal growth factor that expression obtains
Not high enough.
Content of the invention
It is an object of the invention to provide a kind of preparation method of highly active restructuring pig's epidermal growth factor.
The invention provides the nucleotide sequence shown in seq id no.2.
The present invention has also supplied a kind of recombinant vector, and it includes the nucleotide sequence shown in seq id no.2.
Preferably, described recombinant vector is restructured Pichia pastoris in expression carrier ppic9k.
The present invention also supplied a kind of recombinant bacterium it is characterised in that: it includes aforesaid recombinant vector.
Preferably, described recombinant bacterium is recombinant yeast pichia pastoris.
The present invention also supplied a kind of fermentation process it is characterised in that: it is by aforesaid recombinant bacterium, be placed in yeast culture
Cultivate in base, methanol induction is expressed, you can.
Preferably, it comprises the steps:
(1) thalli growth: recombinant bacterium is inoculated in bmgy culture medium, cultivates 36～60h in 25～35 DEG C, be collected by centrifugation
Thalline；
(2) abduction delivering: use bmmy inducing culture suspension thalline, inducing culture 48～120h. at 25～35 DEG C again
It is further preferred that in step (1), cultivation temperature is 30 DEG C, incubation time is 48h；In step (2), culture temperature
Spend for 30 DEG C, induction time is 72h.
The present invention has also supplied the expression product that preceding method prepares, and such as ferment the culture fluid obtaining.
The present invention has also supplied a kind of method of purification of Recombinant pig's epidermal growth factor, and step is as follows:
A, take aforementioned expression product, centrifugation, dialysis；
B, chromatographic isolation, you can.
The method of chromatographic isolation is: first adopt ultrasphere-ods chromatograph post separation, then using in c18 chromatographic column point
From.
The present invention has also supplied the purified product that preceding method prepares.
The present invention also supplied aforementioned expression product, aforementioned purified product preparation improve piglet growth performance medicine or
Purposes in feed additive.
The inventive method is recombinant expressed to obtain pig's epidermal growth factor of recombinating, and its activity is very high, and highest is than commercially available hegf
High 3.97 times, and expression is also higher, recombinate comprising of preparing pig's epidermal growth factor fermentation culture and
Eluent after isolating and purifying, its egf activity is high, and content is also higher, and safety is of high nutritive value, and can be used for preparation and improves piglet
The medicine of growth performance or feed additive.
Obviously, the above according to the present invention, according to ordinary technical knowledge and the customary means of this area, without departing from
Under the premise of the present invention above-mentioned basic fundamental thought, modification, replacement or the change of other various ways can also be made.
The specific embodiment of form by the following examples, remakes further specifically to the above of the present invention
Bright.But this scope being interpreted as the above-mentioned theme of the present invention should not be only limitted to Examples below.All based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description
The pcr result figure of Fig. 1 transformant, wherein, swimming lane 1,3,5,7,8,9,10,11,13,14,15,16,17,19,21,
22 belong to positive colony, and swimming lane 2,4,6,12,18,20,23 belongs to negative clone, and m is marker, and ck is comparison.
The sds-page analysis of Fig. 2 expression product epidermal growth factor；
Fig. 3 elisa standard curve；
Fig. 4 egf elution profile；
The comparison diagram that Fig. 5 the inventive method and pig's epidermal growth factor gene are directly expressed.m:marker；Swimming lane 1,3,4,
7,9,11 is egf expression conditions, swimming lane 2,4, and 6,8,10,12 is egf-n expression conditions；
The chromatogram of Fig. 6 molecular sieve purification, is added maker, is collected the molecular weight of product with the labelling present invention in chromatogram；
The impact to fibroblastic growth rate for Fig. 7 egf；
The microphotograph of Fig. 8 moiety concentrations cell growth status.
