CN102627687B - Eriocheir sinensis GnRH analogue, and preparation method and application thereof - Google Patents
Eriocheir sinensis GnRH analogue, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the fields of molecular biology and biological technology, and discloses Eriocheir sinensis GnRH (gonadotropin-releasing hormone) analogues with respective amino acid sequence of QSYHFSHGWKP and QAYHFSHGWKP . Compared the the amino acid sequences of the two subtypes of Eriocheir sinensis GnRH analogues with those of the GnRH of other species, the results show that the former and the latter have similar amino acid conservative sites; the the sequence structures of the Eriocheir sinensis GnRH analogues are similar to that of invertebrate GnRH; and the Eriocheir sinensis GnRH analogues have similar functions as vertebrate GnRH, can stimulate breakdown of oocyte germinal vesicle and have distinct stimulative effect on crab oocyte maturation and important application value for artificial ripening of the Eriocheir sinensis.
Description
Technical field
The present invention relates to molecular biology and biological technical field, be specially mitten crab Buserein and its preparation method and application.
Background technology
Vertebrate gonadotropin releasing hormone (GnRH) is the nerve polypeptide hormone that hypothalamus is secreted, is made up of ten amino acid, GnRH is as hypothalamic pituitary gonadal axis key message molecule, by combining with the special acceptor of GnRH in pituitary gland, thereby stimulate generation and the release of gonad-stimulating hormone (GSH), follicular stimulating hormone (FSH), lutropin (LH), induction of ovulation, raising pregnancy rate and ovary development decelerating obstacle, play a part to regulate reproduction activity.Nineteen seventies researchist starts the research of GnRH, a kind of hormones of purifying out from the hypothalamus of pig such as Schally in 1971 can promote LH to discharge, and are that aminoacid sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH through this hormone of determining in 6 years
2decapeptide, called after is urged Luteinizing hormone releasing hormone (LHRH) (SCHALLY, ARIMURA, KASTIN, et al.Gonadotropin-releasing hormone:one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones[J] .Science, 1971,173 (4001): 1036-1038.).After decades of research in find successively again multiple GnRH analogue, confirm that it has the FSH release function of promotion, this class polypeptide called after GnRH the most at last simultaneously.
Up to now, in vertebrates, have been found that 14 kinds of GnRH types (O.Kah, C.Lethimonier, G.Somoza, L.G.Guilgur, C.Vaillant, J.J.Lareyre, GnRH and GnRH receptors in metazoa:a historical, comparative, and evolutive perspective, Gen.Comp.Endocrinol.153 (2007) 346-364.), adopt and find that first species name it.These dissimilar mammalss (mGnRH) that are respectively, cavy (gpGnRH), chicken (cGnRH-I), chicken (cGnRH-II), bullfrog (rGnRH), madai (sbGnRH), salmon (sGnRH), whitefish (wfGnRH), blue or green Medaka (mdGnRH or pjGnRH), catfish (cfGnRH), catfish (hrGnRH), shark (dfGnRH), Lampetra japonica (Martens). (lGnRH-I), Lampetra japonica (Martens). (lGnRH-II), Lampetra japonica (Martens). (lGnRH-III).According to the difference of physiology, structure, distribution, vertebrates GnRH is divided into four classes: GnRH-I to be synthesized at the frontal lobe position neurone of looking that is arranged in forebrain, be transported to median rise position, finally discharge at hypothalamus position, by stimulating the synthetic of FSH and LH and discharging, hypothalamic pituitary gonadal axis is carried out to final regulation and control; GnRH-II is synthetic at midbrain, infer that it is used as neurotransmitter (Tsai, P.-S., 2006.Gonadotropin-releasing hormone in invertebrates:structure, function, and evolution.Gen.Comp.Endocrinol.148,48-53.); GnRH-III is synthetic at akrencephalon, participate in the reproductive process (Fernald such as regulation and control mating, R.D., White, R.B., 1999.Gonadotropin-releasing hormone genes:phylogeny, structure, and functions.Front.Neuroendocrin.20,224-240.); The representative type of GNRH-IV is lGnRH-I and lGnRH-III, by diencephalon and the synthetic (Silver in ventricles of the brain position, M.R., Kawauchi, H., Nozaki, M., Sower, S.A., 2004.Cloning andanalysis of the lamprey GnRH-III cDNA from eight species of lamprey representing the three families of Petromyzoniformes.Gen.Comp.Endocrinol.139,85-94.), discharge at hypothalamus position, its function need research.
