CN103468735B - The preparation of magnificent river crab metallothionein(MT) (rcMT) and the monoclonal antibody thereof of recombinating - Google Patents
The preparation of magnificent river crab metallothionein(MT) (rcMT) and the monoclonal antibody thereof of recombinating Download PDFInfo
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Abstract
The invention provides a kind of preparation method of easy and simple to handle, with low cost, purity is high, active good solubility is recombinated magnificent river crab metallothionein(MT) (rcMT), the method, first using phoA plasmid as expression vector, builds phoA-MT recombinant expression vector; Transformation of E. coli BL21 again, builds colibacillus engineering strain phoA-MT/BL21, and is inoculated in LB substratum by engineering bacteria and prepares seed, then dilute bacterium to low-phosphorous substratum induction, express, utilize Ni? sepharose
tM6Fast? Flow affinitive layer purification obtains the magnificent river crab metallothionein(MT) of restructuring.Utilize the magnificent river crab metallothionein(MT) of restructuring to prepare anti-magnificent river crab metallothionein(MT) monoclonal antibody, this antibody susceptibility is high, high specificity, can be applicable to the ELISA detection by quantitative of metallothionein(MT).
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of preparation of recombinate river crab metallothionein(MT) and monoclonal antibody thereof.
Background technology
Metallothionein(MT) (Metallothionein, MT) be that a class is widely distributed, molecular weight (6000 ~ 7000Da), be rich in halfcystine, without die aromatischen Aminosaeuren and Histidine, can by metal inducement and in conjunction with the special non-enzymatic proteins of multiple atoms metal.Margoshes with Vallee of Harvard University was separated first and named from horse kidney nineteen fifty-seven.Research subsequently finds, MT is extensively present in animal, plant and microbial world.Research shows, MT participates in the storage of trace element in body, transhipment and metabolism, there is the effect of antagonism ionizing rays, scavenging free radicals and heavy metal removing toxic substances, also grow with body growth, delay senility and some disease relevant.MT also plays an important role in the generation, development of tumour more to have scholar to find in recent years.As a kind of medical protein resource with broad prospect of application, the additive of a kind of desirable healthcare products, makeup, the mass-producing preparation of metallothionein(MT) is one of focus of application and development always.
Existing metallothionein(MT) extracts mainly through mammalian liver, yields poorly, cost is high, complex process is (see CN1085045A; CN1583790A).Also have report to extract from yeast, neurospora or Cordyceps, but it is lower to be limited by expression level equally, is difficult to obtain desirable yield (see CN1130687A; CN118098A; CN102199645A; CN102206693A).Along with the development of biotechnology, utilizing protein engineering means to express recombinant protein becomes the first-selection obtaining a large amount of MT albumen.Particularly escherichia expression system, the maturation that possesses skills, expression amount be high, be easy to cultivate and operation and the many advantages such as production cost is low.But utilize this expression system, be difficult to the MT obtaining great amount of soluble.Mainly due to MT molecular weight, and be rich in halfcystine, very easily form the inclusion body of inactive.Have bioactive MT for obtaining, have to pass through complicated change, renaturation process, program is loaded down with trivial details, and the target protein of large losses.Simultaneously MT is unstable in intestinal bacteria, and demetalization sulfoprotein, is difficult to obtain higher productive rate to problems such as the tolerance differences of metal ion to the toxicity of cell and cell always.For addressing this problem, also have scholar utilize integration technology by MT gene with have assist albumen correctly to fold fusion rotein if glutathione S-transferase (GST) or small molecules ubiquitin sample modified protein (SUMO) are (see CN1348010A; CN101144093A) amalgamation and expression.Although this method solves the problem of albumen solubility to a certain extent, improve protein yield, due to proteolytic enzyme costly, enzyme cuts the problems such as consuming time, limit it and use.
