CN107880135A - One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof - Google Patents
One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof Download PDFInfo
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- CN107880135A CN107880135A CN201711142717.XA CN201711142717A CN107880135A CN 107880135 A CN107880135 A CN 107880135A CN 201711142717 A CN201711142717 A CN 201711142717A CN 107880135 A CN107880135 A CN 107880135A
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- C07K7/04—Linear peptides containing only normal peptide links
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- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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Abstract
The invention provides breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof, this method includes：(1) cLHRH genes are designed；(2) gene order to design synthesized, annealed, digestion, connection, be then transferred to competent escherichia coli cell again, obtain recombinant plasmid；(3) original bacterium solution is prepared；(4) lactose induces；(5) low temperature osmotic shock isolates and purifies；(6) 10 × hydroformylation salt solution hydroformylation is used；(7) seedling is matched somebody with somebody.The preparation method is simple, and cost is low, whole preparation process be not related to it is any have potential poisonous, harmful to animal or be difficult to the material degraded, obtained vaccine is good to castration effect, convenient use, and stability is good.
Description
Technical field
The invention belongs to castration vaccine technical field, and in particular to a breeder luteinizing hormone releasing hormone recombinant antigen castration
Vaccine and preparation method thereof.
Background technology
Luteinizing hormone releasing hormone (Luteinizing hormore releasing hormore, LHRH) is by animal
The peptide hormone of one kind ten of hypothalamus secretion, its principal biological function are that the synthesis of control anterior lobe of hypophysis cell and secretion rush are yellow
Body hormone (LH) and follicle-stimulating hormone (FSH) (FSH), these hormones can promote the normal development of sexual gland and sexual organ, maintain the life of animal
Grow function.Castration vaccine can immunoregulation animal internal system property associated hormone it is horizontal, suppress animal sexual organ development, make
Animal loses sexual behaviour and sexual function, so as to prevent chaotic mating (causing drove to be degenerated) and fight, promotes growth of animal, improves
Trunk forms and meat, makes Fresh ＆ Tender in Texture and prevents smell of mutton, improves the price of deed.Its mechanism is by LHRH synthetic antigens or again
After group antigen makees vaccine immunity animal, LHRH antibody is formed in vivo, neutralizes endogenous LH RH, so as to significantly reduce testosterone and female
The sex hormone levels such as hormone, reach the purpose of gentle castration.Compared with traditional operation castration, vaccine castration be not only simple it is easy,
Great convenience is brought to manager, is particularly suitable for large-scale cultivation and herds drove, and can prevent operation castration from causing
Bleeding, infection and stress caused by subtract food and Body weight loss, in addition vaccine castration be applicable to big animal or toy, female
Or buck, young age and adults, livestock and poultry and other animals, furthermore vaccine castration can realize invertibity by supporting technology
Control.
But report that is present and having no effective preferably chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine.
The content of the invention
For the above-mentioned problems in the prior art, the present invention provides a breeder luteinizing hormone releasing hormone recombinant antigen
Castration vaccine and preparation method thereof, the preparation method is easy, and cost is low, and obtained vaccine castration effect is preferable, easy to use.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine, its preparation method comprise the following steps：
(1) cLHRH genes are designed, sequence is as follows：
P1:5’-GGCCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGAACGGCGGTGAGCACTGGTCTTACGG
CCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGAACTAATGAACTCGAGCA-3’(SEQ ID
NO:1)；
P2:5’-TGCTCGAGTTCATTAGTTCGGGCGCAGACCGTAAGACCAATGTTCACCGCCACCCGGCTGCAG
GCCGTAAGACCAGAGCTCACCGCCGTTCGGACGCAGACCGTAGGACCAGTGTTCAGCCATGGCC-3’(SEQ ID
NO:2)；
(2) gene order to design synthesized, annealed, digestion, connection, it is thin to be then transferred to E. coli competent again
Born of the same parents, obtain recombinant plasmid；