GnRH is as the key genital regulating factor of one, on its aminoacid sequence composition, has high conservative, and wherein 1,4,9,10 amino acids sequences are identical, 2 and 3 amino acids except gpGnRH all the same with the analogue lGnRH-I.1st～3 be with effector cell on receptors bind point, and through the Ca of cytolemma
2+passage enters cell, and 4-10 amino acids residue participates in receptors bind.Determine its function by the 2nd and the 6th amino acids as main active group, simultaneously, in gonad-stimulating hormone synthesizes and discharges, the 8th amino acids is critical sites (P.S.Tsai, L.Zhang, the The emergence and loss of gonadotropin-releasing hormone in protostomes:orthology of regulation and control, phylogeny, structure, and function, Biol.Reprod.79 (2008) 798-805.).By transformation GnRH polypeptide structure, develop now and exceeded 2000 kinds of GnRH analogues, for example, in animal reproduction control, GnRH-A has started commercialization widespread use.In fish aquaculture, application GnRH induction fish lay eggs becomes the method that Technique in Fishes generally adopts in breeding, for culture fishery has been brought great economic benefit.
GnRH is a kind of ancient peptide family, has found 9 kinds of GnRH analogues in the urochordates of Urochordata, and these GnRH analogues are similarly decapeptide structure and have the conservative position of identical amino acid with vertebrates.Immunological identification research shows all to have GnRH analogue (Fang Yongqiang in the non-vertebratess such as cephalochordate, hemichordate, echinoderms, arthropods, platyhelminth, mollusk, yellow authority, Chen Lei .GnRH is in the distribution [J] of lancelet brain and Hatschek's pit. animal journal, 1999,45 (1): 106-11; RASTOGI R K, DI FIORE M M, D ANIELLO A, et al.GnRH in the invertebrates:onoverview[J] .Prog Brain Res, 2002,141,19-29; ANCTIL M, TEKAYA S.Gonadotropin-releasing hormone-like immunoreactivity in the planarian delloura candida (Platyhelminthes, Tricladida) [J] .Invert Bio, 2005,1 124:11-17.).
GnRH is similar to vertebrates, and non-vertebrates GnRH analogue also has the effect of regulation and control reproduction.In river snail (Helisoma trivolvls), confirm that synthetic mGnRH can stimulate egg laying amount to increase (YOUNG, CHANG, GOLDBERG.Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis:possible involvement in reproduction[J] .J Comp Neurol, 1999, 404 (4): 427-437.), the sexual gland of urochordates (Ciona inte stinalis) adds mGnRH and cGnRH-I can promote the release of Goandal steroid hormone while carrying out isolated culture, tGnRH-II can stimulate hemichordate (Saccoglossus bromophenolosus) gamete release (Di FIORE, RASTOGI, CECILIANI, et al.Mammalian and chicken I forms of gonadotropin-releasing hormone in the gonads of a protochordate, Ciona intestinalis[J] .Proc Natl Acad Sci U S A, 2000, 97 (5): 2343-2348.).LH injection RH in tigar prawn, sGnRH, after lGnRH-I, there is the effect of obvious promotion ovary maturity, infer that the Crustacean development of ovary is also to regulate and control (NGERNSOUNGNERN by GnRH analogue, NGERNSOUNGNERN, WEERACHATYANUKUL, et al.The existence of gonadotropin-releasing hormone (GnRH) immunoreactivity in the ovary and the effects of GnRHs on the ovarian maturation in the black tiger shrimp Penaeus monodon[J] .Aquaculture, 2008, 279 (1-4): 197-203.).
So far at octopus (Octopus vulgaris), sea hare (Aplysia californica), sea urchin (Strongylocentrotus purpuratus), limpet (Lottia gigantea), sea worm (Capitella teleta), leech (Helobdella robusta) and 3 kinds of urochordates (Chelyosoma productum, Ciona intestinalis, Ciona savignyi) in find altogether 15 kinds of non-vertebrates GnRH analogue (ROCH, BUSBY, SHERWOOD.Evolution of GnRH:diving deeper[J] .Gen Comp Endocrinol, 2011, 171 (1): 1-16.).Although find multiple GnRH analogue non-vertebrates, but substantial sepn does not go out polypeptide mostly, only to obtain corresponding sequence in mRNA level to infer aminoacid sequence, only isolate first invertebrates GnRH analogue octGnRH in octopus (Octopus vulgaris) application reverse phase HPLC chromatogram (RP-HPLC) technology, show that through N-end and C-terminal amino acid order-checking this analogue is 12 amino acid short peptides, aminoacid sequence is: pGlu-Asn-Tyr-His-Phe-Ser-Asn-Gly-Trp-His-Pro-Gly-NH
2there is the conservative site of similar amino acid to vertebrates GnRH, the octGnRH of synthetic can promote quail cell LH to discharge, there is vertebrates GnRH functional character (IWAKOSHIE, TAKUWA-KURODAK, FUJISAWAY, et al.Isolation and characterization of a GnRH-like peptide from Octopus vulgaris[J] .Biochem Biophys Res Commun, 2002,291:1 187-1193.).
