CN104152457B - Preparation method of veterinary follicle-stimulating hormone analogue - Google Patents
Preparation method of veterinary follicle-stimulating hormone analogue Download PDFInfo
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Abstract
The invention provides a veterinary follicle-stimulating hormone analogue and a preparation method thereof. A connecting sequence is designed according to length, foldability, hydrophobicity and charge effect of a connecting sequence between an alpha peptide chain and a beta peptide chain, the corresponding nucleotide sequence of the connecting sequence is any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, and respective expression quantity and biological activity are compared by virtue of the study. A fused protein of the prepared veterinary follicle-stimulating hormone analogue is similar to the known fused protein in term of biological functions, the expression quantity of multiple fused proteins is obviously increased, the veterinary follicle-stimulating hormone analogue can be used for replacing natural ovarian-stimulating hormone, has great commercial value and is worthy of wide-range popularization.
Description
Technical field
The present invention relates to genetic engineering field, particularly to a kind of preparation method of follicule-stimulating hormone (FSH) analog for animals.
Background technology
Follicule-stimulating hormone (FSH) (Follicle-stimulating hormone, FSH) is by animal brain anterior pituitary basophil
Cell synthesis and a kind of glycoprotein hormoneses of secretion, are formed with non-covalent bond connection by two polypeptide chains of α and β (or claiming subunit).
Secretion, induced animal heat, promotion follicle maturation of ovum, superovulation and the stimulation spermatogenesis of estrogen can be promoted.
FSH for animals is animal brain's extract at present, and the lutropin not only mixing different content causes super row's effect
Really unstable, also carry the potential danger of biogenic infective agent.FSH for animals is researched and developed by genetic engineering, is current grinding
Study carefully focus.The gene engineering strategy being seen in the relevant FSH of report so far mainly has two kinds.One kind is α and β two
After bar polypeptide chain is expressed respectively, then formation heterodimer is connect by non-covalent bond.This method be subject to this two polypeptide chains that
The efficiency of this connection is limited, thus many researcheres transfer how research is connected to one two polypeptide chains of α and β in gene level
Rise, thus being encoded to the fusion protein of single peptide chain.Between two polypeptide chains of α and β, wherein play the aminoacid sequence of interconnection function
Row are referred to as catenation sequence (Linker).Weenen etc. (J Clin Endocrinol Metab, 2004,89:5204-5212.) with
And U.S. Patent Application Pub.No US 2008/0234186A1 passes through two polypeptide chains of α and β of people FSH and different catenation sequences
Restructuring, compares the activity of formed fusion protein.Introduce respectively in catenation sequence and connect glycosylation position containing 1,2 and 4 N-
Point sequence, or adopt carboxylic end peptide (CTP) of human chorion gonadotrophic hormone beta subunit as catenation sequence.Obtained fusion egg
White FSH-N1, FSH-N2 and FSH-N4 and the degree of glycosylation of FSH-CTP and Half-life in vivo all substantially increase compared with wild type FSH
Plus, but Half-life in vivo and increasing of degree of glycosylation are not in simple linear relationship.This invention highlights glycosylation to length
The effect of effect, but do not fully take into account the impact for expressing fusion protein for the catenation sequence itself.Result shows its expression all
Only within the scope of 1.3 14pmol/ml (J Clin Endocrinol Metab, 2004,89:5204-5212 and the U.S. are special
Profit application bulletin US 2008/0234186 A1).
α and β peptide chain in the characteristics and fusion protein such as the length of catenation sequence, foldability, hydrophobicity, charge effect exists
Correct space structure can be each folded into closely related before connecting each other.Article two, peptide chain and connection sequence therebetween
Row, the mutual relation of this three's space three-dimensional structure directly affects the biological activity of fusion protein, also affects the product of fusion protein
Amount.So being adapted to the selection of catenation sequence base of α and β peptide chain and its putting in order most important.
Content of the invention
It is an object of the invention to provide a kind of follicule-stimulating hormone (FSH) analog for animals and preparation method thereof.For above-mentioned existing
The problem that technology exists, according to the length of α and β peptide interchain catenation sequence, foldability, hydrophobicity, charge effect come the company of design
Connect sequence thus obtaining different follicule-stimulating hormone (FSH) analog for animals.To the different follicle being merged by different catenation sequences
The yield of stimulin analog and biological activity are compared and screen, and therefrom select the optimal sequence of high expression.
