CN103965348A - Monopterus albus estrogen receptor alpha gene, encoding protein and enzyme-linked immunosorbent assay method - Google Patents

Abstract

The invention discloses a monopterus albus estrogen receptor alpha gene, an encoding protein and an enzyme-linked immunosorbent assay method. The enzyme-linked immunosorbent assay method comprises the following steps: obtaining an ER alpha sequence in monopterus albus gonad by adopting an RT-PCR method, selecting cDNA sequence corresponding to an amino acid sequence with stronger antigenicity as a template to clone the sequence of which length is 945bp, cloning the sequence into a pMAL-c2x expression vector, obtaining purer recombinant protein with immunocompetence through IPTG induction and Amylose-sepharose columella purification, and obtaining ER alpha expression protein capable of being used as immunogen by adopting FactorXa protein enzyme digestion after further purification. A mice can obtain antiserums with higher titer after being immunized, and an immunohistochemical research is preliminarily conducted, shows that the antiserums can be peculiarly bond with ER alpha protein in the monopterus albus gonad, shows positive reaction on oocyte membrane, and lays a foundation for further researching functions of estrogen, ER alpha of the estrogen and the like in gender development of the monopterus albus. According to the invention, an ELISA assay kit is further prepared by taking the antibody as the basis.

Description

Swamp eel estrogen receptorαgene, proteins encoded and enzyme-linked immune detection method

Technical field

The present invention relates to genetically engineered field, specifically relate to swamp eel estrogen receptorαgene, proteins encoded and enzyme-linked immune detection method.

Background technology

Swamp eel ( monopterus albus), have another name called yellow eel, mainly live in Asia, China only has a kind at present, has higher nutritive value.Since the seventies starts to propagate artificially, wild swamp eel seed is just caught in a large number, and environmental pollution simultaneously etc. causes swamp eel wild resource day by day to reduce, cultivation seed critical shortage.The phenomenon that in swamp eel growth cycle, existence reverses, while germinating, swamp eel is female, after sexual maturity is laid eggs, male sex-cell germinates, and the sexual stage between formation, develops into gradually male swamp eel.

In swamp eel growth cycle, the phenomenon of sex reversal is that first Liu Jiankang etc. finds (Liu Jiankang, Gu Guoyan. the change of sexual gland tissue when yellow eel sex reversal, hydrobiont journal, 1951,2(1-2): 85-109), then Xiao Yamei (Xiao Yamei, Liu Jun. swamp eel is changed into the RESEARCH ON CELL-BIOLOGY of patrogenesis by a sexual development. aquatic product journal, 1995,19 (4): 297-301; Xiao Yamei. swamp eel reproductive biology research: the 1. female sexual development of swamp eel. Journal of Natural Science of Hunan Normal University. 1995,18 (4): 45-52) etc. detailed research swamp eel sexual gland occur and the process of differentiation, thereby show when swamp eel germinates to be female, after female sexual maturity is laid eggs, progressively abortion of ovum in sexual gland, ovarian structure is degenerated, male sex-cell germinates simultaneously, sexual stage between formation, be transitioned into patrogenesis by this one-phase swamp eel, finally form male swamp eel.Swamp eel sex determining gene or to regulate and control swamp eel sex reversal on gene level be all the focus that investigators pay close attention to all the time, and sex determination is a complicated regulation process that relates to the different space-times of polygene.

Sex steroid hormone (sex steroid hormone) is reaching maturity of adjusting vertebrates hypothalamic pituitary gonadal axis and the important hormone that maintains various functions.Endogenous steroids mainly contains three kinds of male sex hormone, oestrogenic hormon and progestogen in animal body, and their physiological function is all mainly by classical oestrogenic hormon nuclear receptor mediation.Estrogen receptor and estrogen specific ground transcribing in conjunction with rear mediation estrogen response element (estrogen respond elements, EREs) regulatory gene.ER kind and the physiological function etc. of teleostei seem more more complicated than high vertebrates.First fish ER α is separated and is obtained by Pakdel etc. from rainbow trout (Oncorhynchus mykiss) brain, up to now, has more than the 10 ER α that plant fish to be studied report.

The enzyme-linked immunoassay of hormone has no radioactivity pollute, and marker is stable, marker enzyme wide material sources, economy, and the feature such as sensitivity, specificity and ria-determination are suitable, makes the Enzyme immunoassay method of hormone be expected to alternative ria-determination technology.But enzyme-linked immunoassay needs more hormone standard substance, hypophysis that need to be a large amount of with conventional hypophysis extraction technique.

Summary of the invention

The invention provides swamp eel estrogen receptor alpha protein, its aminoacid sequence is as shown in SEQ ID NO.4.

The encode swamp eel estrogen receptorαgene of above-mentioned protein.

The nucleotide sequence of described swamp eel estrogen receptorαgene is as shown in SEQ ID NO.3.

The present invention also provides the carrier that contains above-mentioned swamp eel estrogen receptorαgene.

Above-mentioned carrier, refers to the pMAL that inserts above-mentioned swamp eel estrogen receptorαgene between EcoR I and Hind III site.