Foreign scholar confirms to have GnRH analogue to exist in multiple decapods crustacean application immunohistochemical methods positioning experiment in the recent period.In tigar prawn central nervous system (CNS), find that there is GNRH analogue and have evidence, experiment shows, probably there is oct-GnRH, lGnRH-III analogue (NGERNSOUNGNERN, NGERNSOUNGNERN, KAVANAUGH, et al.The presence and distribution of gonadotropin-releasing hormone-liked factor in the central nervous system of the black tiger shrimp, Penaeus monodon[J] .Gen CompEndocrinol, 2008, 155 (3): 613-622.).In the CNS of Macrobrachium rosenbergii, there are two kinds of GnRH analogues, respectively with lGnRH-III and the similar (NGERNSOUNGNERN of oct-GnRH, NGERNSOUNGNERN, KAVANAUGH, et al.The identification and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii[J] .Invert Neurosci, 2008,8 (1): 49-57.).In Marsupenaeus japonicus by RP-HPLC, time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemical method combine, result is alleged occurrence GnRH analogue (AMANO also, OKUMURA, OKUBO, et al.Biochemical analysis and immunohistochemical examination of a GnRH-like immunoreactive peptide in the central nervous system of a decapod crustacean, the kuruma prawn (Marsupenaeus japonicus) [J] .Zoolog Sci, 2009, 26 (12): 840-845.), but so far at the also report of relevant GnRH separation and purification of crustacean.
Summary of the invention
The present invention aims to provide a kind of gonadotropin releasing hormone (GnRH) analogue, is the Buserein of mitten crab.
The present invention also provides the preparation method and application of the Buserein of above-mentioned mitten crab.
The Buserein of mitten crab, aminoacid sequence is respectively QSYHFSHGWKP and QAYHFSHGWKP.
The Buserein of this mitten crab can be used for artificial ripening crab class ovocyte.
The present invention is first taking reverse high performance liquid chromatography (RP-HPLC) as separation means, from mitten crab (Eriocheir sinensis) cerebral tissue, extract Buserein (GnRH), use octopus gonadotropin releasing hormone antibody (anti-octGnRH) to carry out Nucleolar skeleton experimental identification to separated portion, obtained the immune response separated portion that is positive.Use substance assistant laser desorpted/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to analyze separated portion, find 2 kinds of hypotype GnRH analogues, aminoacid sequence is respectively QSYHFSHGWKP and QAYHFSHGWKP (as shown in sequence table SEQ ID No.1 and No.2).
The method of extracting the above-mentioned GnRH analogue of preparation from mitten crab cerebral tissue comprises the following steps:
(1) get the cerebral tissue crude extract of mitten crab, by RP-HPLC method fractional separation, adopt binary mobile phase linear gradient elution method wash-out, wherein mobile phase A is 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B is 1wt ‰ trifluoroacetic acid acetonitrile solution, and in 0～45 minute, Mobile phase B volume content in moving phase is increased to 35% from 5% linearity; RP-HPLC flow 1ml/ml, 25 DEG C of column temperatures; Collect the component that octopus GnRH immune response is positive; Preferably collect 17th～22min separated portion;
(2) get step (1) product, carry out fractional separation for the second time by RP-HPLC method, adopt binary mobile phase linear gradient elution method wash-out, wherein mobile phase A is 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B is 1wt ‰ trifluoroacetic acid acetonitrile solution, and in 0～50 minute, Mobile phase B volume content in moving phase is increased to 10% from 1% linearity; RP-HPLC flow 1ml/ml, 25 DEG C of column temperatures; Collect the component that octopus GnRH immune response is positive; Preferably collect 28th～29.5min separated portion;
(3) get step (2) product, carry out fractional separation for the third time by RP-HPLC method, adopt binary mobile phase linear gradient elution method wash-out, wherein mobile phase A is 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B is 1wt ‰ trifluoroacetic acid acetonitrile solution, and in 0～50 minute, Mobile phase B volume content in moving phase is increased to 10% from 0% linearity; RP-HPLC flow 1ml/ml, 25 DEG C of column temperatures; Collect the component that octopus GnRH immune response is positive; Preferably collect 33rd～35min separated portion.
The cerebral tissue crude extract preparation method of mitten crab comprises the steps:
Get the healthy mitten crab cerebral tissue of female sexual maturity, add 0～4 DEG C of homogenate of 1wt ‰ trifluoroacetic acid aqueous solution, the centrifugal 2015～20min of 12000～18000rpm, gets supernatant liquor.
The Buserein of these two kinds of mitten crabs that separation obtains and other species GnRH carry out aminoacid sequence and compare, and result shows to have the conservative site of similar amino acid, and sequential structure and invertebrates GnRH are similar.
The Buserein of these two kinds of mitten crabs also can synthetic.