Meanwhile, due to the restriction of quantitative detecting method, the application of MT in medical science, toxicology, environment remediation etc. also fails to develop further.The measuring method of current MT content mainly contains plioweld, electrochemical process, red, orange, green, blue, yellow (ROGBY) etc., because of the singularity of MT albumen, said determination method none not by MT in conjunction with the impact of the factors such as metal species, MT isomorph polymorphism to result produce deviation.Enzyme-linked immunosorbent assay (ELISA) is a kind of detection technique that the high susceptibility of the specificity of antigen antibody reaction and enzymatic reaction is organically combined, the method susceptibility is high, specificity good, simultaneously compared with other detection meanss, operate simple and easy, low cost.The key of ELISA detection method is the preparation of antibody.Also there is ELISA to detect the relevant report of metallothionein(MT) both at home and abroad at present, but all adopt polyclonal antibody to realize.But polyclonal antibody poor specificity, limits throughput, physico-chemical property heterogeneity, be difficult to use in and set up quick, accurate, standardized enzyme-linked immune detection method.
Sinopotamon henanense (Sinopotamonhenanense) is transformed by ocean crab is polynary, belongs to a special branch in Crustachia, Decapoda.As the unicellular lower eukaryote moving in water body stratum basale, river crab is directly in the face of being deposited on the metal ion of water body, and the effect of its metallothionein(MT) more highlights.The present inventor is in early-stage Study, clone Sinopotamon henanense cDNA sequence first, obtain its coding region gene (Ma, W.L., Yan, T., He, Y.J., Wang, L., 2009.PurificationandcDNAcloningofacadmium-bindingmetallo thioneinfromthefreshwatercrabSinopotamonhenanense.Arch.E nviron.Contam.Toxicol.56,747-753.).Result shows, and Sinopotamon henanense MT cysteine content, up to 30.5%, without die aromatischen Aminosaeuren, exists the characteristic sequence Cys-X-Cys of invertebrates MT, 59 amino acid of encoding.As a kind of MT gene deriving from Sinopotamon henanense newly, comparatively Mammals MT compares, and river crab MT, in conjunction with metallic element kind with quantitatively present more outstanding diversity and the simplification of higher structure, has more tempting application prospect.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of easy and simple to handle, with low cost, purity is high, active good solubility is recombinated magnificent river crab metallothionein(MT) (recombinantcrabmetallothionein, rcMT).
Another object of the present invention is to utilize a kind of anti-magnificent river crab metallothionein(MT) monoclonal antibody of restructuring magnificent river crab metallothionein(MT) preparation.The anti-magnificent river crab metallothionein(MT) monoclonal antibody susceptibility utilizing the inventive method to prepare is high, high specificity, can be applicable to the ELISA detection by quantitative of metallothionein(MT).
A kind of solubility is recombinated the preparation method of magnificent river crab metallothionein(MT) (rcMT), comprises the following steps:
1, phoA-MT recombinant expression vector is built
With the carrier T pGEM-MT containing Sinopotamon henanense metallothionein(MT) coding region gene (SEQIDNO:1) for template, utilize Auele Specific Primer: Forwardprimer:5'-G
cCATGGCcCCTGATCCTTGCTGC-3'(is containing NcoI restriction enzyme site) and Reverseprimer:5'-A
gGATCCgGGGCAGCAGGAGCAAG-3'(is containing BamHI restriction enzyme site) pcr amplification; Amplified production and C end introduce the phoA plasmid of 6histag after NcoI and BamHI double digestion, Transformed E .coliDH5 α competent cell after T4DNA ligase enzyme connects, and picking positive colony shakes bacterium, carry out bacterium colony PCR and plasmid enzyme restriction qualification, DNA sequencing;
2, the structure of engineering strain phoA-MT/BL21, induction and expression
The phoA-MT plasmid identified through checking order, according to standard method transformation of E. coli BL21, builds colibacillus engineering strain phoA-MT/BL21; Engineering bacteria is inoculated in LB substratum, 37 DEG C, 165r/min shaking culture 12h prepares seed; According to 2.5% dilution bacterium to low-phosphorous substratum induction, add 1mMZnSO simultaneously
4increase protein stability, 30 DEG C, 165r/min inducing culture 22h;
Described low-phosphorous culture medium prescription is as follows: 2.5g/L Tryptones, the female extract of 0.25g/L enzyme, 3g/L (NH4) 2SO4,5g/LNacl, 1g/LMgSO4,4g/L glucose;
3, rcMT protein purification and qualification
Collect thalline, ice-bath ultrasonic smudge cells, collected after centrifugation supernatant; Nisepharose after 0.22 μM of membrane filtration
tM6FastFlow affinitive layer purification; Target protein is through 3000MWCO super filter tube ultrafiltration and concentration; SDS-PAGE, Westernblot and uv scan qualification.
