CN101748131A - Expression system, production method and applications for recombinant garouper growth hormone gene and corresponding polypeptide - Google Patents
Expression system, production method and applications for recombinant garouper growth hormone gene and corresponding polypeptide Download PDFInfo
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Abstract
The invention discloses a recombinant garouper growth hormone gene. The sequence of the gene is shown as SEQ ID NO:1. The gene of the invention is obtained by using a garouper growth hormone gene sequence as a basic design primer through the primer SEQID NO:2 to SEQ ID NO:11 and PCR amplification. The invention also constructs an expression vector and a recombinant gene engineering strain which comprise the recombinant garouper growth hormone gene. The invention also discloses a method for utilizing the recombinant gene engineering strain to produce the recombinant garouper growth hormone gene and applications thereof. The invention can carry out batch amplification on the recombinant garouper growth hormone gene so as to reduce cost and make the gene source stable. The recombinant garouper growth hormone gene of the invention serves as a growth or reproduction regulation agent, and can obviously improve the growth of garoupers and avoid causing harmful effect on the garoupers.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to expression system, production method and the application of a kind of recombinant garouper growth hormone gene and corresponding polypeptide.
Background technology
(growth hormone GH) is proteohormone by a kind of single peptide chain of Anterior pituitary eosinophil excretory to tethelin, and it is a kind of somatomedin with extensive physiological function.(fish growth hormone fGH) is the synthetic and protein polypeptide of a kind of molecular weight of excretory about 22kD of fish hypophysis to fish growth hormone, and the g and Ds of fish is played an important role.The growth promoting function of GH mainly is to combine back synthesis secretion insulin-like growth factor I (IGF-I) with growth hormone receptor (GHR) in the liver by it to produce.Therefore the mRNA expression amount of IGF-I and GHR has reflected upgrowth situation and the nutritional status of fish to a certain extent in the liver.Found the GHR of two kinds of forms in the cabrilla, difference called after GHR1 and GHR2, but the function difference of these two kinds of GHR is still waiting research aspect growth regulating.
Part fish GH gene has obtained the clone and successful expression in protokaryon or eukaryotic expression system.The tethelin of these reorganization has shown the biological activity identical with the spontaneous growth hormone, can obviously promote the growth of fish after by the method for throwing something and feeding or injecting fish being handled.
Cabrilla is the ocean coral fishes, belongs to Sushi section (Serranidae) Epinephelus (Epinephelus), and kind more than 30 is arranged, and has higher economic value.Its growth hormone gene has obtained the clone, and its aminoacid sequence is that recombinant expressed this gene provides the foundation.Express characteristics such as having the high safety non-toxic of expression amount in the yeast born of the same parents, the expression that utilizes it to carry out garouper growth hormone will have application promise in clinical practice.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of recombinant garouper growth hormone gene is provided.
Another purpose of the present invention is to provide a kind of recombinant garouper growth hormone that is obtained by above-mentioned recombinant garouper growth hormone gene coding.
A further object of the invention is to provide the cloning vector that contains above-mentioned recombinant garouper growth hormone gene.
A further object of the invention is to provide the expression vector that contains above-mentioned recombinant garouper growth hormone gene.
A further object of the invention is to provide the recombinant strain of above-mentioned expression vector conversion.
A further object of the invention provides the production method of above-mentioned recombinant garouper growth hormone.
A further object of the invention provides above-mentioned recombinant garouper growth hormone in the growth of preparation seawater fish use and the application in the breeding adjusting control agent.
Above-mentioned purpose of the present invention is achieved by the following technical programs:
The present invention is a foundation with the garouper growth hormone aminoacid sequence of having announced, has designed the gene fragment of this aminoacid sequence of encoding according to the codon preference of yeast expression, shown in SEQ ID NO:1.This fragment is to be synthesized into the method for bypass method PCR by designing a series of oligonucleotide primers, and described primer sequence is shown in SEQ ID NO:2 ~ SEQ ID NO:11.
Recombinant garouper growth hormone gene of the present invention is connected with cloning vector, has made up the cloning vector and the corresponding recombinant bacterial strain that contain this gene fragment.Wherein, the preferred intestinal bacteria pGEMT-gGH of cloning vector, corresponding recombinant bacterial strain is and changes intestinal bacteria pGEMT-gGH among the intestinal bacteria E.coli.DH5 α formed intestinal bacteria recombinant strain pGEMT-gGH/DH5 α.