According to two kinds of GnRH analogue polypeptides of this analogue aminoacid sequence synthetic, inject its biological function of experimental verification by live body.Result shows, two kinds of analogues can break (GVBD), the maturation of ovocyte is had to obvious promoter action by significant stimulation mitten crab ovocyte germinal vesicle.Although there is not pituitary gland in invertebrates, above results suggest, invertebrates GnRH also plays an important role in gonad development, gamete release process as a kind of regulatory factor, may serve as a kind of upstream regulatory factor, and regulation and control downstream hormone discharges.The acquisition of mitten crab GnRH analogue and the checking of physiological function thereof, lay the foundation for this material is applied to crab class artificial ripening.
GnRH analogue in the present invention has utilized RP-HPLC technology binding immunoassay method isolation identification mitten crab (Eriocheir sinensis) cerebral tissue, be after octopus second at the invertebrates GnRH of protein level isolation identification analogue, also be the GnRH polypeptide obtaining first in Crustacean, lay the foundation for this analogue is further applied to crab class artificial ripening simultaneously.
The method for separating and preparing of mitten crab GnRH analogue is set up in this research, first isolation identification mitten crab GnRH analogue, measure its aminoacid sequence, find the GnRH of two kinds of hypotypes, live body is injected experiments experiment result and is shown, the GnRH of two kinds of hypotypes has the function similar to vertebrate GnRH, can stimulate ovocyte germinal vesicle to break, the final maturation of crab ovocyte is had to remarkable promoter action, there is significant application value for mitten crab artificial ripening.
Brief description of the drawings
Fig. 1 is the color atlas that mitten crab cerebral tissue crude extract separates through RP-HPLC; 1-9 is main detached peaks; No. 7 peaks in square frame, through the immunological identification separated portion that is positive, retention time is 19-20min.
Fig. 2 is the Nucleolar skeleton experiment at mitten crab cerebral tissue crude extract No. 7 peak (retention time: 19-20min) after RP-HPLC separates.
Fig. 3 is that RP-HPLC prepares the separated product that contains GnRH analogue (retention time: 17-22min).
The color atlas that after the first separation of Fig. 4 mitten crab cerebral tissue crude extract, No. 7 peak of gained (retention time: 19-20min) separates through RP-HPLC again; 1-11 is main detached peaks; In square frame, be the immunological identification separated portion that is positive, retention time is 29-30min.
The experimental result (experiment of the immunodotting marking) that Fig. 5 is positive for three separated portions (retention time is respectively: 28-28.5min, 28.5-29min, 29-29.5min) immune response in separating for the second time.
During Fig. 6 separates for the second time, the RP-HPLC of three separated portions (retention time: 28-28.5min, 28.5-29min, 29-29.5min) separates spectrogram, and 1-5 is main detached peaks.Be the immunological identification separated portion that is positive in square frame, retention time is 33-34min.
During Fig. 7 Nucleolar skeleton experimental result shows to separate for the third time, two separated portions (retention time: 33-33.5min, 33.5-34min) immune response is positive.
Fig. 8 is the result that RP-HPLC detects GnRH analogue.
Fig. 9 is the molecular weight of GnRH analogue in one-level mass spectrometric detection RP-HPLC sample separation.
Figure 10 molecular weight is the GnRH analogue second order ms figure of 1373.63Da.
Figure 11 molecular weight is 1356.63DaGnRH analogue second order ms figure.
Figure 12 tests and obtains and the accurate material retention time contrast of synthetic mark GNRH analogue, has identical retention time; Wherein, 1-experiment obtains GNRH analogue, and 2-aminoacid sequence is QSYHFSHGWKP artificial synthetic polypeptide, and 3-aminoacid sequence is QAYHFSHGWKP artificial synthetic polypeptide.
Figure 13 mitten crab GnRH analogue aminoacid sequence represents respectively identical with similar amino-acid residue with other species GnRH aminoacid sequence comparison diagram black with grey color part.
Figure 14 is prematurity and ripe ovocyte, a: immature egg parent cell, and germinal vesicle breaks, and can obviously observe nucleus (N: nucleus); B: mature oocyte, germinal vesicle breaks, and does not observe nucleus.
Figure 15 compares between experimental group (left side histogram is GnRH analogue-I type, and right side histogram is GnRH analogue-II type), negative control group (PBS group), the individual ratio of blank group GVBD after the injection of mitten crab GnRH analogue within experimental period 30 day first stage; Wherein, ordinate zou: GVBD ratio %, X-coordinate: experiment grouping (
*p < 0.05,
*p < 0.01).
Figure 16 compares between experimental group after subordinate phase interior mitten crab GnRH analogue injection 30 day experimental period, negative control group (PBS group), the individual ratio of blank group GVBD, further confirms that GnRH analogue can significantly promote that ovocyte germinal vesicle breaks; Ordinate zou: GVBD ratio %, X-coordinate: component, (
*p < 0.05).
Embodiment
1 materials and methods
1.1 experiment material
Polypeptide separating experiment: mitten crab is purchased from Shanghai Local plant, choose that four limbs are sound, vigor is stronger, the female sexual maturity individuality of body weight 100-150g is supported temporarily in the Ministry of Agriculture of Shanghai Ocean University Fish Germplasm Resources and is utilized emphasis open laboratory, in box for breeding (50cm × 60cm), build hovel, oxygenation in 24 hours, change every other day water, twice, the feed of throwing something and feeding every day.