For reaching above-mentioned purpose, the technical scheme is that：
A kind of follicule-stimulating hormone (FSH) analog for animals, in described follicule-stimulating hormone (FSH) analog for animals, according to α and β peptide interchain even
Connect the length of sequence, foldability, hydrophobicity, charge effect designing catenation sequence, the corresponding nucleotides sequence of its catenation sequence
It is classified as：SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、
SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13、
SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18、SEQ ID NO：
19、SEQ ID NO：Any one in 20.
Further, the corresponding nucleotides sequence of described catenation sequence is classified as：SEQ ID NO 13、SEQ ID NO：14、SEQ
ID NO：15、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：Any one in 18.
Further, described follicule-stimulating hormone (FSH) method for preparing analogue for animals comprises the steps：
Step one, insert by two subunit β of the natural sheep follicule-stimulating hormone (FSH) recorded on Genbank and α gene and betwixt
Known catenation sequence (two ends point containing enzyme action), carries out complete sequence synthesis (SEQ ID NO：1), then with pcDNA3.1 (+) plasmid enters
Row molecular cloning.Respectively
Step one, the gene of natural sheep follicule-stimulating hormone (FSH) β recorded on Genbank and α subunit is contained respectively by two ends
The known catenation sequence of BamHI, SpeI enzyme action point couples together, and carries out complete sequence synthesis, and its sequence is SEQ ID NO：1, two
End I containing Nhe and EcoR I enzyme action point respectively, then with pcDNA3.1 (+) plasmid carries out molecular cloning；Through double digestion, use successively
Following 19 sequences replace above-mentioned known catenation sequence, SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID
NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：
11、SEQ ID NO：12、SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ ID NO：16、SEQ ID
NO：17、SEQ ID NO：18、SEQ ID NO：19、SEQ ID NO：20, to prepare 19 kinds of different new expression plasmids；Will
Gained expression plasmid transfects DH5 escherichia coli, amoxicillin resistance screening, extracts monoclonal bacterium colony plasmid DNA, and enzyme action reflects
It is sequenced after fixed；
After step 2, recombinant plasmid dna sequence are confirmed, carry out single endonuclease digestion, be allowed to linearisation, transfect after linearisation
Chinese hamster ovary celI, through G418 resistance screening, monoclonal, selects high-expression clone by western blotting；
Step 3, amplified step by step Secondary Culture amplification, western blotting detection culture fluid it was demonstrated that restructuring
The high expression of FSH, cell adds antifreezing agent, and subpackage liquid nitrogen is frozen；
Step 4, recovery, through containing blood serum medium culture, serum-free culture is tamed, amplify Secondary Culture amplification, card step by step
The high expression of real Recombinant FSH, as seed cell plus antifreezing agent, subpackage liquid nitrogen is frozen.
Further, in described follicule-stimulating hormone (FSH) method for preparing analogue step one for animals, the restriction endonuclease of double digestion is：Limit
Property restriction endonuclease Nhe I and EcoR I processed.
Further, in described follicule-stimulating hormone (FSH) method for preparing analogue step 2 for animals, the restriction enzyme of single endonuclease digestion
Enzyme is Psp1406I.
Further, in described follicule-stimulating hormone (FSH) method for preparing analogue step 2 for animals, described G418 resistance screening
Concentration is 800 μ g/ml.
Further, in described follicule-stimulating hormone (FSH) method for preparing analogue step 3 for animals, choose 25cm respectively2、75cm2、
175cm2、225cm2Tissue Culture Flask amplified step by step Secondary Culture amplification.
Further, comprise the ovarian stimulation element analog for animals of any of the above-described catenation sequence and containing this ovary thorn for animals
Application in terms of promoting follicular development for the medicine of hormone analogs.
With respect to prior art, beneficial effects of the present invention are：The present invention according to the length of α and β peptide interchain catenation sequence,
Foldability, hydrophobicity, charge effect devise a different set of catenation sequence, thus obtaining a different set of ovary thorn for animals
Hormone analogs, compare its respective expression and biological activity by research.Ovarian stimulation element for animals obtained by the present invention
The fusion protein of analog has similar biological function, and the expression of plurality of fusion protein to known fusion protein
Significantly improve, for natural ovarian stimulation element, there is good vicarious function, there is great commercial value, be worth pushing away on a large scale
Extensively.