The present invention has made up the Yeast expression carrier of recombinant garouper growth hormone gene.This construction of carrier is the common method of this area, be template promptly with cloning vector pGEMT-gGH, behind the pcr amplification recombinant garouper growth hormone gene, carry out that enzyme is cut and purifying, be connected with the corresponding restriction enzyme site (EcoRI and XhoI) of expression vector pPICZ α A, obtain Yeast expression carrier pPICZ α A-gGH.
The present invention has also made up the recombinant strain that contains multiple copied Yeast expression carrier pPICZ α A-gGH.This bacterial strain is to change expression vector pPICZ α A-gGH among the intestinal bacteria E.coli.DH5 α formed intestinal bacteria recombinant strain pPICZ α A-gGH/DH5 α by the method that electric shock transforms, perhaps expression vector pPICZ α A-gGH is changed over to formed recombinant bacterial strain pPICZ α A-gGH/X33 among the pichia spp X33, obtain by the screening of high density microbiotic substratum.
In the method for above-mentioned structure Yeast expression carrier, the preferred 1000ug/mlZeocin of described high density microbiotic.
Utilize described recombinant bacterial strain pPICZ α A-gGH/X33 to produce the method for recombinant garouper growth hormone, comprise the steps:
(1) culture of strains fermentation: recombinant bacterial strain is inoculated in the YPG substratum that contains microbiotic zeocin, and being cultured to OD600 under 28 ℃, 250rpm condition is 2.0;
(2) abduction delivering of bacterial classification: collect bacterium liquid, being resuspended in methyl alcohol is abduction delivering among the substratum YPM of carbon source, and additional methyl alcohol to concentration was 0.5% in per 24 hours, continuous induction 5 days;
(3) with the expression product separation and purification, obtain recombinant garouper growth hormone.
Above-mentioned recombinant garouper growth hormone can be directly used in preparation and be fit to the sea water fish especially growth or the breeding adjusting control agent of Epinephelus use, also can form composition by a certain percentage by appropriate excipients, protective material and use.Preferable methods is in recombinant garouper growth hormone: the weight in wet base ratio of foundation bait is a natural air drying behind 1: 100 the ratio thorough mixing ,-20 ℃ of preservations.Recombinant garouper growth hormone is used for growth and breeding during adjusting control agent, can promotes obviously that the mRNA of IGF-I and GHR2 expresses in the growth of lithosporic fry and the liver, fish body moisture, fat and proteic content are not then made significant difference.
Compared with prior art, the present invention has following beneficial effect:
Recombinant garouper growth hormone gene provided by the invention by with can in corresponding microorganism strains, efficiently express after appropriate carriers is connected, can get highly purified recombinant garouper growth hormone after the separation and purification, and having the expression level height, purifying has a few easily.By the present invention, can produce recombinant garouper growth hormone in a large number easily, reduced production cost, this steady sources, recombinant garouper growth hormone with low cost can further promote the growth of cabrilla can be widely used in culture fishery.
Description of drawings
Fig. 1 is bypass method PCR synthetic lithosporic albonubes somatotropic hormone gene and cloning vector and expression vector establishment process synoptic diagram;
Fig. 2 is the electrophorogram of template amplification gGH with pGEMT-gGH when making up Yeast expression carrier, and wherein: M is a molecular weight standard; 1 and 2 is pcr amplification product; The negative contrast of N;
Fig. 3 is that the PCR of Yeast expression carrier pPICZ α A-gGH identifies electrophorogram, and wherein M is a molecular weight standard; 1 and 2 is pcr amplification product; The negative contrast of N;
Fig. 4 is that the double digestion of Yeast expression carrier pPICZ α A-gGH is identified electrophorogram, and wherein M1 and M2 are molecular weight standard; 1 and the 3 carrier pPICZ α A-gGH that cut for enzyme not; 2 and 4 is corresponding double digestion product;
Fig. 5 is the SDS-PAGE collection of illustrative plates of recombination yeast abduction delivering intracellular protein after 5 days, and wherein M is the molecular weight of albumen standard; N is for transforming the negative control of pPICZ α A; 1-7 is the mono-clonal of the different pPICZ of conversion α A-gGH.The arrow indication is purpose band position;
Fig. 6 is for using black porgy GH antibody as an anti-western blot collection of illustrative plates, and wherein M is the molecular weight of albumen standard; N is for transforming the negative control of pPICZ α A; 1-7 is the mono-clonal of the different pPICZ of conversion α A-gGH.The arrow indication is purpose band position;