Utilize above-mentioned recombinant protein to prepare a kind of anti-magnificent river crab metallothionein(MT) monoclonal antibody, realize by following steps:
1, rcMT protein immunization mouse
Adopt BCA standard measure rcMT albumen; Select water-soluble Quicklyantibodu adjuvant and rcMT albumen to mix, according to 100 μ l, 20 μ g antigen consumption intramuscular injection carry out fundamental immunity to mouse, press the same manner booster immunization after 21d, and 35d afterbody blood sampling ELISA measures; After serum titer reaches requirement, according to 500 μ l, 50 μ g antigen consumption abdominal injections impact immunity;
2, cytogamy and cloning
Choose the mice spleen cell that serum titer is the highest, utilize PEG1500 reagent to carry out external fusion with myeloma cell, utilize the screening of indirect non-competing ELISA method using rcMT as envelope antigen, obtain the hybridoma cell strain of the anti-rcMT of stably excreting through repeatedly subclone;
3, ascites preparation
Using whiteruss as sensitiser, choosing 14 ~ 22 week age BALB/c mouse is laboratory animal, with 0.5ml/ injecting fluid paraffin, after the hybridoma cell strain enlarged culturing obtain screening, with 1 × 10
5~ 1 × 10
7production ascites in the inoculum density inoculation hybridoma cell strain to mouse peritoneal of individual/ml; Ascites is collected, the centrifugal 15min of 3000rpm, results supernatant after 10 ~ 12d;
4, monoclonal antibody-purified, qualification
The ascites supernatant of results, after MEPHyperCel hydrophobic chromatography post is slightly carried, then carries out purifying through ProteinA-SepharoseCL-4B affinity chromatography; Measure antibody purity, titer of ascites, identify its antibody subtype and specificity.
Compared with prior art, the advantage that the present invention has is:
1) alkaline phosphatase promoter (phoA) and signal sequence secreting, expressing China river crab metallothionein(MT) in intestinal bacteria is utilized, by phoA promoter regulation MT albumen soluble-expression between periplasmic, overcome the cytotoxicity defect that MT easily forms inclusion body and causes with melts combine in born of the same parents.While the low-phosphorous abduction delivering of rcMT, add a certain amount of zine ion, improve stability and the purity of metallothionein(MT).The metallothionein(MT) utilizing the present invention to prepare only containing 6his label, is cut process without the need to enzyme, is utilized nickel post single step purification to obtain.
2) adopt above-mentioned solubility rcMT as antigen, the anti-magnificent river crab metallothionein(MT) monoclonal antibody specificity that screening obtains is high.Compared with polyclonal antibody, its physicochemical character height is homogeneous, and biological activity is single, is convenient to artificial process and quality control; The high specificity be combined with antigen, decreases possible cross reaction, makes test-results more credible.According to the monoclonal antibody that the inventive method is obtained, for developing a kind of quick, accurate, standardized MT enzyme-linked immunologic detecting kit.
Accompanying drawing explanation
Fig. 1 is phoA-MT recombinant plasmid bacterium colony PCR and double digestion qualification result.Mr is DNAMarker; 1 is that recombinant plasmid phoA-MT is through BamHI, XhoI double digestion; 2 is engineering bacteria phoA-MT/BL21PCR qualification product.
Fig. 2 is phoA-MT recombinant plasmid partial sequence sequencing result.
Fig. 3 is the SDS-PAGE electrophoretic analysis of phoA-MT/BL21 expression product and purifying.Mr is protein standards; 1 for not induce full bacterium; 2 is the full bacterium of low-phosphorous induction; 3 is the target protein of purifying.