1.2 experimental technique
1.2.1 crab brain crude extract preparation
Choose female sexual maturity healthy individuals, dissect rapidly cerebral tissue on ice, every 50 crab cerebral tissues add 1wt ‰ trifluoroacetic acid aqueous solution 10ml, homogenate on ice, and 4 DEG C of centrifugal 20min of 15000rpm, get supernatant liquor, and concentrated latter-80 DEG C save backup
1.22RP-HPLC separating experiment
Apply Japanese Shimadzu (SHIMADZU) Prominence high performance liquid phase system crab cerebral tissue crude extract is carried out to fractional separation, adopt binary mobile phase linear gradient elution method to carry out wash-out, i.e. mobile phase A: 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B: 1wt ‰ trifluoroacetic acid acetonitrile solution.All supersound process in advance of moving phase used, system flow rate: 1ml/min; Chromatographic column: Shimadzu VP-ODS (150L × 4.6) C-18 chromatographic column, column temperature: 25 DEG C, sample size: 150 μ l.Use automatic component collector to collect respectively per minute separated portion, collect concentrated rear-40 DEG C of preservations of component.
1.2.3 Nucleolar skeleton experiment
Nitrocellulose filter (NC film) is soaked in to EBM damping fluid (2.5 × 10-2mol/L Tutofusin tris, 0.2mol/L glycine, 5.0mol/L methyl alcohol) middle 15min, room temperature is dried after about 5min, by 1 μ l sample successively level point on NC film, sampling point is at a distance of 1cm left and right, and each sample is established 3 parallel laboratory tests; Place drying at room temperature 60min, immerse calf serum (1: 10) sealing 60min, discard serum, with TTBS (0.1mol/L Tutofusin tris, 0.15mol/L sodium-chlor, 2.8 × 10-4mol/L tween 80, regulates pH to 7.5 with hydrochloric acid soln) rinsing 3 times, each 8min; Immerse in the TTBS damping fluid that contains octopus GnRH antibody (being given by Tsai professor Pei-San of Univ Colorado-Boulder USA) and hatch subsequently, be positioned over horizontal shaking table, room temperature low speed rocks and spends the night; Inferior daily TTBS rinsing 3 times, each 8min, adds that to contain HRP mark two anti-, incubated at room 60min; TTBS rinsing 3 times, each 8min, finally uses DAB-H
2o
2colour developing.Negative control group is used normal rabbit serum to replace primary antibodie (octopus GnRH antibody), and blank group is not used any serum.
2 experimental results
2.1 mitten crab cerebral tissue GnRH analogue initial gross separation and immunological identifications
In order to obtain the GnRH analogue in crab cerebral tissue crude extract, this experiment adopts RP-HPLC method to carry out fractional separation to it.Condition of gradient elution to RP-HPLC is optimized (table 1), finally determines that parameter is that Mobile phase B obtains separating effect the best when the volume content from 5% to 35% in mutually increases by linear relationship at wash-out in 50min.RP-HPLC color atlas is mainly divided into 9 peaks (as Fig. 1 after showing crude extract separation, the color atlas that mitten crab cerebral tissue crude extract separates through RP-HPLC, 1-9 is main detached peaks), use automatic component collector to collect per minute RP-HPLC separated product.Each is collected to component and carry out Nucleolar skeleton qualification, result shows to only have 19-20min to collect component immune response to be positive that (Nucleolar skeleton experimental result is as Fig. 2, A: Nucleolar skeleton experimental group; B: blank experimental group; C: negative control experimental group), all the other separated portion immune responses are all negative, and blank group and negative control group immune response are also all negative.No. 7 peak in square frame in the corresponding color atlas of 19-20min separated portion in experiment, this shows that this detached peaks contains GnRH analogue.
Table 1:RP-HPLC experiment parameter
The 2.2 first GnRH of acquisition analogue RP-HPLC separate and immunological identification
According to obtaining data under initial gross separation experiment condition, prepare the separated product that retention time is 17-22min (HPLC is as Fig. 3) by liquid phase systems, to collect by optimizing RP-HPLC separation condition (table 2), separated portion separates, it is best (as Fig. 4 that gradient is adjusted in 45min when Mobile phase B concentration rises to 10% by 1% separating effect, after the first separation of mitten crab cerebral tissue crude extract, gained retention time is the color atlas that No. 7 peak of 19-20min separates through RP-HPLC again), collect the separated product of retention time 25-40min, within every 30 seconds, collect a pipe, collect after component is concentrated into 100 microlitres and carry out Nucleolar skeleton experiment detection, experimental result shows 28-28.5min, 28.5-29min, tri-separated portions of 29-29.5min (being 3～No. 5 peaks in square frame) immune response be positive (immunoblot experiment result Fig. 5), may contain GnRH analogue.