The present invention also provides the enzyme-linked immune detection method for swamp eel estrogen receptorαgene, comprises the steps:

(1) preparation of antigen: by swamp eel estrogen receptorαgene insertion vector, and at expression in escherichia coli, the ER α recombinant protein obtaining, purified rear as antigen;

(2) preparation of antibody: the antigen immune mouse obtaining in (1) is prepared to anti-yellowing eel estrogen receptor Alpha antibodies;

(3) purifying of antibody: the antibody of gained in (2), through centrifugal, precipitation, desalination, is obtained to antibody purification;

(4) mark of antibody purification: with the antibody purification of gained in enzyme labelling (3), obtain enzyme labelled antibody;

(5) drafting of typical curve: use the antigen coated on solid phase carrier of different concns, after being combined by the enzyme labelled antibody obtaining in (4), measure, draw the typical curve of antigen concentration and OD value;

(6) sample is measured: sample to be tested is measured, drawn the content of antigen in sample to be tested by typical curve.

Further, in step (1), be between the site of EcoR I by swamp eel estrogen receptorαgene being inserted into pMAL-c2x, Hind III, transform intestinal bacteria TB1, obtain fusion rotein; To after the MBP proteolytic cleavage of fusion rotein, obtain ER α recombinant protein again.

Further, in described step (4), enzyme used is horseradish peroxidase.

The present invention also provides the enzyme-linked immunologic detecting kit of swamp eel estrogen receptor alpha, include above-mentioned swamp eel estrogen receptor alpha protein and corresponding antibody thereof, described antibody is to prepare in mouse immune reaction by swamp eel estrogen receptor alpha protein.

The present invention also provides the application of swamp eel estrogen receptorαgene in swamp eel ER α acceptor detects.

beneficial effect

This test adopts RT-PCR method to obtain ER α sequence in swamp eel sexual gland, select the corresponding cDNA sequence of aminoacid sequence that its antigenicity is stronger to be cloned into the sequence that is about 945 bp as template, and be cloned in pMAL-c2x expression vector, induce by IPTG, the purifying of Amylose-sepharose post, obtain the purer immunocompetent recombinant protein that has, after adopting Factor Xa proteolytic cleavage, being further purified, obtain and can be used as immunogenic ER alpha expression albumen.After immune mouse, obtain the antiserum(antisera) of higher titre, and tentatively carry out immunohistochemical study, show this antiserum(antisera) can be special with swamp eel sexual gland in ER α protein binding, in oocyte membrane, be positive, lay the foundation for further studying the effect such as oestrogenic hormon and ER α thereof in swamp eel gender development.The present invention has also prepared ELISA detection kit taking this antibody as basis.

Brief description of the drawings

Fig. 1 is RT-PCR product agarose electrophoresis figure, and M swimming lane is marker, and 1 swimming lane is RT-PCR product;

Fig. 2 is the electrophorogram in the restricted enzyme cutting analysis of recombinant plasmid pMAL-ER α, and M swimming lane is marker, and 1 swimming lane is that pMD18-T-ER α is through EcoRI and Hind III double digestion product;

Fig. 3 is the electrophorogram that the SDS-PAGE of ER alpha fusion protein in identification of escherichia coli analyzes, and 1 swimming lane is E.coli TB1; 2-3 swimming lane: TB1/pMAL induces 4h through 0.3M, 1M IPTG respectively; 4 swimming lanes: TB1/pMAL-ER α is through 0.3M IPTG induction 4h.

Fig. 4 is the electrophorogram in protein immunoblot test, and M swimming lane is marker; 1 swimming lane is that MBP albumen 2 swimming lanes of TB1/pMAL abduction delivering are the recombinant proteins of TB1/pMAL-ER α abduction delivering; The TB1 lysate of 3 swimming lane inductions.

Fig. 5 is that the antibody titers after mouse immune ER α and physiological saline distributes

Fig. 6 A～Fig. 6 D is that mouse-anti ER α polyclonal antibody carries out swamp eel sexual gland immunohistochemical analysis, wherein, and Fig. 6 A: ovary, Fig. 6 B: ovary contrast, Fig. 6 C: spermary, Fig. 6 D: spermary contrast (in figure, O: ovum, GF: genital fold, SP: smart folliculus)

Fig. 7 A is the western blot analytical results of the gene protein horizontal expression of ER α in swamp eel gonadal tissue; X-coordinate: 1 ovary. 2. spermary; Ordinate zou: the gray level ratio of gene and corresponding β-actin

Fig. 7 B is the protein expression semi-quantitative analysis result of ER α in swamp eel gonadal tissue; 1 ovary. 2. spermary;

Fig. 8 is the linear relationship chart of test kit linear verification.

Embodiment

By reference to the accompanying drawings the present invention is described in further detail below by embodiment.But it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (for example show with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or condition, " the molecular cloning experiment guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

the clone of embodiment 1 swamp eel estrogen receptor alpha sequence, the expression of albumen

1. material

1.1 experiment materials:

Experiment fish: swamp eel, purchased from jackshaft food market, Wuxi, is organized as sexual gland.Mouse: purchased from the ICR mouse of Zhejiang University's Experimental Animal Center.