Fig. 4 is the Westernblot qualification of recombinant protein rcMT.
Fig. 5 is the ultraviolet spectrometry scanning spectra of recombinant protein rcMT.
Fig. 6 is the SDS-PAGE electrophoretic analysis of the monoclonal antibody-purified sample of anti-rcMT.Mr is protein standards; 1,2 is HCLC column purification sample; 3 is the monoclonal antibody through ProteinA-SepharoseCL-4B column purification;
Embodiment
Embodiment 1 Sinopotamon henanense metallothionein(MT) recombinant expressed.
Step one: the selection of plasmid vector
Alkaline phosphate promotor (phoA) and signal sequence secreting, expressing foreign protein in intestinal bacteria has some advantages (see " Molecular Cloning: A Laboratory guide " third edition), at utmost can keep the native conformation of foreign protein, secretion is more stable to the protein of outer pericentral siphon simultaneously.In phoA system, the alkaline phosphatase of phoA genes encoding intestinal bacteria phosphoric acid scavenge system, suppressed when excess phosphoric acid salt exists, under the state of hunger (bacterial growth enters the lag phase), phoA gene is induced gradually.This regulation and control make foreign protein be progressive in intracellular accumulation, unlike the accumulation rapidly of other promoter regulations.This chronic accumulation reduces bacterial expression system overload and forms the chance of inclusion body.Accumulate at a slow speed toxigenous foreign protein when also making to accumulate rapidly to be alleviated, obtain expression simultaneously.PhoA plasmid has ampicillin resistance gene, introduces 6histag through transformation C section.
Step 2: build phoA-MT recombinant expression vector
With the carrier T pGEM-MT containing Sinopotamon henanense metallothionein(MT) coding region gene (SEQIDNO:1) for template, utilize Auele Specific Primer: Forwardprimer:5'-G
cCATGGCcCCTGATCCTTGCTGC-3'(is containing NcoI restriction enzyme site) and Reverseprimer:5'-A
gGATCCgGGGCAGCAGGAGCAAG-3'(is containing BamHI restriction enzyme site) pcr amplification, amplification condition is as follows: 94 DEG C of denaturation 5min, with 94 DEG C of sex change 30s, 65 DEG C of annealing 50s, 70 DEG C extend 1min, then extend 10min, 3O circulation altogether, last 4 DEG C of preservations, product is through 15g/L agarose gel electrophoresis preliminary evaluation.Identify that correct PCR primer reclaims test kit (love pursue progress Bioisystech Co., Ltd) through glue and reclaims object fragment, utilize Nco I, BamH I double digestion, digestion products is connected by T4 ligase enzyme with the phoA plasmid through same double digestion.Connect product conversion bacillus coli DH 5 alpha competent cell, conversion fluid coats (containing penbritin 50 μ g/ml) on LB agar plate, selects positive colony, after bacterium colony PCR and enzyme cut (see figure 1) qualification, and order-checking.Sequencing result display (see figure 2), magnificent river crab MT gene successful clone to phoA carrier, by this recombinant plasmid called after phoA-MT.
Step 3: the structure of engineering strain phoA-MT/MM294, abduction delivering and purifying
Get above-mentioned plasmid according to standard method transformed competence colibacillus peptide e. coli bl21, build colibacillus engineering strain phoA-MT/BL21.Engineering bacteria is inoculated in LB substratum, 37 DEG C, 200r/min shaking culture 12h prepares seed.According to 2.5% dilution bacterium to low-phosphorous substratum (2.5g/L Tryptones, the female extract of 0.25g/L enzyme, 3g/L (NH
4)
2sO
4, 5g/LNaCl, 1g/LMgSO
4, 4g/L glucose) and induction, add 1mMZnSO simultaneously
4, 30 DEG C, 180r/min inducing culture 22h.Molybdate method surveys phosphorus content, and determine that phosphorus all runs out of, 8000r/min, 25min collect bacterium.Every 1g wet bacterium 5ml broken liquid (20mMTris, 500mM sodium-chlor, 1%Trionton, 10mM imidazoles pH7.8) suspend, ice bath stirs 30min, ice-water bath interval ultrasonic (360 ~ 400w) process is inviscid to bacterium liquid, 4 DEG C, the centrifugal 20min of 8000rpm, get full bacterium and carry out 16.5%Tricine-SDS-PAGE analysis, result is presented at about 8KD place band of expression, with the consistent (see figure 3) of molecular weight of albumen of the rcMT estimated, and target protein is mainly expressed in solvable mode.