Fig. 7 is the growth curve chart with the bait feeding lithosporic fry that contains recombination yeast.Wherein H group throws something and feeds to contain the experimental group of 1% reorganization gGH bait, and L group is the experimental group of 0.1% reorganization gGH bait of throwing something and feeding, and control is the control group of 1% empty carrier recombination yeast of throwing something and feeding.* represent p＜0.05; * represents p＜0.01;
Fig. 8 is the quantitative PCR result of cabrilla liver IGF-I, GHR1 and GHR2 mRNA relative expression after the bait that contains recombination yeast of throwing something and feeding.Wherein Hgroup throws something and feeds to contain the experimental group of 1% reorganization gGH bait, and Lgroup is the experimental group of 0.1% reorganization gGH bait of throwing something and feeding, and control is the control group of 1% empty carrier recombination yeast of throwing something and feeding.* represents p＜0.01;
Embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
The synthetic of embodiment 1 Epinephelus coioide tethelin mature peptide gene
According to the aminoacid sequence of the Epinephelus coioide tethelin mature peptide of having delivered and the codon preference of yeast expression, 5 pairs of the primers of design bypass method PCR, the length of every primer is about 75bp, has the overlap (see figure 1) of 22bp between the primer.The sequence of primer is ID NO:2 ~ SEQ ID NO:11, called after P1 ~ P10 successively from left to right.
Building-up process comprises that 3 take turns the PCR reaction.With P1 and P2, P3 and P4, P5 and P6, P7 and P8, P9 and P10 mixing in twos respectively, be made into 100ul in first round reaction according to the PCR reaction system.Reaction conditions is: (1) 94 ℃ of pre-sex change 5 minutes; (2) 94 ℃ of sex change 45 seconds, 53 ℃ of annealing 45 seconds, 72 ℃ were extended 45 seconds, totally 30 circulations; (3) 72 ℃ were extended 5 minutes.Second to take turns reaction be kernel templates with (P5+P6) product, and (P3+P4), (P7+P8) be two primer, respectively gets 33ul, adds the archaeal dna polymerase of 1U, reaction conditions is the same.Third round reaction is a kernel templates with the product of P3 ~ P8, and (P1+P2), the product of (P9+P10) is the both sides primer, respectively gets 33ul and mixes, and adds the archaeal dna polymerase of 1U, the PCR reaction conditions is the same.Obtain 100ul PCR reaction product.
The structure of embodiment 2 cloning vector pGEMT-gGH and contain the acquisition of the intestinal bacteria transformant pGEMT-gGH/DH5 α of this carrier
The operation instructions of reference reagent box pGEMT Easy Vector Systems is mixed in proportion the PCR product of embodiment 1 gained and is connected Transformed E .coli.DH5 α with cloning vector.Extract the plasmid of positive colony, carry out determined dna sequence with the universal primer of carrier.In full accord by the aminoacid sequence that dna sequence dna is translated into the Epinephelus coioide tethelin mature peptide aminoacid sequence of having announced, show that cloning vector successfully constructs.
The structure of embodiment 3 expression vector pPICZ α A-gGH and contain the acquisition of the intestinal bacteria transformant pPICZ α A-gGH/DH5 α of this carrier
Pcr amplification primer according to a pair of primer target gene of sequences Design of synthetic Epinephelus coioide tethelin mature peptide gene order and expression vector pPICZ α A multiple clone site.The upstream primer sequence is shown in SEQID NO:12; The downstream primer sequence is introduced terminator (TCA) simultaneously shown in SEQ ID NO:13.PGEMT-gGH carrier with structure is a template, carries out pcr amplification with above-mentioned primer epinephelus coioides tethelin mature peptide gene, and the fragment that amplification is come out is about 580bp, with the consistent (see figure 2) of expection PCR product size.
With the gene fragment that reclaims purifying with being connected Transformed E .coli.DH5 α with the linear carrier pPICZ α A of double digestion equally behind EcoRI and the XhoI double digestion.Extract the plasmid of positive colony, carry out the PCR screening, can amplify a characteristic fragment (see figure 4) that conforms to expection size (about 580bp) with above-mentioned primer.Detect recombinant plasmid with EcoRI and XhoI double digestion, enzyme is cut product and is comprised and the consistent fragment of PCR product size (being about 580bp), and the fragment (see figure 3) consistent with linear carrier pPICZ α A size (3.3kb).Show that the gGH gene has inserted among the expression vector pPICZ α A.