(3) recombinant plasmid is inoculated in the LB culture mediums containing Amp, trained under the conditions of 36-37 DEG C, 160-250r/min
Support, obtain original bacterium solution；
(4) it is that 2%-3% is inoculated in the LB culture mediums containing Amp by inoculum concentration by original bacterium solution, in 36-37 DEG C, 160-
Cultivated under the conditions of 250r/min, treat bacterium solution OD600nmIt is worth to add final concentration of 20-30g/L lactose Fiber differentiation during 0.6-0.7
5-6h；
(5) bacterium solution after step (4) Fiber differentiation is centrifuged, abandons supernatant, obtain the first precipitation, the first precipitation is placed in 2-8
It is resuspended in DEG C high sepage, ice bath 10-15min, centrifugation, abandons supernatant, obtain the second precipitation, the second precipitation is placed in 2-8 DEG C of hypotonic medium
Middle resuspension, ice bath 10-15min, centrifugation, collect supernatant；
(6) it is supernatant obtained by step (5) is degerming with 0.22 μm of membrane filtration, then add degerming rear gained liquid liquid 1/10-
10 × hydroformylation salt solution of 1/5 volume, mix, be placed in 2-8 DEG C, hydroformylation processing 72-76h, obtain the protein liquid after hydroformylation；
(7) Tween-80 will be added in the protein liquid after hydroformylation, mixes, obtain aqueous phase, wherein Tween-80 accounts for aqueous phase cumulative volume
4%；
It is 94 by volume by injection white oil and Si Ben -80:6 ratios, mix, 121 DEG C of sterilizing 60min, obtain oil phase；
Oil phase and aqueous phase are pressed 2:1 ratio emulsifies, and water-in-oil emulsion is made, water-in-oil emulsion and 1% tween salt solution are pressed
Volume ratio is 3:7 ratios emulsify, and chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine is made.
Further, Inclusion of Lactose is 20g/L in step (4).
Further, lactose induction time is 6h in step (4).
Further, step (5) middle and high infiltration liquid is prepared by the following method to obtain：Tris 484g, EDTA are weighed respectively
146g and sucrose 40kg, mixing, adds appropriate water for injection, mixes, and then adjusts pH value to continue to add injection to 8.0 with HCl
Water is mixed after 121 DEG C of autoclaving 30min, is made to 200L.
Further, hypotonic medium is prepared by the following method to obtain in step (5)：Tris 484g and EDTA are weighed respectively
146g, mixing, adds appropriate water for injection, mixes, and then adjusts pH value to 8.0 with HCl, continues to add and injects water to 200L,
Mix after 121 DEG C of autoclaving 30min, be made.
Further, 10 × hydroformylation salt solution is prepared by the following method to obtain in step (6)：Sodium chloride is dissolved in injection
With being made into 8.75% salt solution in water, with 121 DEG C of autoclavings 30 minutes, after cooling, 5ml formals are added in every liter of salt solution
Woods, it is well mixed, is made.
Further, 1% tween salt solution is prepared by the following method to obtain in step (7)：Add into every liter of water for injection
Enter sodium chloride 9g, Tween-80 10mL, be well mixed, 121 DEG C of autoclavings 30 minutes, be made.
Breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine provided by the invention and preparation method thereof, have with
Lower beneficial effect：
(1) because cLHRH recombinant antigens are soluble protein, and design and carry specific " signal peptide ", can use and " ooze
The specific process of shock thoroughly ", selectively induces cLHRH fused antigens and secrete from bacterium and discharge, then through centrifugation work
Skill separation of solid and liquid, directly obtain the cLHRH fused antigens of high-purity.With the separation and Extraction of conventional expression of recombinant e. coli albumen
Technique is compared, and the purifying process is not only simple, and is more avoided the trouble that bacteria lysis technique is brought to protein purification, is more avoided
The pollution of bacterial component.
(2) as a result of derivant of the lactose as expression in preparation process, and osmotic shock point is used as by the use of sucrose
The derivant secreted, the whole production process of this product stoste, which is not related to, any to be had potential poisonous, harmful or is difficult to animal
The material of degraded.
(3) preparation method is simple, and cost is low, and the vaccine of preparation is good to castration effect, convenient use, and stability is good.
Brief description of the drawings
Fig. 1 is cLHRH destination protein purity analysis result figures after lactose induction.