Specific embodiment
Embodiment 1 prepares the engineering bacteria containing target gene fragment
First, the acquisition of restructuring pig's epidermal growth factor gene
1st, gene sequencing
Transfer known pig's epidermal growth factor gene from ncbi data base, remove signal peptide in epidermal growth factor simultaneously
Part, the following sequence of acquisition:
aatagttactctgaatgcccgccgtcccacgacgggtactgcctccacggtggtgtgtgtatgtatatt
gaagccgtcgacagctatgcctgcaactgtgtttttggctacgttggcgagcgatgtcagcacagagacttgaaatg
gtgggagctgcgctaa
Sequence amounts to 162bp, the protein containing 53 amino acid residues for the coding:
nsysecppshdgyclhggvcmyieavdsyacncvfgyvgercqhrdlkwwelr
Pig's epidermal growth factor dna sequent synthesis
Pig's epidermal growth factor gene sequence is analyzed, designs new egf gene and synthesized, be named as egf-n
(seq id no.2):
aattcttactctgaatgtccaccttcccacgacggttactgtttgcacggtggtgtttgtatgtatatt
gaagctgttgattcttacgcttgtaactgtgtttttggttacgttggtgaaagatgtcagcatagagatttgaagtg
gtgggaactgagataa
2nd, the structure of recombinant bacterium
The structure of 1.ppic-egf-n carrier
Full genome egf-n is cloned on Pichia sp. constitutive expression carrier ppic9k by ecori/noti site, obtains
To recombinant vector ppic-egf-n, concrete operations are carried out according to " molecular cloning experiment handbook " reorganization operation part.
2. the electroporated and screening of Pichia sp.
Recombinant vector ppic-egf-n is allowed to linearisation with bln i enzyme action, electroporated Pichia sp..Picking is positive to be turned
Beggar.Electricity conversion and jump go the method for positive transformant referring to invitrogen company workbook.
By the method for pcr use egf-n gene special primer (5 ' end gene primers:
gaattcaattcttactctgaatgtcca；3 ' end vector primer 3 ' aox:gcaaatggcattctgacatcc) turn to positive
Beggar carries out pcr detection.
As shown in the pcr result of Fig. 1, no target gene fragment in negative transformants, contains target base in positive transformant
Because of fragment egf-n, illustrate that the present invention has obtained the positive transformant containing target fragment egf-n: recombinant yeast pichia pastoris.
Embodiment 2 adopts the recombinant expressed pig's epidermal growth factor of recombinant yeast pichia pastoris of the present invention
1st, express
(1) expression
The recombinant yeast pichia pastoris that Example 1 builds in 30 DEG C of shaking table cultures 48h in 5ml bmgy culture medium, receive by centrifugation
Collection thalline, adds 1ml bmmy methanol induction culture medium suspension thalline, continues inducing culture 96h at 30 DEG C, and period is spaced 12h
Sampling detection.
Bmgy culture medium: 2% peptone, 1% yeast extract, 100mm kaliumphosphate buffer (ph6.0), 1.34%
Ynb, 4 × 10-5% biotin, 1% glycerol；
Bmmy inducing culture: 2% peptone, 1% yeast extract, 100mm kaliumphosphate buffer (ph6.0),
1.34%ynb, 4 × 10-5% biotin, 0.5% methanol.
(2) detect
The sds-page analysis of 2.1 expression product epidermal growth factors
Take the culture fluid after induction 24-96 hour, centrifuging and taking 20 μ l supernatant carries out tricine-sds-page analysis.
Experimental result as shown in Fig. 2 by tricine-sds-page identify recombinant human epidermal growth factor albumen expression,
It can be seen that the epidermal growth factor of Pichia anomala expression has 2 protein bands from sds-page electrophoresis, its apparent molecular weight is
About 6.5kd about, with the theoretical molecular (6.5kd) being inferred to from aminoacid sequence quite, illustrate that expression has obtained Recombinant Swine
Epidermal growth factor.
It can further be seen from figure 2 that after induction 24h, restructuring pig's epidermal growth factor has obvious expression, as time went on,
Expression gradually steps up, and the preferably abduction delivering time is 72-96h.
2.2 expression water product examines are surveyed
The pig egf detection kit being produced using cloud clone company of the U.S., egf is in culture fluid (abduction delivering for detection
Culture fluid after 72h) in content.Operating procedure is referring to kit specification.
Draw concentration standard curve, standard curve, such as Fig. 3 by standard substance.
| Egf standard substance curve (pg/ml) | 250 | 125 | 62.5 | 31.75 | 15.875 |
| od450 | 1.3564 | 0.7235 | 0.4386 | 0.2958 | 0.1842 |
Using standard curve as reference, according to extension rate, culture fluid sample is carried out with elisa detection, determine expression water
Flat.
Computing formula is egf content=(202.92* (aSample-ags115) -24.66) * extension rate (100 times)
By detection, 1.501 μ g/ml of culture fluid egf content.