Recombinant plasmid is carried out two-way order-checking, and its sequence is in full accord with expection, base mutation do not occur, and reading frame is correct.
The structure of embodiment 4 restructured Pichia pastoris in expression bacterial strain pPICZ α A-gGH/X33
Get 20ug expression vector pPICZ α A-gGH and carry out enzyme with SacI and cut, enzyme cuts full back and reclaims test kit with glue and carry out purifying, is dissolved in the 15ul distilled water.Get 80ul pichia spp X33 competent cell, the carrier that cuts with enzyme mixes on ice, leaves standstill 5 minutes.Change mixture over to the electric shock cup, 2000V voltage electric shock transforms.Electric shock finishes the back and adds 1ml 1M sorbital rapidly, and 30 ℃ leave standstill and are coated on YPDS flat board (1000ug/ml Zeocin after 2 hours
TM).Cultivate after 3 days for 30 ℃ and grow yeast mono-clonal pPICZ α A-gGH/X33 with resistance.
The methyl alcohol of embodiment 5 recombination yeasts utilizes type to identify
The mono-clonal corresponding points on MDH and MMH flat board, are put MDH earlier, back point MMH.The all good clone of growth is the fast type of methyl alcohol utilization on two kinds of flat boards, and only well-grown clone utilizes type at a slow speed for methyl alcohol on the MDH flat board.The result shows that transformant is the quick type of methyl alcohol utilization.
The abduction delivering of embodiment 6 garouper growth hormones in pichia spp and the screening of high expression level transformant
The inoculation of 7 well-grown target gene yeast transformants of picking and 1 negative control transformant (pPICZ α A/X33) earlier with bacterial classification to containing microbiotic Zeocin
TMIn the BMGY substratum (500ug/ml), 28 ℃, 250 rev/mins are cultured to OD600 is 2.0.Centrifugal collecting cell is resuspended in that to contain with methyl alcohol be the substratum BMMY abduction delivering of carbon source.Additional methyl alcohol to final concentration was 0.5% in per 24 hours, continuous induction 5 days.Centrifugal collecting cell at smudge cells on ice, gets that supernatant carries out SDS-PAGE and western blot analyzes with acid-treated granulated glass sphere after centrifugal.Western blot used one is anti-to be the anti-black porgy GH of rabbit polyclonal antibody, and two anti-ly mark goat anti-rabbit igg for horseradish peroxidase.With the BCA method supernatant is carried out the concentration determination of total protein simultaneously.
SDS-PAGE (see figure 5) as a result shows that the band of an about 22kDa has all appearred in 7 target gene transformants, be consistent with expection, and negative control does not occur.Western blot (see figure 6) as a result is also consistent with SDS-PAGE result.By software SDS-PAGE is carried out scanning analysis, the exogenous protein expression amount that draws No. 6 transformants is the highest, accounts for 8.4% of the interior solubility total protein of born of the same parents.In conjunction with the detected result of total protein content, the expression of recombinant proteins amount is about 850mg/l.
Contain the interpolation of reorganization Epinephelus coioide tethelin in embodiment 7 bait
5 days recombinant garouper growth hormone yeast of centrifugal collection abduction delivering, in the ratio of weight in wet base 1% and 0.1% respectively with the foundation bait thorough mixing.Contrast bait mixes 1% empty carrier recombination yeast.The bait natural air drying ,-20 ℃ frozen.
Embodiment 8 contains the growth promoting function of the bait of reorganization Epinephelus coioide tethelin to the lithosporic fry
The promotes growth experiment is carried out in Dayawan Aquatic Products Experimental Center, Guangdong Prov., and the time is that in the beginning of July, 2008 is to mid-September.During raising and train, it is in the bucket of 140L that the experiment fish is raised and train in volume respectively for 3 groups by every group 35 tail branch component, natural light cycle, 24h flowing seawater and inflation.Seawater temperature 28-30 ℃, salinity 26-30 ‰.Throw something and feed twice in 8:30 and 17:00 with foundation bait every day, and till the fed to appetite, the time of raising and train is 10 days.Throw something and feed to testing fish respectively with different bait after raising and train, time and the method for throwing something and feeding is consistent when raising and train, and throws something and feeds 8 weeks by a definite date time, and the body weight of per two all experiments of measuring fishes once.Experimental result shows (see figure 7), with containing 1% recombinant garouper growth hormone zymic bait feeding fry (Hgroup) tangible promotes growth effect is arranged.(control) compares with control group, and significant difference (p＜0.05) appears in body weight after 6 weeks, and 8 all backs differences are (p＜0.01) extremely significantly, and this moment, H group rate of body weight gain was higher by 36.6% than control.Containing 0.1% recombinant garouper growth hormone zymic bait (L group) compares with control and does not have tangible promotes growth effect.