Embodiment
The structure of the cLHRH-RA-RS1 strain recombinant expression plasmids of embodiment 1
1st, the design of cLHRH genes and artificial synthesized
Amino acid sequence and corresponding large intestine bar of the design of genetic fragment according to chicken luteinizing hormone releasing hormone (cLHRH)
Bacterium preference codon, and design two complementary cLHRH gene orders the characteristics of combination pET-32c plasmids itself.Ensuring to insert
Under being inscribed before reading frame is correct after carrier, ' held with 3 ' 5 and add two restriction enzyme sites of Ncol and Xhol, digestion position respectively in end
Add two protectiveness bases before point, it is as follows with automatic dna synthesizer synthetic gene, gene order:
P1:5’-GGCCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGAACGGCGGTGAGCACTGGTCTTACGG
CCTGCAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGAACTAATGAACTCGAGCA-3’(SEQ ID
NO:1)
P2:5’-TGCTCGAGTTCATTAGTTCGGGCGCAGACCGTAAGACCAATGTTCACCGCCACCCGGCTGCAG
GCCGTAAGACCAGAGCTCACCGCCGTTCGGACGCAGACCGTAGGACCAGTGTTCAGCCATGGCC-3’(SEQ ID
NO:2)
2nd, the annealing of cLHRH genetic fragments
It is 100pmol/ μ l (suitable that artificial synthesized P1 and P2 genetic fragments are dissolved into concentration with sterilizing ultra-pure water respectively
In 1.82 μ g/ μ l) solution, respectively take 2 μ l add 72 μ l ultra-pure waters in mix, put in boiling water bath and slowly cool to room after 2 minutes
Temperature, -20 DEG C freeze it is standby.
3rd, the digestion of cLHRH genes
Double digestion is carried out to pET-32c plasmids with NcoI and XhoI, concrete operations use with reference to TaKaRa companies restriction enzyme
Specification is carried out.
4th, connect
DNA coupled reactions are carried out with reference to TakaRa company DNA ligation kit (DNA Ligation Kit) operation instruction.
5th, convert
The connection products of 10 μ 1 are taken to convert 200 μ l DH5a competence bacteriums.Take 200 μ l converted products coating μ containing Amp50 g/
Ml LB agar plates, while set the impression of unconverted competence bacterial controls (negative control) and the conversion of pET-32c plasmids
State bacterial controls (amp resistances Quality Control control).Flat board is put in 37 DEG C of incubators and cultivated 18 hours.
Negative control Sterile is born length, and positive control bacterium colony is in " babysbreath " shape, illustrates to convert successfully.
6th, the digestion screening of recombinant plasmid
PET-32c plasmid multiple cloning sites have EcoRI and the restriction enzyme sites of Hand III, after cLHRH genes are inserted the two
Restriction enzyme site should disappear, and NcoI and XhoI restriction enzyme sites still have.Principle accordingly, the single bacterium colony after picking conversion,
It is inoculated with after the LB meat soups containing Amp expand culture and extracts plasmid, carries out single endonuclease digestion with Hand III, EcoRI, NcoI and XhoI respectively,
Hand III and EcoRI restriction enzyme sites is selected to disappear and positive colony that NcoI and XhoI restriction enzyme sites still suffer from.
The recombinant plasmid identified through digestion is selected, using T7Terminater primer as sequencing primer, it is surveyed
Sequence.Sequencing result is identical with implementation sequence.
It is that recombinant antigen is referred to as cLHRH-RA-RS1 strains by above-mentioned recombinant plasmid, in 2017.9.28 preservations to China
Type Tissue Collection, preservation address are Wuhan University, and deposit number is CCTCC NO：M2017564.
Embodiment 2 prepares original bacterium solution
According to protein expression characteristic and the immunogenicity of expressing protein, screen anti-for the restructuring of chicken luteinizing hormone releasing hormone
The original strain of former castration vaccine development, detailed process are as follows：
1st, convert
By cLHRH/pET-32c recombinant plasmid transformeds e. coli bl21 (DE3) competence bacterium, meanwhile, with pET-32c
Plasmid converts Escherichia coli, makees negative vector control.200 μ l transformed bacteria solutions are taken, the LB agar for being coated on the g/ml of μ containing Amp50 is put down
Plate, negative control is made with unconverted competence bacterium, put 37 DEG C and cultivate 18 hours.2-8 DEG C is stored in, treats picking colony, is expanded
After increasing screening, optimal bacterium colony is chosen, establishes primordial seed batch.
2nd, original bacterium solution is prepared
Picking cLHRH/pET-32c recombinant plasmid transformed E. coli clones, are inoculated in 10ml Amp+LB culture mediums, put
37 DEG C of shaking tables, concussion and cultivate is overnight, obtains original bacterium solution.
The determination of the optimum inoculation amount of embodiment 3
The original bacterium solution that embodiment 2 is obtained is inoculated in containing 250ml Amp-LB fluid nutrient medium 1000ml conical flasks,
Inoculum concentration is 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% respectively, under the same terms
In triplicate, respectively first group, second group, the 3rd group, with 36-37 DEG C, 230r/min shake cultures.Record thalline culture
Growth curve, and 1 hour after culture, sampling in 2 hours, determine OD600nmValue, it the results are shown in Table 1-3.