1.2 reagent: T4 DNA ligase, Taq archaeal dna polymerase, gel reclaim test kit, EcoR I, Hind III etc. purchased from the precious biotech firm in Dalian, polyacrylamide, RNase inhibitor etc. are purchased from Shanghai Shen Neng lottery industry bio-engineering corporation, Amylose-sepharose post, Factor Xa, anti-maltose binding protein (MBP) monoclonal antibody are all purchased from Niu Yinglun biotechnology (Beijing) company, and other biochemical reagents are domestic analytical pure.Fu Shi is complete, Freund's incomplete adjuvant, and ELISA Sptting plate etc. are all purchased from Shanghai Sheng Gong biotechnology company limited.

1.3 instruments: PCR instrument is purchased from BioRad company, and what experimental result record and analysis of protein used is the gel imaging system of Kodak, and DNA sequence analysis uses DNAstar software, and full-automatic examining order is completed by Shanghai Bo Shang bio-engineering corporation.Enzyme mark determinator is purchased from TECAN company.

1.4 plasmids and bacterial strain: pMD18-T carrier is purchased from the precious biotech firm in Dalian, and expression plasmid pMAL-c2x, acceptor bacterium TB1 and JM109 are for preserving this chamber.

2 methods

Steroid hormone 17-β estradiol (estradiol, E2) is the important regulatory factor of the different physiological roles such as mediation reproduction, cell proliferation, differentiation, apoptosis, inflammation, metabolism, homeostasis.What mediation E2 participated in these functions is exactly estrogen receptor.Estrogen receptor alpha (ER α) is that the purifying from human breast cancer cell such as Green in 1986 is shared and obtained, and is studied at first a kind of estrogen receptor.And in bony fish, first fish ER α be from rainbow trout ( oncorhynchus mykiss) in separate and obtain.Up to now rainbow trout ( oncorhynchus mykiss), goldfish ( carassius auratus), zebra fish ( danio rerio), golden head porgy ( sparus auratus), Japanese eel ( anguilla japonica), Europe sole ( solea solea), hyoid tooth perch ( dicentrarchus labrax) with the research and the report that in the fishes such as Oreochromis aureus, have carried out the aspects such as the separating of ER α gene, clone and tissue expression.The ER α of swamp eel does not also have bibliographical information at present, and therefore we,, according to the sequence of other fish ER α genes, have designed pair of degenerate primers.

2.1 design of primers are reacted with RT.PCR: adopt Primer 5.0 to design pair of primers:

Upstream primer: 5'-gactacatgtgccctgctacaaaycartgyac-3'(SEQ ID NO.1)

Downstream primer: 5'-catgctgtacaggtgctccatnccytt-3'(SEQ ID NO.2)

Y is t or c

Reverse transcription primer AP is purchased from Shanghai Sheng Gong bio-engineering corporation.

Template is for extracting from the total RNA of swamp eel sexual gland, carry out synthetic the first chain cDNA of reverse transcription, taking reverse transcription product as template and synthetic upstream and downstream primer carry out pcr amplification, reaction conditions is: 94 DEG C of denaturation 3min, 94 DEG C of denaturation 30s, 59 DEG C of annealing 45s, 72 DEG C are extended 1.5min, 30 circulations, 72 DEG C are extended 7min.

The cloning and identification of 2.2 PCR products:

PCR product is identified through 0.8% agarose gel electrophoresis, recovery meets expects the fragment of object, 4 DEG C, recovery product and pMD18-T carrier is connected and spent the night, connect product and transform e. coli jm109, random picking bacterium is extracted plasmid, after EcoR I, the qualification of Hind III double digestion, positive colony is delivered to Shanghai Bo Shang biotech firm and carry out DNA sequencing.For sequencing result, the software such as DNAstar, CLUSTAL X is analyzed.

The agarose gel electrophoresis of RT-PCR product shows the object fragment of amplification to about 945bp, and as shown in Figure 1, this conforms to expection size electrophorogram.To reclaim product connects after pMD18-T carrier, transform e. coli jm109 bacterial strain, random picking 10 clones extract plasmid and carry out EcoRI and Hind III double digestion, enzyme is cut the band that result shows to have after pMD18-T-ER α enzyme is cut a treaty 945bp, show that ER α is successfully cloned in T carrier, send the order-checking of order-checking company by recombinant plasmid pMD18-T-ER α, sequencing result is as shown in SEQ ID NO.3, and sequencing result shows that the fragment that RT-PCR increases is ER α equally.

The sequence of relatively guarding by be cloned into the part of ER α gene from the ovary of swamp eel, homology comparison shows that the ER α gene of this sequence and other fish has higher homology, therefore can confirm that this gene is exactly the ER α gene of swamp eel.

The structure of 2.3 prokaryotic expression carriers

PMD18-T-ER α clone and prokaryotic expression plasmid pMAL-c2x that forward is connected use respectively EcoR I, 37 DEG C of double digestions of Hind III, electrophoresis reclaims ER fragment and linearizing pMAL-c2x plasmid, according to the specification sheets design ligation of T4 DNA ligase, construction expression plasmid pMAL-ER α, transform e. coli jm109, random 5 clones of picking carry out enzyme and cut qualification: after extracting plasmid, carry out double digestion again with above-mentioned enzyme, enzyme is cut result as shown in Figure 2, the visible object fragment that has size to be about 945bp, shows successfully to have built expression vector pMAL-ER α.