Brief description
Fig. 1：It is the expression figure monitoring Recombinant FSH using western blotting sxemiquantitative, Recombinant FSH is carried out engineering
The monoclonal of cell is selected.1：Protein Marker；2：The Chinese hamster ovary celI culture fluid (negative control) of untransfected, sample-adding amount is
20μl；3-8；Six Chinese hamster ovary celI culture fluid cloning transfection, sample-adding amount is respectively 15 μ l.Sentenced according to the intensity of band development
Disconnected expression.Band above master tape is the dimer of recombiant protein, and the band below master tape is catabolite.
Fig. 2：For comparing the ovarian weight augmentation functional diagram of follicule-stimulating hormone (FSH) analog.Standard substance and 7 parts are measured by test product all embodiments
Effect relation, substantially in linear relation.Wherein by the catenation sequence of known catenation sequence (Linker 1) and present invention design
The slope of (Linker 16) and standard substance three basically identical (k is respectively 21.782 (Linker1), 21.785
(Linker16), and 21.783 (standard substance).
Specific embodiment
Below in conjunction with the accompanying drawings and specific embodiment is described in further details to the present invention program：
Embodiment 1：The structure of expression plasmid and the foundation of engineering cell
1. according to the aminoacid sequence of natural sheep FSH recorded on Genbank and nucleotide sequence (http:// www.ncbi.nlm.nih.gov/sites/entrez？Cmd=Retrieve＆db=nucleotide＆dopt=GenBank＆ List_uids=57619323, andhttp://www.ncbi.nlm.nih.gov/sites/entrez？Cmd=Retrieve＆ Db=nucleotide＆dopt=GenBank＆list_uids=1365), design and carry out full genome SEQ ID NO：1 conjunction
Become, containing known catenation sequence (Linker 1):ggatccggct ccaacgccac cggctccggc tccaacgcca
Cctccggctc cactag sequence (J Clin Endocrinol Metab, 2004,89:5204-5212).
2. construction expression plasmid：By double-strand SEQ ID NO 1 and pcDNA3.1 (+) plasmid is through respectively through double digestion
(NheI and EcoRI), 1% agar electrophoresises separate, extraction.By the SEQ processing through double digestion ID NO 1 and pcDNA3.1
(+) plasmid is connected by ligase ligase.By standard CaCl2Method transfects DH5 α escherichia coli, and amoxicillin resistance is sieved
Choosing, 37 DEG C of cultures.Extract monoclonal bacterium colony plasmid DNA by the description of plasmid extraction kit (Quiagen), be named as
pcDNA3.1-FSH1.Enzyme action identification after carry out sequencing confirm, standby.By pcDNA3.1-FSH1 through the double enzyme of BamHI+SpeI
Cut, separate through 1% agar electrophoresises, reject Linker 1, extraction obtains linearizing pcDNA3.1-FSH, standby.Double-strand
SEQ ID NO：2(Linker 2)、SEQ ID NO：3(Linker 3)、SEQ ID NO：4(Linker 4)、SEQ ID NO：5
(Linker 5)、SEQ ID NO：6(Linker 6)、SEQ ID NO：7(Linker 7)、SEQ ID NO：8(Linker 8)、
SEQ ID NO：9(Linker 9)、SEQ ID NO：10(Linker 10)、SEQ ID NO：11(Linker
11)、SEQ ID NO：12(Linker 12)、SEQ ID NO：13(Linker 13)、SEQ ID NO：14(Linker 14)、
SEQ ID NO：15(Linker 15)、SEQ ID NO：16(Linker 16)、SEQ ID NO：17(Linker 17)、SEQ
ID NO：18(Linker 18)、SEQ ID NO：19(Linker 19)、SEQ ID NO：20 (Linker 20) are (hereinafter referred to as
Linker 2～20), respectively through BamHI+SpeI double digestion, separate respectively through 1% agar electrophoresises, extract.Will be through double
The Linker 2～20 of enzyme action is connected with pcDNA3.1-FSH respectively, 19 parts of recombiant plasmid of acquisition (pcDNA3.1-FSH2～
PcDNA3.1-FSH20), for transfecting DH5 α escherichia coli.Amoxicillin resistance screening, extracts monoclonal bacterium colony respectively
Plasmid DNA, obtains corresponding 19 parts of recombiant plasmid pcDNA3.1-FSH2～pcDNA3.1-FSH20.Surveyed after enzyme action identification
Sequence confirms.
3. set up engineering cell：
3.1.CHO cell attachment growth：The previous day of transfection, with 3～4x 105The cell density inoculation 6 hole culture of/ml
Plate, culture medium full nutrient chemical DMEM containing 10% Ox blood serum.Set 21 holes altogether.