The inoculum concentration of table 1 and OD600nmValue relation (first group)
The inoculum concentration of table 2 and OD600nmValue relation (second group)
The inoculum concentration of table 3 and OD600nmValue relation (the 3rd group)
Test result indicates that each group inoculum concentration is in 2%-3%, and strain culturing 2 hours, bacterial cultures OD600nmValue exists
Between 0.6-0.7, meet Product Process requirement.
The lactose of embodiment 4 induces cLHRH recombinant antigens
1st, the determination of lactose induction optimum concentration
Detailed process is：
(1) the bacterium solution 5ml for meeting production technology that Example 3 obtains, is inoculated in 6 bottles of 250ml containing ampicillin
LB fluid nutrient medium conical flasks, 37 DEG C, with 230r/min shake cultures, are worked as OD600nmLactose is added when value reaches 0.6 and induces its table
Up to cLHRH destination proteins, lactose is added by 10g/L, 20g/L, 30g/L, 40g/L, 60g/L, 80g/L respectively, 37 DEG C of cultures 6 are small
Shi Hou, 6 sample bacterium solution 10ml are taken respectively, thalline is collected by centrifugation.
(2) thalline obtained by step (1) is suspended in the high sepages of 4ml 2-8 DEG C, ice bath is handled 10 minutes, 10000rpm from
The heart 10 minutes, collect thalline.
The preparation of high sepage：Tris 484g, EDTA 146g, sucrose 40kg are weighed respectively, are mixed, are added appropriate injection
Water, mix, then adjust pH value to 8.0 with HCl, continue to add and inject water to 200L, mix and divide after 121 DEG C of autoclavings 30
Clock.
(3) thalline obtained by step (2) is suspended in 4ml 2-8 DEG C hypotonic mediums, ice bath is handled 10 minutes, 10000rpm from
The heart 10 minutes, collect supernatant.Target protein content, record data are surveyed with AAS.
The preparation of hypotonic medium：Tris 484g, EDTA 146g are weighed respectively, mixes, adds appropriate water for injection, are mixed,
Then pH value is adjusted to 8.0 with HCl, continue to add and inject water to 200L, mix after 121 DEG C of autoclavings 30 minutes.
Repeat experiment and carry out 3 groups of parallel laboratory tests simultaneously, collect cLHRH destination protein contents under different lactose induced concentrations
Data, the relation of analysis lactose induced concentration and cLHRH destination protein expression quantity, the results are shown in Table 4.
The lactose final concentration of table 4 and the relation of cLHRH destination proteins expression yield
As shown in Table 4, lactose concn is in 20g/L-30g/L, cLHRH destination proteins expression yield highest.
2nd, the determination of lactose Fiber differentiation Best Times
Detailed process is：
(1) bacterium solution for meeting production technology that Example 3 obtains is inoculated in 250ml benzyls containing ammonia green grass or young crops by inoculum concentration 2.5%
The LB fluid nutrient medium conical flasks of mycin, 37 DEG C, with 230r/min shake cultures, are worked as OD600nmLactose is added when value reaches 0.6 to lure
Lead it and express cLHRH destination proteins.Since the timing adding lactose, sample 25ml, thalline be collected by centrifugation per hour.
(2) thalline obtained by step (1) being suspended in the high sepages of 5ml 2-8 DEG C respectively, ice bath is handled 10 minutes,
10000rpm is centrifuged 10 minutes, collects thalline.
(3) thalline obtained by step (2) is suspended in 5ml 2-8 DEG C hypotonic mediums, ice bath is handled 10 minutes, 10000rpm from
The heart 10 minutes, collect supernatant, as cLHRH destination proteins liquid.With metric measurement destination protein content, number is recorded
According to.
(4) different induction time osmotic shocks are extracted into cLHRH destination proteins, carry out electrophoresis, and with Coomassie brilliant blue and
Highly sensitive kinds of staining methods for protein-argentation dyes respectively, analyzes cLHRH destination protein purity, as a result sees Fig. 1.
3 batch different experiments are carried out according to the method described above, are designated as A experiments, B experiments, C experiments respectively, are collected different lactose
The cLHRH destination protein contents (unit mg/ml) of Fiber differentiation time, analysis lactose Fiber differentiation time and cLHRH purposes
The relation of expressing quantity, the results are shown in Table 5.