Expression, the qualification of 2.4 goal gene in intestinal bacteria

Expression plasmid pMAL-ER α and empty carrier plasmid pMAL-c2x are transformed respectively to intestinal bacteria TB1, and transformed bacteria is spending the night containing 37 DEG C of shaking culture in 2 × YT substratum of penbritin (100 μ g/ml).Within second day, press 1:100 inoculation enlarged culturing, 37 DEG C of shaking culture to OD600 values reach 0.6 left and right, add inductor IPTG to be respectively 0.3 or 1 mmol/L to final concentration, continue 37 DEG C of gentle agitations and cultivate 4 hours, 4 DEG C centrifugal (5000rpm, 5min) collects thalline.Adopt 1 × PBS washing precipitation twice, finally add 1 appropriate × PBS by thalline Eddy diffusion.In the suspended substance of thalline, add isopyknic 2 × sds gel sample loading buffer (100mM Tris-HCl pH6.8; 200mM dithiothreitol (DTT); 4%SDS; 0.2% tetrabromophenol sulfonphthalein; 20% glycerine).Sample is placed in to boiling water and boils 5 minutes, smudge cells, makes the complete sex change of protein, then carries out the SDS-PAGE electrophoresis of 12% concentration, after electrophoresis, in coomassie brilliant blue R_250, dyes.Electrophoresis result as shown in Figure 3.

As shown in Figure 3, add after IPTG induction, proceed to and in the total bacterial protein extract of carrier pMAL, expressed molecular weight and be about the protein band (Fig. 3 swimming lane 2,3) of 42.5kD, occurred that molecular weight is about the new protein of 78.1kD (Fig. 3 swimming lane 4) and proceed in the total bacterial protein extract of recombinant plasmid.Because the MBP molecular weight of pMAL expression plasmid coding itself is 42.5kD, and ER alpha molecule amount is about 35.6kD, and therefore the molecular weight of fusion rotein should be about 78.1kD in theory.This is consistent with electrophoresis result, shows that recombinant expression vector is after IPTG induction, and target protein is expressed, and there is no corresponding protein expression in intestinal bacteria TB1.Target protein expression amount, through software analysis, has approximately reached 42.3% of bacterial expression Tot Prot.The Western blot test of carrying out taking the antiserum(antisera) of anti-MBP albumen shows that in size be 42.5 and visible two protein bands (swimming lane 2,3 in seeing Fig. 4) clearly in 78.1KD place.The aminoacid sequence that SEQ ID NO.3 derives is as shown in SEQ ID NO.4.

Acquisition, purifying and the cracking of 2.5 fusion roteins

Expression plasmid pMAL-ER α and empty carrier plasmid pMAL-c2x are transformed respectively to intestinal bacteria TB1, and transformed bacteria is spending the night containing 37 DEG C of shaking culture in 2 × YT substratum of penbritin (100 μ g/ml).The 10ml bacterium enlarged culturing of getting again incubated overnight in 1L LB(containing glucose) in, 37 DEG C of shakes are cultivated after 3h, add IPTG to final concentration be 0.3mM, then continue to cultivate 2h, the centrifugal 20min of 4000g collects thalline, is resuspended in 50ml post damping fluid.Adding final concentration is 1mg/mL N,O-Diacetylmuramidase, ice bath 30min.Finally adding final concentration is 5 μ g/mL DNA enzyme and RNA enzymes, hatches 10min for 4 DEG C.The centrifugal 30min of 9000g, shifts supernatant for subsequent use, contains the recombinant protein of expression in supernatant.

Supernatant is splined on to the Amylose-sepharose affinity column that balance is good, and level pad is 1 × PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na ₂hPO ₄, 1.8 mM KH ₂pO ₄pH 7.3), balance is to baseline, with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) wash-out, in elutriant, be the fusion rotein after purifying, last SDS-PAGE electrophoresis detection, only having molecular weight is the single band of 78.1kD left and right, obviously bigger than normal than the molecular weight of MBP albumen, show that the fusion rotein effect that purifying obtains is better.

Get that in the recombinant protein that purifying is good, to add Factor Xa be 200 μ g/mL to final concentration, room temperature effect 4h, removes bmp protein, the same Amylose-sepharose affinity column of crossing, use level pad wash-out, make equally SDS-PAGE electrophoresis detection, being single molecular weight is 35.6kD band.

Correlative study shows that the albumen of exogenous gene expression in intestinal bacteria mainly exists with inclusion body form, in order to obtain the active soluble proteins high compared with strongly expressed amount, the process that need to carry out a series of denature and renature to inclusion body could obtain the target protein of certain activity, the prokaryotic expression system that this research adopts is commercial pMAL-c2x carrier, this belt carrier is conducive to the MBP albumen of purifying, thereby this label protein is easy to be separated by proteolytic enzyme cutting, its commercial Amylose-sepharose purifying pillar purification efficiency is very high simultaneously, therefore we have obtained purer recombinant expression protein.

the preparation of embodiment 2 ER α receptor protein detection kit

The sero-fast preparation of 2.1 expression product

After the ER α recombinant protein reclaiming is mixed with 1 appropriate × PBS, get the antigen of 0.2ml(approximately 20 μ g) 6 female BALB/c mouse of abdominal injection (4-5 age in week), before immune mouse, first collecting part blood sample, as negative serum.Immunity in every two weeks once, starts blood sampling (mouse a few hours before blood sampling be should give fasting) for the first time after immunity.Blood sample collection be collected in through sterilizing, not containing being positioned over 4 DEG C of refrigerator overnight in the centrifuge tube of antithrombotics, make it to solidify, separate out faint yellow antiserum(antisera).Antiserum(antisera) is transferred in another pipe, and blood clot is with the centrifugal 10min of 1500g, and sucking-off supernatant liquor is incorporated in the antiserum(antisera) pipe of collecting.Antiserum(antisera) part is held in-70 DEG C of refrigerators for subsequent use.