Table 2:RP-HPLC experiment parameter
2.3 crab cerebral tissue GnRH analogues obtain and immunological identification
During acquisition is separated for the second time, obtaining 28-28.5min, 28.5-29min, tri-separated portions of 29-29.5min carries out again RP-HPLC according to optimization experiment condition (table 3) after concentrated and separates and (separate spectrogram as Fig. 6,1-5 is main detached peaks), collect the separated portion in 30-45min interval, within every 30 seconds, collect a pipe, every pipe separated product is concentrated into 100 microlitres and carries out Nucleolar skeleton experiment, experimental result shows to be positive at 33-33.5min and 33.5-34min separated portion (separated portion in Fig. 6 square frame) immune response, as Fig. 7.
Table 3:RP-HPLC experiment parameter
The similar class preparation of 2.4 mitten crab GnRH and tandem mass spectrum qualification amino acid structure
According to obtaining data under fractional separation experiment condition, preparation GnRH analogue component, this component is carried out RP-HPLC detection (Fig. 8) after having prepared, and result is shown as obvious single peak value, there is no other assorted peaks, and retention time is consistent with immune positive component.
2.5 mitten crab GnRH analogue MALDI-TOF/TOF MS measure aminoacid sequence and structure verification
MALDI-TOF/TOF MS measures peptide molecule weight range (Fig. 9) in RP-HPLC sample separation, one-level mass spectrometric detection result display separation component is mainly made up of 3 kinds of micromolecule polypeptides, molecular weight is respectively: 1356.63Da, 1373.63Da, 1801Da, select 1356.63Da, two peaks of 1373.63Da to carry out tandem mass spectrum detection, obtain the rear aminoacid sequence that obtains of second order ms figure (Figure 10, Figure 11) by software analysis and be respectively QAYHFSHGWKP and QSYHFSHGWKP.According to obtaining aminoacid sequence artificial synthetic polypeptide, RP-HPLC detection (Figure 12) shows to obtain GnRH analogue and has identical retention time with artificial synthetic polypeptide, illustrates that both amino acid structures are consistent.
2.6 mitten crab GnRH analogue aminoacid sequence comparisons
The aminoacid sequence acquiring and other species GnRH analogue are compared, and result is as follows:
The contrast of each sequence amino acid difference as shown in figure 13.
Result shows, this analogue has the typical structure of invertebrates GnRH, compare same two amino acid (Figure 13, wherein black represents respectively identical with similar amino-acid residue with grey color part) that have more after the first amino acids with analogue in vertebrates.Meanwhile, this analogue has and the identical conserved amino acid of vertebrates GnRH family site, wherein Q
1, H
4, F
5, S
6, G
8, P
11gnRH is identical with vertebrates.Wherein one type very similar to the amino acid structure of extra large worm, only in the tenth amino acids, have difference, be respectively K
10with F
10.Given this, can judge that separating the polypeptide obtaining is crab GnRH analogue.
In vertebrates, in GnRH sequence, part amino acid sites plays most important effect for polypeptide active, and existing result of study shows, Q
1be and the key factor of GnRH through cytolemma, the variation in this site directly has influence on the activity of peptide section, obtains sequence and all have this site in this experiment.H in receptor binding site
4and G
8amino acid determines the function of peptide section as main active group, this experiment obtains polypeptide at H
4, F
5, S
6, G
8the amino acid of four positions is consistent with vertebrates, and this result shows that mitten crab GnRH analogue probably has similar physiological action, has regulating and controlling effect to the crab development of ovary.
The function of embodiment 2 mitten crab Busereins
1 first stage experimental verification GnRH analogue activity
1.1 materials and methods
11.1 experiment materials
Mitten crab is purchased from Shanghai Local plant, choose that four limbs are sound, vigor is stronger, the female sexual maturity individuality of body weight homogeneous cultivates in Shanghai Ocean University's aquatic products and cultivation center, Life Sciences, adopt circulating water culture system to cultivate, in each box for breeding (70cm × 50cm × 40cm), build hovel, the feed oxygenation in twice, 24 hour of throwing something and feeding every day, recirculated water in periodic replacement cultivating system, keeps water body clean.
1.1.2 artificial synthetic polypeptide
Entrust gill biochemical company limited in Shanghai according to mitten crab GnRH analogue aminoacid sequence synthetic GnRH, Syrups by HPLC purity is greater than 99.5%, and mass spectroscopy molecular weight and theoretical molecular match.Mitten crab GnRH analogue-I type aminoacid sequence is QAYHFSHGWKP, and mitten crab GnRH analogue-II type aminoacid sequence is QSYHFSHGWKP.