3.2. the linearisation of plasmid and liposome：After recombiant plasmid sequence confirms through sequencing, using restricted interior
Enzyme cutting Psp1406I carries out linearization for enzyme restriction respectively.Linearized for 2 μ l (1 μ g/ μ l) recombinant plasmid dna is dissolved in 200 μ l
In serum-free medium, mix；In addition 20 μ l liposome Lipofectin (Invitrogen) are dissolved in 200 μ l serum-free cultures
In base, gently mix, then the DNA preparing is gently mixed uniformly with liposome, room temperature places 25～30min, stand-by.According to
Same method prepares 21 parts of mixed solutions, and wherein 1 part eliminates plasmid, as negative control.
3.3. the transfection of cell：Chinese hamster ovary celI contains the culture medium adhere-wall culture of calf serum to 85- in 6 well culture plates
95%, rinsed after twice with serum-free medium, add 2ml serum-free medium.Above-mentioned 21 parts of mixed solutions are separately added into
In 21 holes, gently shake up.At 37 DEG C, 5% CO2, it is incubated 12h in saturated humidity, change the full culture containing 10% Ox blood serum
Base continues culture 48～72h.
3.4. the screening of resisting cell：Transfectional cell is carried out passing on after growth 24h, is replaced by selective medium (5%
Calf serum simultaneously contains 600 μ l G418) continue culture.Interval 2～3d changes liquid once, when negative control cell is all dead,
Collect the Chinese hamster ovary celI system of G418 resistance.0.25% pancreatin acts on the cell of monolayer adherence, under the conditions of 37 DEG C, digests 1-2
Minute.And dispelled with culture fluid, accurate counting.Respectively with 2x10^6/ml density inoculating cell adhere-wall culture, using after three days
Know radioimmunology (J Clin Endocrinol Metab, 2004,89:5204-5212.) detect each culture fluid supernatant
The concentration results of middle follicule-stimulating hormone (FSH) analog such as table 1 (pRIA, table 1).
The concentration of follicule-stimulating hormone (FSH) analog in each culture fluid supernatant of table 1.
4. the cloning of transfectional cell：By the cell line of wherein 6 high expression (Linker 13-18) and comparison (Linker
1) cell line monoclonal.0.25% pancreatin acts on the cell of monolayer adherence, under the conditions of 37 DEG C, digests 1-2 minute.And
Dispelled with culture fluid, after accurate counting, be serially diluted and contain 10 cells to every ml, then add big 96 hole cultures with 100 μ l/ holes
After 6 days in plate, observe and count monoclonal cell hole, take monoclonal hole cell culture fluid 15 μ l, according to above-mentioned Western blot's
Method monitoring FSH expression.Western blot detects FSH albumen：The polyacrylamide concentration glue of configuration 4% and 12.5% divide
From glue, take 15 μ l culture fluid to be specimen, carry out SDS-PAGE by the description of Bio-Rad company protein electrophoresises system.After electrophoresis
Electricity is needed on nitrocellulose filter again, plus one and resists in 37 DEG C of incubation 1h, wash buffer three times；Again plus horseradish peroxidase
The two of labelling resist in 37 DEG C of incubation 1h, are taken pictures with chemoluminescence method colour developing, as shown in Figure 1.
Continuous three time clonings, select expression highest clone, further culture amplification.Each cell line respectively stays portion
The monoclonal cell culture of highest expression, totally 7 parts.With the culture of 2x10^6/ml density inoculating cell, pass through after seven days to put and exempt from method inspection
Survey the concentration (mRIA, table 1) of follicule-stimulating hormone (FSH) analog in each culture fluid supernatant.
Test example 2：Ovarian weight augmentation is tested
1. adopt anti-FSH Alpha antibodies chromatographic column (CNBr-activated Sepharose 4B, Amersham
Biosciences) affine separation and Extraction is carried out to above-mentioned 7 parts of monoclonal cell culture fluid supernatant.Purity 24-26% it
Between.
2. basis《Veterinary drug national standard collects》In 3rd, hypophysis follicle stimulating hormone biologic assay measures each follicle to stimulate
The potency of plain analog.
The preparation of 2.1 standard solutions (S):The sign potency of menotrophin standard substance is 190IU/ ampoule.The test same day,
Take ampoule standard substance, plus 19ml normal saline fully dissolves, and is configured to the highly concentrated solution of 10IU/ml.Take 3ml 10IU/ml
Highly concentrated solution dilutes the middle strength solution of 5 times of one-tenth 15ml 2IU/ml；The middle strength solution taking 3ml 2IU/ml again dilutes 5 times
Become 15ml 0.4IU/ml low concentration solution, that is, be made into high, medium and low Three doses concentration (dS3, dS2, dS1：10IU/ml,2IU/
Ml, 0.4IU/ml) standard solution, the potency ratio of adjacent solutions is equal to be 1:0.2.