Embodiment 9 contains the bait of reorganization Epinephelus coioide tethelin to lithosporic fry moisture, the influence of fatty and proteic content
After 8 weeks threw something and fed, every experimental group took by weighing 3 tail fishes at random and carries out chemical composition analysis.With its oven dry, its moisture content was calculated in the back of weighing after fish put to death.Dried fish is pulverized, taken by weighing on a small quantity, carry out the mensuration of total lipid content with sherwood oil total fat of extracting in cable-styled extractor; Claim to carry out with kjeldahl apparatus with sulfuric acid digestion back on a small quantity the mensuration of total protein in addition.Measurement result (seeing Table 1) shows that these three kinds of compositions do not have significant difference between experimental group and control group, and the bait that showing throws something and feeds contains reorganization Epinephelus coioide tethelin does not change the chemical ingredients of cabrilla.
The table 2 lithosporic fry body fluids after the bait that contains recombination yeast of throwing something and feeding, fatty and proteic content
Embodiment 10 contains reorganization gGH zymic bait to lithosporic fry liver IGF-I, the influence of GHR1 and GHR2 mRNA expression level
After 8 weeks threw something and fed, every experimental group was got the liver sample of 8 tail fishes, put into liquid nitrogen rapidly and preserved.Extract liver total RNA, reverse transcription becomes cDNA.With 18S is that confidential reference items are quantitative PCR detection IGF-I, the mRNA expression level of GHR1 and GHR2.18S upstream primer sequence is shown in SEQ ID NO:14, and the downstream primer sequence is shown in SEQ ID NO:15; IGF-I upstream primer sequence is shown in SEQ ID NO:16, and the downstream primer sequence is shown in SEQ ID NO:17; GHR1 upstream primer sequence is shown in SEQ ID NO:18, and the downstream primer sequence is shown in SEQ ID NO:19; GHR2 upstream primer sequence is shown in SEQ ID NO:20, and the downstream primer sequence is shown in SEQ ID NO:21.Be reflected in Roche lightcycle 480 quantitative PCR detection systems and carry out, reaction system is 10ul, and reaction conditions is: (1) 94 ℃ of pre-sex change 3 minutes; (2) 94 ℃ of sex change 20 seconds, 55 ℃ of annealing 20 seconds, 72 ℃ were extended 30 seconds, collected fluorescence 1 second for 84 ℃, totally 45 circulations.
The result shows, can obviously improve the expression of IGF-I and GHR2 in its liver with containing 1% reorganization gGH zymic bait feeding fry, and GHR1 expression amount there was no significant difference; Then the expression amount of these three growth related genes is not all made significant difference with containing 0.1% reorganization gGH zymic bait feeding.The result of quantitative PCR and somatotrophic effect show consistence, and IGF-I and GHR2 play a significant role in the promotes growth effect of external source gGH to cabrilla (Fig. 8) are described.