2.2 sero-fast mensuration

2.2.1 directly enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA)

ER α recombinant protein (antigen) is diluted in to coated damping fluid (0.1M Na ₂cO ₃, PH9.6) in, final concentration is 5ng/ μ l, every hole adds 50 μ l.And set up blank group (only adding coated damping fluid).After 4 DEG C of coated spending the night.With 1 × PBST(1 × PBS+ (0.1%) Tween 20) under room temperature, wash each 5min three times.Then every hole adds 200 μ l confining liquids (10% calf serum is dissolved in 1 × PBS), in 4 DEG C of refrigerators, seals and spends the night.After 1 × PBST washing three times, add primary antibodie (serum sample by 1:100 or 1:200 doubling dilution in 1 × PBS), every hole 100 μ l, negative hole adds 100 μ l 1:200 negative serums, and blank well adds 100 μ l 1:100 sample serum.Hatch 2 hours for 37 DEG C.Adopt 1 × PBST washing three times.Add ELIAS secondary antibody, ELIAS secondary antibody by specification is diluted in 1 × PBS as 1:1000, every hole 50 μ l, 37 DEG C, 2h.Colour developing, nitrite ion (5mg OPD (O-Phenylene Diamine); 4 μ l 30% H ₂o ₂; 10ml substrate buffer solution (0.2M Na ₂hPO ₄, 0.1M Trisodium Citrate)), every hole 100 μ l, 37 DEG C, develop the color after 10 minutes, add 50 μ l 2M H ₂sO ₄termination reaction.Sentence read result in microplate reader.

To ICR mouse immune originality test-results: just exempt from after 1 week, the specific antibody of the protein induced generation of ER α in the serum of immune group mouse, just can be detected, after booster immunization, the 7th week antibody titers rises to 1.158 ± 0.232(Fig. 5), reach maximum, and blank group (injecting normal saline group) can not produce specific antibody, and immune group and blank group antibody titer significant difference (P<0.05).

Detect the recombinant antigen of preparation and the cross reaction of antibody with conventional Salmonella method.The recombinant expression protein obtaining with this research is with the coated solid enzyme target of pH 9.5 carbonate buffer solution dilution, fully after washing, using the antibody of commercially available people, mouse, mandarin fish and above-mentioned purifying as primary antibodie, carry out ELISA reaction, 37 DEG C of reactions are after 1 hour, react with corresponding ELIAS secondary antibody, 100 μ 1/ holes, 37 DEG C of reactions are after 1 hour, and automatic washer is washed plate three times, 5 minutes/time, carry out color reaction.Result shows that people, mouse, mandarin fish estrogen receptor antibody can not react with the recombinant antigen of preparation, and the ELISA value of mensuration is 0, and the recombinant antibodies of preparation can react with recombinant antigen, its result and negative control significant difference.

2.2.2 immunohistochemical analysis

Adopt SuperPicTureTM method (the brown reaction method of DAB) to carry out image formation, mouse-anti swamp eel estrogen receptor antibody is by preparing in step 2.1, extent of dilution is 1:100, and the extent of dilution of the sheep anti-mouse antibody (green skies biotechnology research institute) of horseradish peroxidase-labeled is 1:150.DAB colouring reagents box is purchased from the green skies, Jiangsu biological reagent company, and control experiment sheet substitutes first antibody with normal mouse serum and hatches, and carries out Hematorylin and redye.

Application immunohistochemical methods SuperPicTure ^tMthe section of method (the brown reaction method of DAB) dyeing, the colourless or light brown of background.ER alpha immunization reacting positive material is the brown and brown yellow granule (shown in Fig. 6 A, C) that the depth does not wait, mainly be positioned on cytolemma, fine particulate, and in control group, the nucleus of cell is redyed into light blue violet (Fig. 6 B) by Hematorylin, there is not brown particle positive signal, consistent with potential result. again illustrate that ER Alpha antibodies is specific.

The immunohistochemical methods location preliminary study that adopts the polyclonal antibody of preparation to carry out, result shows that ER α is all positive in swamp eel ovary spermary tissue slice, therefore show preparation polyclonal antiserum can with swamp eel ER α albumen generation immune response, further observation can be found each interim immune positive reaction that is all such as oocyte membrane, oocyte nuclei in ovary.