The Fiber differentiation time of table 5 and the relation of cLHRH destination proteins expression yield
| Time (hour) | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| A | 0.134 | 0.383 | 0.727 | 1.049 | 1.195 | 1.182 | 1.223 |
| B | 0.101 | 0.490 | 0.627 | 1.162 | 1.235 | 1.209 | 1.199 |
| C | 0.159 | 0.513 | 0.786 | 1.218 | 1.299 | 1.264 | 1.231 |
From table 5 and Fig. 1, lactose Fiber differentiation 5-6 hours, cLHRH destination proteins expression yield highest, continue to cultivate
Protein expression yield no longer improves.Expressing protein is analyzed through electrophoresis dying, and culture 6 is small after especially argentation shows lactose induction
When purity of protein higher than lactose induction after cultivate 7 hours, 8 hours.Result of the test shows, in large-scale production, after lactose induction
The Best Times of fermented and cultured are 6 hours.
The low temperature osmotic of embodiment 5 shock isolates and purifies cLHRH recombinant antigens (fusion protein)
1st, small sample is tested
The bacterium solution for meeting production technology that embodiment 3 obtains is inoculated in 200mlAmp-LB cultures by inoculum concentration for 2.5%
Base, 37 DEG C of shaking table cultures to OD600nmWhen value reaches 0.6, lactose is added to final concentration 20g/L, after continuing culture 5 hours, culture
Thing is divided into two parts, each 100ml.A ultrasonication, another isolates and purifies cLHRH recombinant antigens by low temperature osmotic shock
(fusion protein).
Ultrasonication：Supernatant, whole bacterial sediments 10mlPBS (pH7.4) weights are abandoned into the centrifugation of 100ml bacterial cultures
It is outstanding, ultrasonic disruption (ice bath) 5 minutes, ultrasonic power 240W, supernatant is transferred in another container after centrifugation, precipitation is used
10ml PBS are resuspended, and respectively take 50 μ l supernatants and precipitate the isometric 2X loading buffers of suspension addition, carry out SDS-PAGE.To solidifying
Glue carries out the percentage composition that gray scale scanning determines destination protein.
Low temperature osmotic is suffered a shock：100ml bacterial cultures, survey OD600nm, 10000r/min centrifugation 1 minute, supernatant is abandoned, is precipitated
It is resuspended with 20ml high-permeation solutions, ice bath 10 minutes, 10000r/min is centrifuged 1 minute, abandons the hyposmosis that supernatant adds same volume
Liquid is resuspended, and ice bath 10 minutes is simultaneously shaken frequently, and 10000r/min is centrifuged 1 minute, and supernatant is transferred in another centrifuge tube, precipitation
It is resuspended with the hypotonic medium of same volume.Precipitation carries out SDS-PAGE after supernatant is resuspended.If the whole cell control of non-osmotic shock.
Gray scale scanning is carried out after electrophoresis to gel and determines the percentage composition of destination protein, and with the albumen in metric measurement supernatant
Content.
2nd, osmotic shock is carried out under the conditions of scale-up process to isolate and purify
(1) bacterium solution culture：LB fluid nutrient mediums and appropriate defomaing agent are added in culture tank, 121 DEG C sterilize 15 minutes.Treat
Add Amp by culture medium final concentration 200ug/ml when culture medium is cooled to 36-37 DEG C, added by the 2%-3% of culture medium weight real
The bacterium solution for meeting production technology of the acquisition of example 3 is applied, in 36-37 DEG C of continuous culture, works as OD600nmWhen value reaches 0.5-0.6, by 2%-
3% inoculum concentration carries out tank body and expands culture 2 hours, works as OD600nmWhen value reaches 0.6-0.7, the inducible protein expression of addition lactose, lactose
Final concentration of 2%, continue 5 hours harvest bacterium solutions of culture, determine OD600nmIt is worth, then separation of solid and liquid culture bacterium solution carries out continuous stream
Centrifugation, 16000r/min, collect thalline.
(2) high osmotic treatment：By thalline obtained by step (1) be suspended in the high sepage of 2-8 DEG C of precooling-in, ice bath handles 10 points
Clock.
High sepage dosage：Zymocyte liquid volume × OD600nmValue/5.