The impact to fibroblastic growth for 2.3 Activity determination restructuring pig's epidermal growth factor (ssegf)
2.3.1 experimental technique
By Mus balb/c 3t3 cell (purchased from invitrogen company), subpackage to 96 orifice plates, every hole 5*103Individual cell,
100ul complete medium, CO2 gas incubator is overnight；Cell changes liquid, change into maintenance culture medium (maintain culture medium: 4%fbs,
1%penicillin/streptomycin, dmem-high glucose), CO2 gas incubator is overnight；With maintaining culture medium
Prepare plus sample as shown in table 1, wherein hegf is people restructuring egf, purchased from peprotech company, ssegf is prepared by the present embodiment
The present invention restructuring pig's epidermal growth factor (ssegf) culture fluid (abduction delivering 72h)；Sample-adding product, and it is right to arrange three blank
According to being respectively: a. is acellular, plus maintain culture medium；B. cell, plus maintain culture medium；C. cell halves, plus maintains culture medium,
CO2 gas incubator cultivates 72h；5mg/ml mtt now joins, molten with maintaining culture medium to add 900 μ l mtt according to 9ml culture medium
Liquid, prepares solution a, draws old culture medium, and every hole adds 110 μ l solution a, and CO2 gas incubator cultivates 4h；Draw solution a, plus
150 μ ldmso, vibrate 5min, survey 490nm light absorption value, and 570nm light absorption value compares；Calculate and draw growth curve.
Table 1 egf Concentraton gradient
| Concentration (ng/ml) | 1 | 3 | 4 |
| hegf | 128 | 1 | 0.25 |
| ssegf | 128 | 1 | 0.25 |
2.3.2 experimental result
Experimental result is as shown in table 2, Fig. 7～8:
Table 2 variable concentrations egf is to fibroblastic growth rate
| Concentration (ng/ml) | 128 | 1 | 0.25 |
| hegf | 119.7 | 19.16 | 25.98 |
| ssegf | 340.2 | 76.11 | 45.14 |
Test result indicate that, the restructuring pig's epidermal growth factor that the inventive method prepares, it is given birth to fibroblast
Long rate remarkably promotes effect, and with the rising of egf concentration, fibroblastic rate of growth is stepped up；And, with commercially available
Hegf compares, and the activity of the restructuring pig's epidermal growth factor that the inventive method prepares significantly improves, and wherein, in concentration is
In the case of 1ng/ml, its activity is hegf3.97 times, achieves completely unexpected technique effect.
2nd, isolate and purify
Take the culture fluid that step 1 fermentation obtains, 12000rpm, 4 DEG C of centrifugation 5min obtain supernatant and load 3500da dialysis
By the buffer [1%cf of 10 times of volumes in bag3Cooh, 5%hcooh (vol/vol), 1%nacl, 1m hcl] at 4 DEG C
Dialyzed overnight.All separation are carried out at low ambient temperatures, and constant flow rate 1ml/min, and operational factor is by being equipped with 2 milliliters of samples
The altex model 324-40 chromatograph of injection ring controls.The protein of dialysis can by using ldc spectromonitor iii
Become the detection of wavelength spectrophotometer.The fragment collected in preparation process all passes through sterilized water can be effective by the dilution proportion of 1:1
Minimizing acetonitrile concentration so that can more preferable hydrophobic interaction.Then according to 2ml aliquot is reprinted and is filled with
In the stainless steel column of ultrasphere-ods, (internal diameter is 15cm*4.6mm, and uses the hyptafluorobutyric acid of main solvent 0.2% in advance
(c3f7cooh)/h2O, ph2.3 balance).After loading, chromatograph is in main solvent and secondary solvent (50% acetonitrile/50%h2o/
0.2%c3f7Cooh, wherein, acetonitrile and h2The volume ratio of o is 50:50, c3h7Cooh acetonitrile and h2The 0.2% of o cumulative volume) between
Carried out by running linear 50 minutes gradients, eluting material is recovered between 36 40min.By salvage material in internal diameter
In 30cm*9mm c18 (waters company) semi-preparative column, in 32.5% acetonitrile/67.5%h2O/0.2%c3h7Cooh is (wherein,
Acetonitrile and h2The volume ratio of o is 32.5:67.5, c3h7Cooh is acetonitrile and h2The 0.2% of o cumulative volume) and 40% acetonitrile/60%
h2O/0.2%c3h7Pass through 50 minutes linear gradient elutions, in such a system, efg is eluted in 28.7min, must wash between cooh
De- liquid, figure such as Fig. 4 of eluting, in eluent, the concentration of egf is 43 μ g/ml, and purity can reach more than 80%.