11.3 experiment first stage polypeptide injection solution preparations
Polypeptide stoste: synthetic polypeptide uses PBS (sodium-chlor: 8mg/ml, Repone K: 0.2mg/ml, Repone K: 0.2mg/ml, Sodium phosphate dibasic 1.44mg/ml, dipotassium hydrogen phosphate 0.24mg/ml) solution dissolving, compound concentration is 1mg/ml, solution preparation is rear-20 DEG C of preservations well.
In experiment, stoste is diluted, be mixed with different concns injection liquid.Experimental design is three concentration: 10ng/g, 50ng/g, 500ng/g, and solution preparation mode is in table 4.
Table 4 injection polypeptide preparation table
1.2 grouping experiment
Mitten crab supports temporarily that after 30 days, to choose Individual Size close, carries out grouping experiment in the female sexual maturity individuality of same etap.Experiment is divided into eight groups, 15 mitten crabs of each experimental group, and injected dose is shown in preparation table, and experimental group is in the 1st, 10,20 days injection polypeptide of experiment, and it is the 5th step base pitch that every animal is injected 100 microlitre injection sites.The same experimental group of negative control group (PBS group) injection all the other steps of 100 microlitres.Blank group (not injecting any material) is observed germinal vesicle situation in experiment first day, adopts the transparent liquid of crab class ovum (formaldehyde: dehydrated alcohol: glacial acetic acid ratio is 30: 30: 1) to observe oocyte maturation degree.Break (GVBD) as oocyte maturation mark using germinal vesicle (nucleus), immature egg parent cell has obvious nucleus, germinal vesicle breaks, and (Figure 14 a), mature oocyte germinal vesicle breaks, and (Figure 14 b), does not observe nucleus.
1.3 observe and statistics
Within the 15th day, start observation experiment group, PBS group, blank group germinal vesicle situation from testing, within every 5 days, get at random 3 individualities for each group and observe, to the experiment end of the 30th day first stage.Experiment finishes rear all individualities in experimental group, PBS group, blank group samples to the observation germinal vesicle situation of breaking.After Experiment Data Records, adopt SPSS Statistics software analysis.
1.4 experimental result
Before experiment, check oocyte maturation degree, select germinal vesicle that the individual injection of skew polypeptide occurs.In whole experimentation, progressively move to edge the biochemical bubble of blank group position, but GVBD does not occur, and this experimental result reflects ovocyte natural maturity process under this experiment condition, show that the cultivating system and the breeding way that in experimentation, adopt are suitable simultaneously.Compared with PBS group and GnRH analogue injection experimental group, one has 19 individual germinal vesicles and breaks, wherein the 500ng/g experimental group of GnRH analogue-I type effect compared with other experimental group is more obvious, it is higher that GVBD individuality accounts for the individual ratio of experimental group, and PBS group only has 1 individual germinal vesicle to break, in blank group, there is no the individual germinal vesicle break (Figure 15) that occurs, use SPSS Statistics software to carry out comparing check analysis between two, result shows that the GVBD ratio between experimental group and blank group has significant difference (p < 0.05), wherein the GVBD ratio between 500ng/g experimental group and the control group of GnRH analogue-I type has extremely significant difference (p < 0.01), show that mitten crab GnRH analogue can significantly promote ovocyte finally ripe.
2 subordinate phase tests are checking GnRH analogue activity further
First stage obtains experimental data and shows, the effect obtaining in the time that injection concentration is 500ng/g is than obvious.Therefore in subordinate phase experiment, this injection concentration is further verified.Two peptide species are carried out to hybrid injection, the impact on germinal vesicle under research two peptide species actings in conjunction simultaneously.
2.1 subordinate phase experiment polypeptide injection solution preparations
Polypeptide stoste: synthetic polypeptide uses PBS solution to dissolve, and compound concentration is 1mg/ml, solution preparation is rear-20 DEG C of preservations well.In experiment, be divided into GnRH analogue-I type, GnRH analogue-II type and two kinds of hypotype hybrid injection groups, solution preparation mode is in table 5.
Table 5 injection polypeptide preparation table
2.2 experiment groupings
Mitten crab supports temporarily that after 30 days, to choose Individual Size close, carries out grouping experiment in the female sexual maturity individuality of same etap.Experiment is divided into five groups, 10 mitten crabs of each experimental group, and injected dose is shown in preparation table, and experimental group is in the 1st, 10,20 days injection polypeptide of experiment, and it is the 5th step base pitch that every animal is injected 100 microlitre injection sites.The same experimental group of PBS group injection all the other steps of 100 microlitres.
2.3 observe and statistics
Within the 10th day, start observation experiment group from testing, PBS group, negative control group germinal vesicle situation, got at random 3 individualities for every 10 days for each group and observe, to subordinate phase experiment end in the 30th day.Experiment finishes rear all individualities in experimental group, PBS group, negative control group samples to the observation germinal vesicle situation of breaking.After Experiment Data Records, adopt SPSS Statistics software analysis.