The expression system of a kind of recombinant garouper growth hormone gene and corresponding polypeptide, production method and application sequence table
SEQUENCE?LISTING
＜110〉Zhongshan University
＜120〉expression system of a kind of recombinant garouper growth hormone gene and corresponding polypeptide, production method and application
<130>
<160>21
<170>PatentIn?version?3.2
<210>1
<211>564
<212>DNA
＜213〉artificial sequence
<400>1
caaccaatca?ctgacggtca?aagattgttc?tccatcgctg?tttctagagt?tcaacacttg 60
cacttgttgg?ctcaaagatt?gttctccgac?ttcgagtcca?ctttgcaaac?tgaggagcaa 120
agacaattga?acaagatctt?cttgcaagac?ttctgtaact?ctgactacat?catctcccca 180
atcgacaagc?acgagactca?aagatcctcc?gttttgaagt?tgttgtccat?ctcctacaga 240
ttggttgagt?cctgggagtt?cccatctaga?tccttgtccg?gtggttctgc?tccaagaaac 300
caaatttctc?caaagttgtc?tgagttgaag?actggtatct?tgttgttcat?cagagctaac 360
caagacggtg?ctgagttgtt?cccagactcc?tccgctttgc?aattggctcc?atacggtaac 420
tactaccaat?ctttgggtgc?tgacgagtct?ttgagaagaa?cttacgagtt?gttggcttgt 480
ttcaagaagg?acatgcacaa?ggttgagact?tacttgactg?ttgctaagtg?tagattgtct 540
ccagaggcta?actgtacctt?gtag 564
<210>2
<211>75
<212>DNA
＜213〉artificial sequence
<400>2
caaccaatca?ctgacggtca?aagattgttc?tccatcgctg?tttctagagt?tcaacacttg 60
cacttgttgg?ctcaa 75
<210>3
<211>74
<212>DNA
＜213〉artificial sequence
<400>3
gaattgtctt?tgctcctcag?tttgcaaagt?ggactcgaag?tcggagaaca?atctttgagc 60
caacaagtgc?aagt 74
<210>4
<211>75
<212>DNA
＜213〉artificial sequence
<400>4
ctgaggagca?aagacaattg?aacaagatct?tcttgcaaga?cttctgtaac?tctgactaca 60
tcatctcccc?aatcg 75
<210>5
<211>75
<212>DNA
＜213〉artificial sequence
<400>5
ctgtaggaga?tggacaacaa?cttcaaaacg?gaggatcttt?gagtctcgtg?cttgtcgatt 60
ggggagatga?tgtag 75
<210>6
<211>75
<212>DNA
＜213〉artificial sequence
<400>6
ttgttgtcca?tctcctacag?attggttgag?tcctgggagt?tcccatctag?atccttgtcc 60
ggtggttctg?ctcca 75
<210>7
<211>75
<212>DNA
＜213〉artificial sequence
<400>7
tcaacaacaa?gataccagtc?ttcaactcag?acaactttgg?agaaatttgg?tttcttggag 60
cagaaccacc?ggaca 75
<210>8
<211>75
<212>DNA
＜213〉artificial sequence
<400>8
gactggtatc?ttgttgttca?tcagagctaa?ccaagacggt?gctgagttgt?tcccagactc 60
ctccgctttg?caatt 75
<210>9
<211>75
<212>DNA
＜213〉artificial sequence
<400>9
tcttctcaaa?gactcgtcag?cacccaaaga?ttggtagtag?ttaccatatg?gagccaattg 60
caaagcggag?gagtc 75
<210>10
<211>75
<212>DNA
＜213〉artificial sequence
<400>10
ctgacgagtc?tttgagaaga?acttacgagt?tgttggcttg?tttcaagaag?gacatgcaca 60
aggttgagac?ttact 75
<210>11
<211>70
<212>DNA
＜213〉artificial sequence
<400>11
ctacaaggta?cagttagcct?ctggagacaa?tctacactta?gcaacagtca?agtaagtctc 60
aaccttgtgc 70
<210>12
<211>37
<212>DNA
＜213〉artificial sequence
<400>12
cggcgaattc?aataatgtct?caaccaatca?ctgacgg 37
<210>13
<211>28
<212>DNA
＜213〉artificial sequence
<400>13
gcggctcgag?tcacaaggta?cagttagc 28
<210>14
<211>23
<212>DNA
＜213〉artificial sequence
<400>14
cctgagaaac?ggctaccaca?tcc 23
<210>15
<211>26
<212>DNA
＜213〉artificial sequence
<400>15
agcaacttta?gtatacgcta?ttggag 26
<210>16
<211>20
<212>DNA
＜213〉artificial sequence
<400>16
gacattgccc?gcatctcatc 20
<210>17
<211>22
<212>DNA
＜213〉artificial sequence
<400>17
aaaagcctct?ctctccacac?ac 22
<210>18
<211>19
<212>DNA
＜213〉artificial sequence
<400>18
gcgactccat?cttcattca 19
<210>19
<211>18
<212>DNA
＜213〉artificial sequence
<400>19
gcatcctcag?catccacc 18
<210>20
<211>17
<212>DNA
＜213〉artificial sequence
<400>20
gacgctgctg?aatgtga 17
<210>21
<211>18
<212>DNA
＜213〉artificial sequence
<400>21
acccgaacct?cgtgaatg 18
Claims (10)
1. recombinant garouper growth hormone gene, the sequence that it is characterized in that this gene is shown in SEQ IDNO:1.