Experimental result illustrates, the inventive method can be expressed and obtain restructuring pig's epidermal growth factor (ssegf), its activity
Height, and expression is also higher, the culture fluid being obtained using engineering bacterium fermentation of the present invention and the eluent after isolating and purifying
Egf activity high, content is also higher, and safety is of high nutritive value, and can be used for preparing the medicine improving piglet growth performance or raises
Feed additives.
The ferment tank of embodiment 3 engineering bacteria of the present invention
1st, fermentation process
The recombinant yeast pichia pastoris that Example 1 builds, are fermented in 50l fermentation tank.The fermentation of recombination yeast is height
Cell density fed-batch fermentation, sweat is divided into strain culturing stage, carbon source feeding period and abduction delivering stage, concrete grammar
See " pichia fermentation process guidelines " operating instruction, version b053002, the 1-7 page
(https://tools.thermofisher.com/content/sfs/manuals/pichiaferm_
prot.pdf).
In Induction Process, every 12h sampling once measures the accumulation of egf of expression and carries out expressing protein
tricine-sds-page.
2nd, detect
Test result indicate that: select the high Pichia sp. recombinant bacterial strain p.pastoris egf-14 of shaking table horizontal expression amount
Carry out 50l ferment tank research.
Egf is can't detect as shown in Figure 5, luring with methanol in thalli growth stage fermentation supernatant before induction
Lead, in supernatant, egf content dramatically increases, and albumen constantly accumulates.After methanol induction 72h, egf content (fermentation titer) reaches 5 μ
g/ml.
Experimental result illustrates, adopts the inventive method specifically, can to adopt with high efficient expression restructuring pig's epidermal growth factor
50l ferment tank culture available 250000ug restructuring pig's epidermal growth factor.
The comparison that embodiment 4 the inventive method and natural pig's epidermal growth factor gene are directly expressed
Egf and egf-n genetic fragment of the present invention are cloned into Pichia sp. constitutive expression by ecori/noti site
On carrier pgap α, obtain recombinant vector pgap-egf and pgap-egf-n.Reorganization operation Main Basiss " the molecular cloning of dna
Laboratory manual " carry out.
Recombinant vector pgap-egf and pgap-egf-n is allowed to linearisation with bln i enzyme action, electroporated complete red ferment
Female.Picking positive transformant.Electricity conversion and screening technique are referring to invitrogen company workbook.
Pgap-egf and pgap-egf-n positive colony is inoculated in liquid ypd culture medium, 30 DEG C, 220rpm, training
Support 60 hours, using trichloroacetic acid method (according to " fine works protein science experiment guide ") by the albumen precipitation in fermentation liquid, and
Expression by two kinds of sequences of protein electrophoresis.
As shown in fig. 6, using the gene expression of natural pig's epidermal growth factor, pig's epidermal growth factor (at 6.5kd) table
The level that reaches is low, and adopts sequence egf-n expression of the present invention, and pig's epidermal growth factor (at 6.5kd) expression increases substantially.
Experimental result illustrates, using natural pig's epidermal growth factor gene cannot high efficient expression in vitro, and adopt this
The gene egf-n of bright design then can be with high efficient expression pig's epidermal growth factor.
Beneficial effects of the present invention are described with the mode of experimental example below:
The restructuring pig's epidermal growth factor (ssegf) of experimental example 1 the inventive method preparation is to child care phase weaned piglets
Impact
By adding present invention restructuring pig's epidermal growth factor (ssegf) of embodiment 2 preparation in child care piglet diet
Culture fluid (abduction delivering 72h) and the pic9k carrier without egf gene convert the fermentation liquid of strain gs115-9k as right
According to the animal using effect of checking pig's epidermal growth factor.
1 test material and method
1.1 experimental animal
This test fully comply with random selection, body weight mutually not etc., child care stage that to observe healthy development situation better young
Pig.
1.2 test periods and place
Test period is on January 31,29 days to 2015 December in 2014,34d altogether.Test piglet, No. 1 hurdle is 50
Age starts to 84 ages in days；No. 2 hurdles to No. 6 hurdles be 45 ages in days to 79 ages in days.Test site is Sichuan Dayi pig farm.
1.3 EXPERIMENTAL DESIGN
Randomly choose 134 ablactational baby pig, if 12 process, be divided into test group 1, test group 2, test group 3, test group 4,
Test group 5, test group 6.It is 1 that test group 1 feeding feed stuff is numbered, and it is 2 that test group 2 feeding feed stuff is numbered, test group 3 feeding feed stuff
Numbering is 3, the like.It is shown in Table 1.