2.2.3 Western blot analyzes

Extract respectively the total protein (spermary and ovary are respectively got 6 samples) of swamp eel spermary, ovary tissue, measure protein concentration according to Bradford method, when SDS-PAGE electrophoresis, adopt the applied sample amount of 100 μ g.Primary antibodie was by dilution in 1: 500, and 4 DEG C of hybridization are spent the night, and spent the night by 4 DEG C of hybridization in 1: 3000 purchased from β-actin primary antibodie of Sigma company, anti-ly hybridized 1h by 1: 1000 at 4 DEG C purchased from two of the anti-mouse of Santa Cruz company.The concrete grammar of Western blot is shown in molecular cloning.Utilize gray analysis software UnScanIt gel 6.1 to carry out semi-quantitative analysis to Fig. 7 A, by calculating the gray level ratio of specific gene and corresponding β-actin in swimming lane, and draw accordingly the column type figure (Fig. 7 B) of genetic expression, experimental result shows, ER α gene all has expression in swamp eel spermary and ovary, strong in expression ratio ovary in spermary, mean ratio in spermary is 0.507 ± 0.03, mean ratio in ovary is 0.394 ± 0.02, has significant difference (P<0.05).

More much higher clone's antiserum(antisera) can obtain tiring after the recombinant protein immune mouse that use obtains with this expression system in this research, detect this antiserum(antisera) through western blot and can be good at identifying ER α albumen, show that it has good immunologic opsonin.Certainly in prokaryotic expression system, expressing eukaryotic gene also there is certain risk, as after transcribing, can not carry out glycosylation, the modification such as methylate, thereby there is no activity after causing a lot of Eukaryotic genes not express or express, if its wild-type protein molecular weight of the structural protein VP28 of white spot syndrome virus (WSSV) is 28kDa, the molecular weight of the VP28 albumen of prokaryotic expression is 22kDa, the reason making a difference may be that wild-type has the glycosylation modified effect of Denging, and the posttranslational modification of the VP28 albumen of prokaryotic expression is incomplete (Madhabananda S, Frank W. Differential expression of estrogen receptor-β and estrogen receptor-α in the rat ovary [J]. Endocrinology, 1999, 140 (2): 963 971.).Next is that different expression systems has the preferences of selecting different codons, thereby causes many genes can not express in prokaryotic system, is difficult to obtain experimental result.But this test carry out process in be not subject to the impact of these unfavorable factors, recombinant protein has good antigenicity, has finally obtained antiserum(antisera).

The purifying of 2.3 antibody

Get the serum obtaining in 4.2m1 step 2.1, add equal-volume 4.2 m1 PBS (0. 01M, pH7.8), mix.Add ammonium sulfate saturated solution 8.4 m1 while stirring, make into 50% saturation ratio, put 60 minutes for 4 DEG C.Centrifugal 20 minutes of 4000 rpm, abandon supernatant, with volume ratio be saturated ammonium sulphate: the mixed solution washing and precipitating of PBS=4.2:4.2 secondary, remove supernatant.Precipitation is dissolved in 4.2 m1 PBS, drips saturated ammonium sulphate solution 2.1 ml, make into 30% saturation ratio, put 30 minutes for 4 DEG C, the centrifugal supernatant that goes, repeats this step once.Desalination: precipitation is dissolved in 0.84 m1 physiological saline, moves in dialysis tubing, dialyses and there is no SO4 in physiological saline to lye ^2-, use 1% BaC1 ₂solution titration detects SO4 ^2-.Change again dialyzate one time, make antibody purification crude product 1.5m1.

The mark of 2.4 antibody purifications

10 mg horseradish peroxidases are dissolved in to 0.2 ml 1.25% glutaraldehyde-0.1 M PBS (pH 6.8), room temperature effect 18 hours, glutaraldehyde is gone in dialysis.Add physiological saline to 1 ml, add the antibody purification preparing in 5 mg steps 2.3, add 0.1 ml 1M carbonate buffer solution (pH 9.6), put 24 hours for 4 DEG C.Add 0.1 m1 0.2M Methionin, room temperature effect 2 hours, in 0.15 M PBS (pH7.2), fully dialysis.The centrifugal precipitation of going, supernatant is the antibody of horseradish peroxidase-labeled.

The preparation of 2.5 test kits

2.5.1 coated: dilute aforementioned purifying traget antibody (1:800) with pH 9.5 carbonate buffer solutions, coated 96 orifice plates, 100 μ 1/ holes, 4 DEG C of coated spending the night, negative control hole is established 4/plate, is coated with normal mouse serum.4 holes/plate is established in blank hole, adds coating buffer 100 μ 1.Coating buffer: 0.1 M Na ₂cO ₃buffered soln (pH 9. 6).

2.5.2 wash plate, wash plate three times with automatic washer, 5 minutes/time, washings was TTBS (20 mM Tris-0.8% NaC1-0. 1% Tween 20, (pH 7. 5).

2.5.3 sealing: with there is no the site in conjunction with upper antibody on 1% BSA sealase target.200 μ l/ holes, room temperature effect 1 hour, washes plate.

2.5.4 add ER α recombinant protein or standard protein diluent sample diluting liquid: 1%BSA-TTBS, room temperature effect 1 hour.Negative control hole adds respectively 100,11. 1,1.235 ng/m1 standard protein solution.Wash plate; In every hole, add again enzyme labelled antibody (1:600), 100 μ 1/ holes.

2.5.5 add substrate: in substrate, contain: 0. 1 M-citrate buffers-0. 05% (wt/v O-Phenylene Diamine-0. 025% (V/V) H ₂o ₂dark place reaction 20 minutes, adds 2 N H ₂sO ₄termination reaction, 50 μ l/ holes, read OD490 after 10 minutes, blank tube zeroing.