The supernatant that upper step obtains utilizes Nisepharose after 0.22 μM of membrane filtration
tM6FastFlow affinity chromatography column separating purification.First level pad (20mMTris, 500mM sodium-chlor, 20mM imidazoles pH7.8) is used to balance Nisepharose
tM6FastFlow post, supernatant upper prop after then filtering.Baseline is washed till with level pad again after loading, then use elution buffer (20mMTris, 500mM sodium-chlor, 50mM imidazoles pH7.8) wash-out foreign protein, finally use elution buffer (20mMTris, 500mM sodium-chlor, 250mM imidazoles pH7.8) wash-out target protein peak.Elution peak component through 3000MWCO super filter tube ultrafiltration and concentration to storage liquid (0.01MTris-HCl, 0.2mMPMSF, pH7.8).
Step 4: the qualification of recombinant protein and concentration determination
Purified target protein is analyzed as the single band (see figure 3) of about 8KD through 16.5%Tricine-SDS-PAGE, and display has obtained the higher rcMT of purity.After purifying protein electrophoresis, with electroporation (Bio-Rad), the albumen in SDS-PAGE glue is gone on pvdf membrane, close with confining liquid (PBST+5% skim-milk) and spend the night, PBST washes film 4 times, 10min/ time, add Anti-6Histag rabbit source how anti-as primary antibodie, 4 DEG C are spent the night, and after PBST washes film 4 times, add two anti-(goat-anti rabbit AP traget antibody) room temperature reaction 2h, after washing film 4 times with PBST, finally with the colour developing of BCIP/NBT colouring reagents box lucifuge.Result shows, and the rcMT of purifying is because band 6his label is in 8KD place colour developing (see figure 4).
The rcMT ultrafiltration and concentration sample of purifying adopt UNICOUV-2102PC type ultraviolet-visible pectrophotometer under carry out full wavelength scanner (190nm-1100nm).Result shows, and rcMT has an absorption peak because being combined in 225nm place with zinc, and at 280nm without conventional protein characteristic absorption peak, meets Zn-MT ultra-violet absorption spectrum feature (see figure 5).
The preparation of the anti-magnificent river crab metallothionein(MT) monoclonal antibody of embodiment 2
Step one: mouse immune
The rcMT of purifying, using BSA as standard protein, utilizes BCA method to measure protein concentration.
(1) fundamental immunity:
Get and be stored in damping fluid (0.01MTris-HCl, 0.2mMPMSF, pH7.8) rcMT in is diluted to 120 μ g/ml, volume ratio according to 1: 1 fully mixes with Quicklyantibody water-soluble adjuvant (purchased from Kang Bi spring bio tech ltd, Beijing), intramuscular injection immune mouse, injection volume 0.1ml/ only.Immunity amount 0.1ml, 20 μ grcMT/ only, to sterilize injection site with cotton ball soaked in alcohol after injection, protect from infection.(2) booster immunization: after 21d, second immunisation mouse, immunity amount and immunization method the same.(3) immunity is impacted: 35d afterbody blood sampling ELISA measures.After serum titer reaches requirement, according to 500 μ l, 50 μ g antigen consumption abdominal injections impact immunity.To sterilize injection site with cotton ball soaked in alcohol after injection, protect from infection.
Step 2: cytogamy and cloning
Immunity impacts latter 3rd day, get the splenocyte of immune mouse, after conventionally carrying out cytogamy with myeloma cell, add 96 orifice plates (100 μ l/ hole) that the appropriate HAT containing 20% foetal calf serum is dispensed into the feeder cell of preparation gently after suspendible.Put 37 DEG C, cultivate in CO2 case.Cultivate after 5 days, liquid is changed by HAT substratum half amount, within 7 ~ 10 days, change liquid with HT substratum afterwards, cultivate with common perfect medium after 14 days, when hybridoma grows to hole floorage 1/3, adopt indirect non-competing ELISA method to detect, retain the best hybridoma cell strain of a strain after positive aperture three limited dilution clonings, i.e. anti-Sinopotamon henanense metallothionein(MT) monoclonal antibody hybridoma strain.