(3) measure of hypotonic medium dosage：Take high sepage bacterial suspension 1.0ml, 12000r/min to centrifuge 3 minutes, abandon supernatant.
By thalline be suspended in the 1.0ml hypotonic mediums of 2-8 DEG C of precooling-in, ice bath is handled 10 minutes.12000r/min is centrifuged 3 minutes, is taken
Clearly, using spectrophotometry sample shock protein concentration.By 54% × hypertonic liquid product × sample shock protein concentration/5,
Calculate hypotonic medium dosage.
(4) bacterium solution that step (2) is handled well is separated, 16000r/min continuous flow centrifugations collect thalline.
(5) Hypotonic treatment：Thalline obtained by step (4) is suspended in 2-8 DEG C of hypotonic medium, ice bath effect 10 minutes, then
Supernatant is collected with 16000r/min continuous flow centrifugations, obtains cLHRH fusion protein liquid.
Above-mentioned gained cLHRH fusion protein liquid is diluted to cLHRH fusion protein liquid 0.1mg/ml, with the purifying of nickel zygostyle
Carrier protein carries out SDS-PAGE electrophoresis detections, protein staining observation protein band together, and detects egg through gel gray scale scanning
Bai Chundu.
As a result：
(1) sample test product
Ultrasonication：Thalline after lactose is induced 5 hours, respectively to supernatant and heavy after being centrifuged with ultrasonic disruption
Form sediment and carry out SDS-PAGE, and compared with 5 hours with being induced without lactose parallel culture whole cells, Commassie dyeing can
See that molecular weight about 22kDa major protein bands are come across in ultrasonic treatment liquid supernatant, show cLHRH fusion proteins through breast
It is in solubility expression after sugar induction.
Gel gray scale scanning result shows that the cLHRH fusion proteins of expression account for the 73.95% of thalline total soluble protein
(table 6).
Low temperature osmotic is suffered a shock：Osmotic shock processing gained supernatant and thalline are subjected to SDS-PAGE, Commassie dyeing can
See that cLHRH fusion proteins can be discharged into outside thalline by osmotic shock.Gel gray scale scanning result shows (referring to table 6), stops
The high purity 94.5% of cLHRH fusion protein master tapes in gram supernatant, and the cLHRH fusion proteins remained in thalline only account for bacterium
The 2.05% of body total protein.The solubility of expressing protein and can secretory, isolate and purify cLHRH fusion proteins for osmotic shock method
Prepare vaccine protein stoste (semi-finished product) and provide foundation.
The main albumen of the SDS-PAGE of table 6 (22kDa) gel gray scale scanning result
Swimming lane 2 and 3 in table 6 centrifuges supernatant (soluble protein) for full bacterium thalline ultrasonic treatment liquid after lactose induction；4
It is full bacterium thalline ultrasonic treatment liquid centrifugation after lactose induces with 5；6 and 7 be that osmotic shock centrifuges supernatant, in about 19kDa
It can be seen that the cLHRH fusion protein bands isolated and purified；8 and 9 be osmotic shock centrifugation (mycoprotein)；10 and 11 be lactose
Full bacterium cellular lysate liquid control before induction.
(2) large-scale production
Through SDS-PAGE electrophoresis Coomassie orchid protein stainings, it is seen that the cLHRH fusion proteins of high-purity, molecular weight is about
22kDa.In addition to the catabolite of a small amount of relatively small molecular weight, foreign protein band is not evident that.
Gel gray scale scanning result shows, gained semi-finished product cLHRH fusion protein purity between 80.4%-88.7%,
Average value is 84.58%；Its purity, than carrier protein sample (average value 67.62%) the also height of nickel zygostyle affinity purification.Statistics
Analysis result shows that standard deviation is 3.64 between semi-finished product purity batch, the coefficient of variation (CV) 4.30%, coincidence rate 95.70% between batch,
Show, the purity of different batches semi-finished product, repeatability between there is good batch, referring to table 7.
The osmotic shock of table 7 isolates and purifies the gel gray scale scanning result of cLHRH recombinant antigens
Under the conditions of scale production process, 6 batches of chicken luteinizing hormone releasing hormone recombinant antigens, each batch weight are continuously manufactured experimently
Group antigen (semi-finished product) repeats to obtain the cLHRH weights of high-purity through SDS-PAGE electrophoresis and gel gray scale scanning test sensitivity
Group antigen, demonstrates validity and stability of the purifying process under the conditions of large-scale production.