Table 1 EXPERIMENTAL DESIGN
| Group | Feedstuff is numbered | Packet | Sample situation |
| Test group 1 | 1 | 11 piglets | Egf compares |
| Test group 2 | 2 | 11 piglets | Egf compares |
| Test group 3 | 3 | 11 piglets | egf |
| Test group 4 | 4 | 12 piglets | egf |
| Test group 5 | 5 | 11 piglets | Blank |
| Test group 6 | 6 | 12 piglets | Blank |
1.4 test daily rations
Test Diet Formula, based on this pig farm child care material, adds egf sample liquid and egf sample liquid in original child care material
Comparison, addition is respectively 1 ‰.
1.5 feeding and management
Test pig house by the way of high bed stable breeding, free choice feeding, free water, indoor temperature controls at 22-32 DEG C, wet
Degree 80%-85%；Daily time sight feeding (7:00,11:00,15:00,18:00), feeding is with slightly clout in feed trough every time
It is advisable；Other raising is carried out by pig farm routine operation program with immune programme for children；In treatment, few treatment as far as possible, does not adopt this everyday
The mode having an injection carries out health care.
1.6 test testing indexs
Average daily gain (adg): weigh respectively at test the 1st age in days, the 34th age in days morning empty stomach, calculate growth stage
adg；
Average daily gain (adfi): daily in units of repeating observation piglet search for food situation, then in morning on every Sundays
Calculate feed consumption rate in units of repeating, and calculate the adfi of each repetition；
Feed-weight ratio (f/g): f/g is calculated according to weightening and feed intake；
Piglet diarrhea rate: daily before sweeping colony house (daily 8:00 and 14:00) to repeat head every for unit observed and recorded
The situation of diarrhea of pigs, diarrhea frequency=diarrhoea piglet head number/(pig total head number × time) × 100%；
Death rate (%)=extremely wash in a pan pig's head number/total pig's head number × 100%.
2 result of the tests
Test result indicate that, restructuring pig's epidermal growth factor (ssegf) culture fluid being obtained using the inventive method expression
Weaned piglets can be effectively improved, have obvious effect for the weightening of child care piglet.
To sum up, the present invention builds and has obtained the recombinant yeast pichia pastoris containing egf-n, and adopts this restructured Pichia pastoris in expression
Obtain highly active restructuring pig's epidermal growth factor (ssegf), fibroblastic growth can have been effectively facilitated, improve pig life
Produce performance, application prospect is good.
Claims (10)
- Nucleotide sequence shown in 1.seq id no.2.
- 2. a kind of recombinant vector it is characterised in that: it includes the nucleotide sequence shown in seq id no.2；Preferably, described heavy Group carrier is restructured Pichia pastoris in expression carrier ppic9k.
- 3. a kind of recombinant bacterium it is characterised in that: it includes restructuring described in claim 2 and carries；Preferably, described recombinant bacterium is attached most importance to Group Pichia sp..
- 4. a kind of method of Prepare restructuring pig's epidermal growth factor it is characterised in that: it is by the restructuring described in claim 3 Bacterium, is placed in culture in microzyme culture medium, and methanol induction is expressed, you can.
- 5. method according to claim 4 it is characterised in that: it comprises the steps:(1) thalli growth: recombinant bacterium is inoculated in bmgy culture medium, cultivates 36～60h in 25～35 DEG C, bacterium is collected by centrifugation Body；(2) abduction delivering: use bmmy inducing culture suspension thalline, inducing culture 24～96h at 25～35 DEG C again.
- 6. method according to claim 5 it is characterised in that: in step (1), cultivation temperature is 30 DEG C, and incubation time is 48h；In step (2), cultivation temperature is 30 DEG C, and induction time is 72h.
- 7. the expression product that claim 4～6 any one methods described prepares.
- 8. a kind of method of purification of Recombinant pig's epidermal growth factor it is characterised in that: step is as follows:A, take expression product described in claim 7, centrifugation, dialysis；B, chromatographic isolation, you can.
- 9. the purified product that claim 8 methods described prepares.
- 10. purified product described in expression product, claim 9 described in claim 7 improves the medicine of piglet growth performance in preparation Purposes in thing or feed additive.
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| CN112538483A (en) * | 2020-12-29 | 2021-03-23 | 芜湖英特菲尔生物制品产业研究院有限公司 | Preparation method and application of recombinant porcine epidermal growth factor |
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