Utilize the sample of different ER α recombinant protein concentration to measure after OD490, draw the typical curve of relation between antigen concentration of specimens and OD490 value, then testing sample is measured in the same method, look into typical curve and obtain the concentration of antigen in sample to be tested.

Detect the estrogen receptor alpha albumen of different fish bodies with the test kit of preparation.Get the homogenate buffer (homogenate buffer: 1 mol/L Tris-HCl (pH=7.6) 10mL, NaCl 2.925 g that the sexual glands such as 5g tilapia, jian carp, mandarin fish, hybridized prussian carp, swamp eel, liver, kidney etc. add 5mL precooling; EDTA:0.05 g and 100 mmol/L PMSF 0.5 mL, be settled to 500 mL with distilled water; ) ice bath homogenate, (centrifugal force=12 000 × g) get the detection liquid of supernatant liquor as ELISA after 30 min centrifugal at 4 DEG C, detected result show test kit can only detect swamp eel sexual gland, liver, nephridial tissue in estrogen receptor alpha, the estrogen receptor alpha in tilapia, jian carp, mandarin fish, hybridized prussian carp can not be detected, show that test kit specificity is stronger, can not produce cross reaction with other fish, show that the ELISA test kit that this experiment is set up has higher reliability and stability simultaneously.

2.6 test kits detect performance verification

2.6.1 repeated

The ER α recombinant protein extracting respectively after the purifying obtaining in ER α albumen and embodiment 1 in the gonadal tissue of 3 parts of same swamp eel 5g detects according to the method for step 2.5 respectively, OD value result is as table 1, statistical study shows that the relative standard deviation of each class value is all less than 2%, shows that the test kit of preparation has good repeatability.

Table 1 test kit replica test result

2.6.2 linear

ER α recombinant protein after purifying is mixed with to 5 concentration gradients of 0.3,0.6,1.2,2.4,4.8 μ g/mL, measures according to the indirect competitive ELISA condition after optimizing, ordinate zou is OD value, and drawing standard curve (see figure 8) repeats 5 times.The regression equation of ELISA detection method and the index of correlation are: y=0.0005x-0.0535, r ²=0.9979, linearity range is 0.05～2.2 μ g/L.

2.6.3 accuracy

The accuracy of test kit represents by the interpolation rate of recovery.Restructuring ER α albumen is added respectively in the fewer muscle tissue of swamp eel ER α protein expression, make its concentration reach 0. 5 μ g/kg and 1. 0 μ g/kg, after sample pre-treatments, measure with this test kit, every batch of 5 repetitions, 3 batches of replications, calculate recovery rate and variability.The results are shown in Table 2 demonstrations, this test kit in the rate of recovery of each tissue all in 69. 8%～118. 6% scopes, in batch with interassay coefficient of variation <22. 3%.

Table 2 test kit accuracy testing result

SEQUENCE LISTING

<110> China Aquatic Science Research Academy Fresh Water Fishery Research Center

<120> swamp eel estrogen receptorαgene, proteins encoded and enzyme-linked immune detection method

<130> none

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 32

<212> DNA

<213> artificial sequence

<400> 1

gactacatgt gccctgctac aaaycartgy ac 32

<210> 2

<211> 27

<212> DNA

<213> artificial sequence

<220>

<221> misc_feature

<222> (22)..(22)