The preparation of the castration vaccine finished product of embodiment 6
1st, detailed process is as follows：
(1) protein liquid hydroformylation
The cLHRH fusion proteins liquid that embodiment 5 is obtained is degerming using 0.22 μm of membrane filtration, then adds degerming rear gained
10 × hydroformylation salt solution of the volume of protein liquid 1/10, fully mix, be placed in 2-8 DEG C, hydroformylation is handled 72 hours.
The preparation of 10 × hydroformylation salt solution：Sodium chloride is dissolved in water for injection and is made into 8.75% salt solution, with 121 DEG C of high pressures
Sterilizing 30 minutes, after cooling, 5ml formalin (36%-40% formalins) is added in every liter of salt solution, is well mixed, i.e.,
For 10 × hydroformylation salt solution.
(2) seedling is matched somebody with somebody
The preparation of aqueous phase：Tween-80 is added in protein liquid after hydroformylation, is fully mixed, Tween-80 accounts for aqueous phase cumulative volume
4%.
The preparation of oil phase：It is 94 by volume by injection white oil and Si Ben -80:6 ratios, uniformly mixing, 121 DEG C of sterilizings
60 minutes.
It is 2 by volume by oil phase and aqueous phase:1 ratio emulsifies, and water-in-oil emulsion is made.Water-in-oil emulsion and 1% are told
Warm saline is 3 by volume:7 ratios emulsify, and castration vaccine finished product is made；
The wherein preparation process of 1% tween salt solution：Sodium chloride 9g, Tween-80 10mL are added into every liter of water for injection, is mixed
Close uniform, 121 DEG C of autoclavings 30 minutes, be made.
2nd, finished product is tested
Outward appearance：Milky emulsion.
Formulation：Water-in-oil-in water, a cleaning suction pipe is taken, draw a little vaccine and drip in cold water surface, spread in cloud.
Stability：Draw vaccine 10ml to add in centrifuge tube, centrifuged 15 minutes with 3000r/min, the aqueous phase that ttom of pipe separates out
No more than 0.5ml.
Viscosity：By existing《Chinese veterinary pharmacopoeia》Annex is measured, and meets regulation.
Steriling test：By existing《Chinese veterinary pharmacopoeia》Annex is tested, asepsis growth.
Safety verification：With 15~20 age in days SPF chickens 10, each intramuscular injection vaccine 1.0ml, observe 14 day by day, all
It is strong to live, without locally or systemically adverse reaction.
Efficacy test：With 15~20 age in days SPF chickens 10, each intramuscular injection vaccine 0.5ml.It is in immune first 2 days and immune
Gather within 15th blood respectively afterwards, separate serum, determine ELISA antibody titers.Immune chicken serum ELISA antibody is the positive (P/N
>=2.1, N of value is preimmune serum OD450nmMeasured value), and geometrical mean is not less than 5126.
Residual formaldehyde determines：By existing《Chinese veterinary pharmacopoeia》Annex is measured, and meets regulation.
Endotoxin content detects：By existing《Chinese veterinary pharmacopoeia》Annex is tested, and 500EU is not higher than per plumage part.
Effect and purposes：Castration for chicken.Duration of immunity is 3 months.
Usage and dosage：Intramuscular injection.15~20 age in days chickens, 0.5ml/ plumages.
Storage：2-8 DEG C of preservation.