The preparation of 2.2 need testing solutions (T):Take extract respectively analog containing 30ng, be diluted to respectively with normal saline
The highly concentrated solution of 20ng/ml (estimation potency is 30IU/ml), then dilutes, prepares high, medium and low Three doses concentration altogether
Need testing solution [dT3 (20ng/ml), dT2 (4ng/ml), dT1 (0.8ng/ml)], the potency ratio of adjacent diluent is 1:
0.2.Checking method:Take the Sprague Dawley rat of 19-22 age in days, body weight is 45 ± 5g, is randomly divided into 24 groups, every group 6
Rat.Rat subcutaneous injection standard substance or test sample respectively.Standard solution per injection 0.5ml, one time a day, continuously injects 3
Day.Test sample only injected 0.5ml in the 1st day.After last administration 18 hours, animal is put to death, weighs, dissect.Take out
Ovary, peels off surrounding tissue and fat, press dry on filter paper, weighs (precision to 0.1mg), is converted into the ovary of every 100g body weight
Weight.
2.3 experimental result：As table 2
Table 2. standard substance or test sample each group rat every 100g body weight ovary average weight (mg)
By comparing the ovarian weight augmentation function of follicule-stimulating hormone (FSH) analog it was demonstrated that standard substance and 7 parts are measured by test product all embodiments
Effect relation, (Fig. 2) substantially in linear relation.Wherein by the connection sequence of known catenation sequence (Linker 1) and present invention design
The slope of row (Linker 16) and standard substance three basically identical (k is respectively 21.782 (Linker1), 21.785
(Linker16), and 21.783 (standard substance).
2.4 calculating potency：
In formula：DS is contained unitss (accumulated dose) in the high dose of standard substance, is 15IU.
DT is for calculating contained unitss (accumulated dose) by its labelled amount or estimation potency in the high dose of test sample；For
15IU.
S, T are the meansigma methodss of standard substance or test sample each group every 100g body weight ovary weight, and group sequence 1,2,3 refers to low dosage
Organize high dose group；
I is the logarithm of adjacent two dose ratio:I=log5=0.69897
The potency being calculated test sample is equivalent to the percent (table 3) estimating potency
The potency of the test sample that table 3. is merged by 6 different catenation sequences is equivalent to the percent estimating potency
3. monoclonal (Linker 16) cell of high expression high-titer is through culture bottle (25cm2、75cm2、175cm2、
225cm2) amplifying Secondary Culture amplification step by step, Western blot detection culture fluid is it was demonstrated that Recombinant FSH height is expressed.Cell adds anti-
Freeze agent DMSO, subpackage liquid nitrogen is frozen.
Test example 3：Prepare seed cell
1st, tame engineering cell：Engineering cell (monoclonal pcDNA3.1-FSH16 transfectional cell) (contains in DMEM culture medium
5% calf serum, 1% mycillin and 1%GlutaMax), 37 DEG C, 5% CO2, cultivate in saturated humidity, gradually reduce little
Ox blood serum content (changing culture medium daily, calf serum content reduces 1% in every two days), to containing 1%, is replaced by serum-free training then
Foster base Hyclone (containing 1% mycillin and 1%GlutaMax), engineering cell suspension growth.
2nd, serum-free culture 7d, collects culture fluid, and centrifugal filtration is standby.
3rd, detection (method such as embodiment 1) the Recombinant FSH expression of destination protein expression is 210pmol/ml
4th, laboratory animal is the young rat of Sprague Dawley19-22 age in days, body weight 43-50g, all female, 20,
It is randomly divided into 2 groups, every group 10, first group is matched group, with serum-free medium as control sample；Second group is experiment
Group, expression FSH cell strain is cultivated 7 days with serum-free medium, takes culture fluid as test sample.Every rat abdomen of laboratory sample
Subcutaneous injection 0.5ml.Put to death animal after four days, weigh, dissect.Take out ovary, peel off surrounding tissue and fat, on filter paper
Press dry, weigh (precision to 0.1mg), be converted into the ovary weight of every 100g body weight.
Result is as shown in table 4：
The FSH of expression can promote rat ovary to increase weight.Control rats average weight 19.1mg, experimental group rat ovary is put down
All weight 32.0mg, compared with matched group weightening 67.5%.