2.4 experimental result
In subordinate phase experiment, in each experimental group, all there is GVBD individuality to occur, GVBD ratio is respectively 20%, 20%, 30%, and PBS group does not all have the individual appearance of GVBD with blank group.Blank group does not have the individual appearance of GVBD in whole experimentation.Experimental data is used SPSS software to carry out comparing check analysis between two, and result shows that between experimental group and PBS group (negative control group), blank group, GVBD ratio exists significant difference (p < 0.05) (Figure 16).The GVBD ratio of I+II type hybrid injection group is higher than other two experimental group, although do not have significant difference between experimental group, but there is first GVBD individuality on the 10th day in experiment in I+II type hybrid injection group, other experimental group just has the individual appearance of GVBD in the time that within the 30th day, experiment finishes, and this result further confirms that mitten crab GnRH analogue can obviously shorten the oocyte maturation time.In conjunction with 60 days two stages experimental result, we confirm that mitten crab GnRH analogue can significantly promote GVBD that (being that germinal vesicle breaks) occurs, and obviously accelerates oocyte maturation.
Claims (4)
1. mitten crab Buserein, is characterized in that, aminoacid sequence is as shown in sequence table SEQ ID No.1 or SEQ ID No.2.
2. the preparation method of mitten crab Buserein described in claim 1, is characterized in that, comprises the following steps:
(1) get the cerebral tissue crude extract of mitten crab, by RP-HPLC method fractional separation, adopt binary mobile phase linear gradient elution method wash-out, collect the component that the octopus GnRH immune response of 17th～22min is positive; Wherein mobile phase A is 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B is 1wt ‰ trifluoroacetic acid acetonitrile solution, and in 0～50 minute, Mobile phase B volume content in moving phase is increased to 35% from 5% linearity; RP-HPLC flow 1ml/min, chromatographic column is Shimadzu VP-ODS(150L × 4.6) C-18 chromatographic column, 25 DEG C of column temperatures, UV-detector wavelength 238nm;
(2) get step (1) product, carry out fractional separation for the second time by RP-HPLC method, adopt binary mobile phase linear gradient elution method wash-out, the component that the octopus GnRH immune response of collection 28th～29.5min is positive; Wherein mobile phase A is 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B is 1wt ‰ trifluoroacetic acid acetonitrile solution, and in 0～45 minute, Mobile phase B volume content in moving phase is increased to 10% from 1% linearity; RP-HPLC flow 1ml/min, chromatographic column is Shimadzu VP-ODS(150L × 4.6) C-18 chromatographic column, 25 DEG C of column temperatures, UV-detector wavelength 214nm;
(3) get step (2) product, carry out fractional separation for the third time by RP-HPLC method, adopt binary mobile phase linear gradient elution method wash-out;
Wherein mobile phase A is 1wt ‰ trifluoroacetic acid aqueous solution; Mobile phase B is 1wt ‰ trifluoroacetic acid acetonitrile solution, and in 0～50 minute, Mobile phase B volume content in moving phase is increased to 10% from 0% linearity; RP-HPLC flow 1ml/min, chromatographic column is Shimadzu VP-ODS(150L × 4.6) C-18 chromatographic column, 25 DEG C of column temperatures, UV-detector wavelength 214nm;
Collect 33rd～33.5 and the component that is positive of the octopus GnRH immune response of 33.5-34min, one-level mass spectrometric detection result shows, in 3 kinds of small-molecular peptides, molecular weight is two peaks of 1356.63Da and 1373.63Da, is respectively the mitten crab Buserein described in SEQ ID No.2 and SEQ ID No.1.
3. the preparation method of mitten crab Buserein described in claim 2, is characterized in that, the cerebral tissue crude extract preparation method of described mitten crab comprises the steps:
Get the healthy mitten crab cerebral tissue of female sexual maturity, add 0～4 DEG C of homogenate of 1wt ‰ trifluoroacetic acid aqueous solution, the centrifugal 20min of 12000～18000rpm, gets supernatant liquor.
4. the application of mitten crab Buserein aspect artificial ripening crab class ovocyte described in claim 1.
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| US5378688A (en) * | 1989-02-23 | 1995-01-03 | Colorado State University Research Foundation | GnRH analogs for destroying gonadotrophs |
| US6214969B1 (en) * | 1988-10-21 | 2001-04-10 | The Administrators Of The Tulane Educational Fund | Luteinizing hormone releasing hormone analogs with cytotoxic moiety |
| CN1328061A (en) * | 2000-06-08 | 2001-12-26 | 中国科学院上海生物工程研究中心 | Application of genetically engineered fish growth hormone in aquatic product culture |
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| US5378688A (en) * | 1989-02-23 | 1995-01-03 | Colorado State University Research Foundation | GnRH analogs for destroying gonadotrophs |
| CN1328061A (en) * | 2000-06-08 | 2001-12-26 | 中国科学院上海生物工程研究中心 | Application of genetically engineered fish growth hormone in aquatic product culture |
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