Step 3: prepared by ascites
Using whiteruss as sensitiser, choose BALB/c mouse in 14 ~ 22 week age, with 0.5ml/ injecting fluid paraffin, after the hybridoma cell strain enlarged culturing obtain screening, with 1 × 10
5~ 1 × 10
7production ascites in the inoculum density inoculation hybridoma cell strain to mouse peritoneal of individual/ml.Ascites is collected, the centrifugal 15min of 3000rpm, results supernatant after 10 ~ 12d.
Step 4: monoclonal antibody-purified, qualification
The ascites supernatant of results, the method set up with reference to Zhao Fengmei etc. carries out purifying (Zhao Fengmei, Qi Yanhong, what Yongji, Deng. a kind of research utilizing the anti-rhTF243 monoclonal antibody methodology of HCIC fast purifying. China Immunology Journal, 2010,26(6): 548-551).The sample of purifying analyzes the higher (see figure 6) of its purity through 12%SDS-PAGE.Conventional ELISA method detects antibodies specific, the antibody of result display purifying and coating protein rcMT specific effect, and the Recombulin somatomedin prepared with this laboratory of reference protein 6his-IGF() do not react.Be that envelope antigen measures and utilizes indirect non-competing method ELISA to measure antibody titer with rcMT, result shows this monoclonal antibody and tires and reach 1 × 10
5.Utilizing mouse monoclonal hypotype qualification ELISA kit (cellway-lab company) to analyze this monoclonal antibody is IgG1.
Claims (1)
1. solubility is recombinated a preparation method for magnificent river crab metallothionein(MT), it is characterized in that, comprises the following steps:
1) using containing phoA promotor and the phoA plasmid that can secrete to the signal sequence of periplasmic as expression vector, build the phoA-MT recombinant expression vector containing Sinopotamon henanense metallothionein(MT) coding region gene;
2) according to standard method transformation of E. coli BL21, colibacillus engineering strain phoA-MT/BL21 is built; Engineering bacteria is inoculated in LB substratum and prepares seed, then induce to low-phosphorous substratum according to 2.5% dilution phoA-MT/BL21 bacterium, add 1mMZnSO simultaneously
4, 30 DEG C, 165r/min inducing culture 22h;
Described low-phosphorous culture medium prescription is as follows: 2.5g/L Tryptones, the female extract of 0.25g/L enzyme, 3g/L (NH
4)
2sO
4, 5g/LNaCl, 1g/LMgSO
4, 4g/L glucose;
3) thalline is collected, ice-bath ultrasonic smudge cells, collected after centrifugation supernatant; Nisepharose
tM6FastFlow affinitive layer purification.
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| CN104357474A (en) * | 2014-10-17 | 2015-02-18 | 四川农业大学 | Method for preparing in vitro expression and polyclonal antibody of porcine Sox6 protein |
| CN110438131B (en) * | 2019-07-23 | 2022-09-13 | 江西农业大学 | Prokaryotic expression vector of cucumber metallothionein gene CsMT4 and application thereof |
| CN111499740A (en) * | 2020-04-01 | 2020-08-07 | 山西省农业科学院农产品加工研究所 | Tartary buckwheat metallothionein FtMT (FtMT) resistant monoclonal antibody as well as preparation method and application thereof |
| CN111363048B (en) * | 2020-04-01 | 2023-07-28 | 山西省农业科学院农产品加工研究所 | Soluble recombinant tartary buckwheat metallothionein FtMT with membrane penetrating activity and preparation method thereof |
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| CN1360022A (en) * | 2000-12-19 | 2002-07-24 | 中国科学院上海生物化学研究所 | Recombinant human epidermal growth factor and its preparing process and medicinal composition |
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