<223> n is a, c, g, or t

<400> 2

catgctgtac aggtgctcca tnccytt 27

<210> 3

<211> 945

<212> DNA

<213> ERa

<400> 3

gggtctggag gggtagtcac tgtggacttc ctggaaggga cgtatgacta tgctgctcca 60

acccctgctt cgactcctct ctacaaccac tcatccactg gctactactc tgctcctcta 120

gatgcccccg gaccactctc tgatggcagc ctccagtccc tgggcagtgg gtctgcaagt 180

cctcttgtgt ttgtgccctc tagcactcag ctcagcccat ttatgcaccc acctagtcag 240

cactctctgg agaccacctc aacatccgtc tacaggtcca gtcagcagcc ggtatccagg 300

gaggaccagc gtggcaccgg tgacgactcg cacggtgtgg gggagtcggg ggctggagct 360

gagaacaggg ggtttgaaat ggccaaagag acacgtttct gtgccgtgtg cagtgacttt 420

gcctctgggt accactatgg ggtgtggtcc tgtgagggct gcaaggcctt cttcaagagg 480

agcatccagg gtcacaatga ctatatgtgc ccagcaacca atcagtgtac catagacaga 540

aatcggagga agagctgcca ggcttgtcgt cttaggaagt gttatgaagt gggcatgatg 600

aaaggaggtg tgcgtaagga ccgcagccgt gttttgcggc gtgacaaaca acggatttgc 660

accagtgaga gagtcaaggc ctctaaggac ctggagcaca gaacagcgcc tcctcaggat 720

ggaaggaaac agagcagaag taataatggc ggtggtgaag gaggaagatc attgtttact 780

gccctgcgtc ctgaccaggt gctcctcatg ccccagggtg cagagccccc aatactctgc 840

tcccgtcaga agctgaacca accgtacact gagatcacca tgatgagcct gcttaccaac 900

atggccgaca aggagctggt ccacatgatt gcttgggcca agaag 945

<210> 4

<211> 315

<212> PRT

<213> ER receptor

<400> 4

Gly Ser Gly Gly Val Val Thr Val Asp Phe Leu Glu Gly Thr Tyr Asp

1 5 10 15

Tyr Ala Ala Pro Thr Pro Ala Ser Thr Pro Leu Tyr Asn His Ser Ser

20 25 30

Thr Gly Tyr Tyr Ser Ala Pro Leu Asp Ala Pro Gly Pro Leu Ser Asp

35 40 45

Gly Ser Leu Gln Ser Leu Gly Ser Gly Ser Ala Ser Pro Leu Val Phe

50 55 60

Val Pro Ser Ser Thr Gln Leu Ser Pro Phe Met His Pro Pro Ser Gln

65 70 75 80

His Ser Leu Glu Thr Thr Ser Thr Ser Val Tyr Arg Ser Ser Gln Gln

85 90 95

Pro Val Ser Arg Glu Asp Gln Arg Gly Thr Gly Asp Asp Ser His Gly

100 105 110

Val Gly Glu Ser Gly Ala Gly Ala Glu Asn Arg Gly Phe Glu Met Ala

115 120 125

Lys Glu Thr Arg Phe Cys Ala Val Cys Ser Asp Phe Ala Ser Gly Tyr

130 135 140

His Tyr Gly Val Trp Ser Cys Glu Gly Cys Lys Ala Phe Phe Lys Arg

145 150 155 160

Ser Ile Gln Gly His Asn Asp Tyr Met Cys Pro Ala Thr Asn Gln Cys

165 170 175

Thr Ile Asp Arg Asn Arg Arg Lys Ser Cys Gln Ala Cys Arg Leu Arg

180 185 190

Lys Cys Tyr Glu Val Gly Met Met Lys Gly Gly Val Arg Lys Asp Arg

195 200 205

Ser Arg Val Leu Arg Arg Asp Lys Gln Arg Ile Cys Thr Ser Glu Arg

210 215 220

Val Lys Ala Ser Lys Asp Leu Glu His Arg Thr Ala Pro Pro Gln Asp

225 230 235 240

Gly Arg Lys Gln Ser Arg Ser Asn Asn Gly Gly Gly Glu Gly Gly Arg

245 250 255

Ser Leu Phe Thr Ala Leu Arg Pro Asp Gln Val Leu Leu Met Pro Gln

260 265 270

Gly Ala Glu Pro Pro Ile Leu Cys Ser Arg Gln Lys Leu Asn Gln Pro

275 280 285

Tyr Thr Glu Ile Thr Met Met Ser Leu Leu Thr Asn Met Ala Asp Lys

290 295 300

Glu Leu Val His Met Ile Ala Trp Ala Lys Lys

305 310 315

Claims (9)

1. swamp eel estrogen receptor alpha protein, its aminoacid sequence is as shown in SEQ ID NO.4.

2. the encode swamp eel estrogen receptorαgene of protein claimed in claim 1, its nucleotide sequence is as shown in SEQ ID NO.3.

3. contain the carrier of swamp eel estrogen receptorαgene claimed in claim 2.

4. carrier according to claim 3, refers to the pMAL that inserts above-mentioned swamp eel estrogen receptorαgene between EcoR I and Hind III site.

5. for the enzyme-linked immune detection method of swamp eel estrogen receptorαgene, it is characterized in that, comprise the steps:

(1) preparation of antigen: by swamp eel estrogen receptorαgene insertion vector, and at expression in escherichia coli, the ER α recombinant protein obtaining, purified rear as antigen;

(2) preparation of antibody: the antigen immune mouse obtaining in (1) is prepared to anti-yellowing eel estrogen receptor Alpha antibodies;

(3) purifying of antibody: the antibody of gained in (2), through centrifugal, precipitation, desalination, is obtained to antibody purification;

(4) mark of antibody purification: with the antibody purification of gained in enzyme labelling (3), obtain enzyme labelled antibody;

(5) drafting of typical curve: use the antigen coated on solid phase carrier of different concns, after being combined by the enzyme labelled antibody obtaining in (4), measure, draw the typical curve of antigen concentration and OD value;

(6) sample is measured: sample to be tested is measured, drawn the content of antigen in sample to be tested by typical curve.

6. the enzyme-linked immune detection method for swamp eel estrogen receptorαgene according to claim 5, it is characterized in that: in step (1), be between the site of EcoR I by swamp eel estrogen receptorαgene being inserted into pMAL-c2x, Hind III, transform intestinal bacteria TB1, obtain fusion rotein; To after the MBP proteolytic cleavage of fusion rotein, obtain ER α recombinant protein again.

7. the enzyme-linked immune detection method for swamp eel estrogen receptorαgene according to claim 5, is characterized in that: in described step (4), enzyme used is horseradish peroxidase.

8. the application of swamp eel estrogen receptorαgene claimed in claim 2 in swamp eel ER α acceptor detects.

9. the enzyme-linked immunologic detecting kit of swamp eel estrogen receptor alpha, it is characterized in that: include swamp eel estrogen receptor alpha protein claimed in claim 1 and corresponding antibody thereof, described antibody is to prepare in mouse immune reaction by swamp eel estrogen receptor alpha protein.