Sequence table
<110>Magnificent growth Engineering Group Co., Ltd
<120>One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 127
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggccatggct gaacactggt cctacggtct gcgtccgaac ggcggtgagc actggtctta 60
cggcctgcag ccgggtggcg gtgaacattg gtcttacggt ctgcgcccga actaatgaac 120
tcgagca 127
<210> 2
<211> 127
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tgctcgagtt cattagttcg ggcgcagacc gtaagaccaa tgttcaccgc cacccggctg 60
caggccgtaa gaccagagct caccgccgtt cggacgcaga ccgtaggacc agtgttcagc 120
catggcc 127
Claims (8)
1. the preparation method of a breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine, it is characterised in that including following step
Suddenly：
(1) cLHRH genes are designed, sequence is as follows：
P1:5’-GGCCATGGCTGAACACTGGTCCTACGGTCTGCGTCCGAACGGCGGTGAGCACTGGTCTTACGGCCTG
CAGCCGGGTGGCGGTGAACATTGGTCTTACGGTCTGCGCCCGAACTAATGAACTCGAGCA-3’；
P2:5’-TGCTCGAGTTCATTAGTTCGGGCGCAGACCGTAAGACCAATGTTCACCGCCACCCGGCTGCAGGCCG
TAAGACCAGAGCTCACCGCCGTTCGGACGCAGACCGTAGGACCAGTGTTCAGCCATGGCC-3’；
(2) gene order to design synthesized, annealed, digestion, connection, be then transferred to competent escherichia coli cell again,
Obtain recombinant plasmid；
(3) recombinant plasmid is inoculated in the LB culture mediums containing Amp, cultivates, obtain under the conditions of 36-37 DEG C, 160-250r/min
Obtain original bacterium solution；
(4) it is that 2%-3% is inoculated in the LB culture mediums containing Amp by inoculum concentration by original bacterium solution, in 36-37 DEG C, 160-250r/
Cultivated under the conditions of min, treat bacterium solution OD600nmIt is worth to add final concentration of 20-30g/L lactose Fiber differentiation 5-6h during 0.6-0.7；
(5) bacterium solution after step (4) Fiber differentiation is centrifuged, abandons supernatant, obtain the first precipitation, the first precipitation is placed in 2-8 DEG C of height
It is resuspended in sepage, ice bath 10-15min, centrifuges, abandon supernatant, obtain the second precipitation, the second precipitation is placed in weight in 2-8 DEG C of hypotonic medium
It is outstanding, ice bath 10-15min, centrifugation, collect supernatant；
(6) it is supernatant obtained by step (5) is degerming with 0.22 μm of membrane filtration, then add degerming rear gained liquid liquid 1/10-1/5 bodies
Long-pending 10 × hydroformylation salt solution, mix, be placed in 2-8 DEG C, hydroformylation processing 72-76h, obtain the protein liquid after hydroformylation；
(7) Tween-80 will be added in the protein liquid after hydroformylation, mixes, obtain aqueous phase, wherein Tween-80 accounts for aqueous phase cumulative volume
4%；
It is 94 by volume by injection white oil and Si Ben -80:6 ratios, mix, 121 DEG C of sterilizing 60min, obtain oil phase；
It is 2 by volume by oil phase and aqueous phase:1 ratio emulsifies, and water-in-oil emulsion is made, by water-in-oil emulsion and 1% tween salt
Water is 3 by volume:7 ratios emulsify, and chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine is made.
2. the preparation method of chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine according to claim 1, its feature
It is, Inclusion of Lactose is 20g/L in step (4).
3. the preparation method of chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine according to claim 1, its feature
It is, lactose induction time is 6h in step (4).
4. the preparation method of chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine according to claim 1, its feature
It is, step (5) middle and high infiltration liquid is prepared by the following method to obtain：Tris 484g, EDTA 146g and sucrose are weighed respectively
40kg, mixing, adds appropriate water for injection, mixes, and then adjusts pH value to 8.0 with HCl, continues to add and injects water to 200L,
Mix after 121 DEG C of autoclaving 30min, be made.
5. the preparation method of chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine according to claim 1, its feature
It is, hypotonic medium is prepared by the following method to obtain in step (5)：Tris 484g and EDTA 146g are weighed respectively, are mixed, are added
Add appropriate water for injection, mix, then adjust pH value to 8.0 with HCl, continue to add and inject water to 200L, mix after 121 DEG C
Autoclaving 30min, it is made.
6. the preparation method of chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine according to claim 1, its feature
It is, 10 × hydroformylation salt solution is prepared by the following method to obtain in step (6)：Sodium chloride is dissolved in water for injection and is made into
8.75% salt solution, with 121 DEG C of autoclaving 30 minutes, after cooling, 5ml formalin is added in every liter of salt solution, is well mixed,
It is made.
7. the preparation method of chicken luteinizing hormone releasing hormone recombinant antigen castration vaccine according to claim 1, its feature
It is, 1% tween salt solution is prepared by the following method to obtain in step (7)：Sodium chloride 9g is added into every liter of water for injection, is told
- 80 10mL of temperature, it is well mixed, 121 DEG C of autoclavings 30 minutes, is made.
8. using chicken luteinizing hormone releasing hormone recombinant antigen castration made from the preparation method described in claim any one of 1-7
Vaccine.
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Cited By (1)
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| CN108904788A (en) * | 2018-06-14 | 2018-11-30 | 广州源博医药科技有限公司 | GnRH- alexin recombinates castration vaccine and its preparation |
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