Table 4 rat every 100g body weight ovary weight (mg)
Conclusion：The follicule-stimulating hormone (FSH) analog of the high expression of the present invention has the biological activity similar to standard substance.
5. confirm the high expression of feature follicule-stimulating hormone (FSH) analog, as seed cell plus antifreezing agent, subpackage liquid nitrogen freezes
Deposit.
The present invention devises one group according to the length of α and β peptide interchain catenation sequence, foldability, hydrophobicity, charge effect
Different catenation sequences, thus obtaining a different set of ovarian stimulation element analog for animals, compares its respective table by research
The amount of reaching and biological activity.The fusion protein of ovarian stimulation element analog for animals obtained by the present invention and known fusion protein tool
There is similar biological function, and the expression of plurality of fusion protein significantly improves, natural ovarian stimulation element is had very
Good vicarious function, has great commercial value, is worth promoting on a large scale.
The above, the only specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any
The change or replacement expected without creative work, all should be included within the scope of the present invention.Therefore, the present invention
Protection domain should be defined by the protection domain that claims are limited.
Claims (3)
1. a kind of follicule-stimulating hormone (FSH) analog for animals is it is characterised in that described follicule-stimulating hormone (FSH) analog for animals is by connecting
Follicule-stimulating hormone (FSH) α and β peptide chain that sequence is connected, according to the length of α and β peptide interchain catenation sequence, foldability, hydrophobicity,
Designing catenation sequence, this follicule-stimulating hormone (FSH) analog for animals is prepared charge effect by following preparation method：
Step one, the gene of natural sheep follicule-stimulating hormone (FSH) β recorded on Genbank and α subunit is contained respectively by two ends
The known catenation sequence of BamHI, SpeI enzyme action point couples together, and carries out complete sequence synthesis, and its sequence is SEQ ID NO：1, two
End I containing Nhe and EcoR I enzyme action point respectively, then with pcDNA3.1 (+) plasmid carries out molecular cloning；Through double digestion, use successively
Following 5 sequences replace above-mentioned known catenation sequence, SEQ ID NO：13、SEQ ID NO：14、SEQ ID NO：15、SEQ
ID NO：16、SEQ ID NO：18, to prepare 5 kinds of different new expression plasmids；Gained expression plasmid is transfected DH5 large intestine bar
Bacterium, amoxicillin resistance screening, extracts monoclonal bacterium colony plasmid DNA, is sequenced after enzyme action identification；
After step 2, recombinant plasmid dna sequence are confirmed, carry out single endonuclease digestion, be allowed to linearisation, after linearisation, transfection CHO is thin
Born of the same parents, through G418 resistance screening, monoclonal, select high-expression clone by western blotting；
Step 3, amplified step by step Secondary Culture amplification, western blotting detection culture fluid it was demonstrated that Recombinant FSH is high
Expression, cell adds antifreezing agent, and subpackage liquid nitrogen is frozen；
Step 4, recovery, through containing blood serum medium culture, serum-free culture tame, step by step amplify Secondary Culture amplification it was demonstrated that weight
The high expression of group FSH, as seed cell plus antifreezing agent, subpackage liquid nitrogen is frozen；
In described follicule-stimulating hormone (FSH) method for preparing analogue step one for animals, genes of interest complete sequence insertion pcDNA3.1 (+) institute
The restriction endonuclease of double digestion needing is：Restricted enzyme Nhe I and EcoR I；Replacing the restriction endonuclease needed for catenation sequence is：
BamHI and SpeI；
In described follicule-stimulating hormone (FSH) method for preparing analogue step 2 for animals, the restricted enzyme of single endonuclease digestion is Psp1406I.
2. according to claim 1 follicule-stimulating hormone (FSH) analog for animals it is characterised in that described follicule-stimulating hormone (FSH) for animals be similar to
In thing preparation method step 2, the concentration of described G418 resistance screening is 800 μ g/ml.
3. according to claim 1 follicule-stimulating hormone (FSH) analog for animals it is characterised in that described follicule-stimulating hormone (FSH) for animals be similar to
In thing preparation method step 3, choose 25cm respectively2、75cm2、175cm2、225cm2Tissue Culture Flask amplified biography step by step
Culture expands.
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| CN102225968A (en) * | 2011-05-09 | 2011-10-26 | 中山大学 | Recombinant anguilla marmorata follicle stimulating hormone FSH beta alpha and preparation method and application thereof |
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