2. a recombinant garouper growth hormone is characterized in that this hormone is obtained by the described recombinant garouper growth hormone gene coding of claim 1.
3. a cloning vector is characterized in that this cloning vector contains the described recombinant garouper growth hormone gene of claim 1.
4. cloning vector according to claim 3 is characterized in that described cloning vector is escherichia coli cloning carrier pGEMT-gGH.
5. a recombinant strain is characterized in that this bacterial strain is to change claim 3 or 4 described cloning vectors among the intestinal bacteria E.coli.DH5 α formed intestinal bacteria recombinant strain.
6. recombinant strain according to claim 5 is characterized in that this bacterial strain is to change the described cloning vector pGEMT-gGH of claim 3 among the intestinal bacteria E.coli.DH5 α formed intestinal bacteria recombinant strain pGEMT-gGH/DH5 α.
7. expression vector pPICZ α A-gGH in the primary yeast born of the same parents is characterized in that described expression vector contains the described recombinant garouper growth hormone gene of claim 1.
8. a recombinant escherichia coli strain is characterized in that this bacterial strain is to change expression vector pPICZ α A-gGH in the described yeast born of the same parents of claim 7 among the intestinal bacteria E.coli.DH5 α formed intestinal bacteria recombinant strain pPICZ α A-gGH/DH5 α.
9. a recombinant pichia yeast strain is characterized in that this bacterial strain is to change expression vector pPICZ α A-gGH in the described yeast born of the same parents of claim 7 among the pichia spp X33 formed recombinant bacterial strain pPICZ α A-gGH/X33.
10. the described recombinant garouper growth hormone of claim 2 is in the growth of preparation ablen use or the application in the breeding regulation and control preparation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242129A (en) * | 2011-05-24 | 2011-11-16 | 福州大学 | Optimized sequence of pseudosciaena crocea growth hormone (pcGH) gene and application of optimized sequence |
| CN103014058A (en) * | 2012-12-03 | 2013-04-03 | 广东现代农业集团研究院有限公司 | Method for industrially producing grouper growth hormone recombination gene protein |
| CN103211099A (en) * | 2013-04-09 | 2013-07-24 | 长沙学院 | Muscle growth promoter for juvenile mandarin fish |
| CN104152461A (en) * | 2014-08-05 | 2014-11-19 | 中山大学 | Orange-spotted grouper ctrp9 gene, encoding protein and application thereof |
| CN106260759A (en) * | 2016-08-15 | 2017-01-04 | 共鳞实业(深圳)有限公司 | A kind of special little peptide functional collaboration feedstuff of cabrilla |
| CN107356766A (en) * | 2017-05-25 | 2017-11-17 | 中山大学 | A kind of Growth Op Tilapia hormone immue quantitative detection reagent box based on Time-resolved fluoroimmunoassay and heterogenetic antibody |
-
2009
- 2009-11-06 CN CN200910193720A patent/CN101748131A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102242129A (en) * | 2011-05-24 | 2011-11-16 | 福州大学 | Optimized sequence of pseudosciaena crocea growth hormone (pcGH) gene and application of optimized sequence |
| CN103014058A (en) * | 2012-12-03 | 2013-04-03 | 广东现代农业集团研究院有限公司 | Method for industrially producing grouper growth hormone recombination gene protein |
| CN103211099A (en) * | 2013-04-09 | 2013-07-24 | 长沙学院 | Muscle growth promoter for juvenile mandarin fish |
| CN104152461A (en) * | 2014-08-05 | 2014-11-19 | 中山大学 | Orange-spotted grouper ctrp9 gene, encoding protein and application thereof |
| CN106260759A (en) * | 2016-08-15 | 2017-01-04 | 共鳞实业(深圳)有限公司 | A kind of special little peptide functional collaboration feedstuff of cabrilla |
| CN107356766A (en) * | 2017-05-25 | 2017-11-17 | 中山大学 | A kind of Growth Op Tilapia hormone immue quantitative detection reagent box based on Time-resolved fluoroimmunoassay and heterogenetic antibody |
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