CN103468735A - Preparation methods of recombinant sinopotamon metallothionein (rcMT) and monoclonal antibody thereof - Google Patents
Preparation methods of recombinant sinopotamon metallothionein (rcMT) and monoclonal antibody thereof Download PDFInfo
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Abstract
The invention provides a preparation method of soluble recombinant sinopotamon metallothionein (rcMT), and the preparation method has the advantages of simplicity and convenience in operation, low cost, high purity and good activity. The method comprises the following steps: firstly constructing a phoA-MT recombinant expression vector by taking a phoA plasmid as an expression vector; further transforming escherichia coli BL21, constructing an escherichia coli engineering strain phoA-MT/BL21, and inoculating the engineering strain into an LB (Luria-Bertani) culture medium to prepare seeds, then diluting bacteria to a low-phosphorus culture medium for induction and expression, and utilizing Ni sepharoseTM6 Fast Flow affinity chromatography for purification so as to get the recombinant sinopotamon metallothionein. An anti-sinopotamon metallothionein monoclonal antibody prepared by utilizing the recombinant sinopotamon metallothionein has high sensitivity and strong specificity and can be applied to ELISA (enzyme-linked immunosorbent assay) quantitative assay of the metallothionein.
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 be by metal inducement and in conjunction with the special non-enzyme protein of a plurality of atoms metals.The Margoshes of Harvard University separated first and named with Vallee from the horse kidney nineteen fifty-seven.Research discovery subsequently, MT extensively is present in animal, plant and microbial world.Research shows, MT participates in storage, transhipment and the metabolism of trace element in body, has the antagonism ionizing rays, removes free radical and to the effect of heavy metal detoxification, also with body growth, grow, delay senility and some disease relevant.More there is in recent years the scholar to find that MT also plays an important role in the generation of tumour, development.As a kind of medical protein resource with broad prospect of application, a kind of desirable healthcare products, the additive of makeup, the mass-producing preparation of metallothionein(MT) is one of focus of application and development always.
Existing metallothionein(MT) mainly extracts by mammalian liver, yields poorly, cost is high, complex process is (referring to CN1085045A; CN1583790A).Also have report to extract from yeast, neurospora or Cordyceps, but it is lower to be limited by equally expression level, is difficult to obtain desirable yield (referring to CN1130687A; CN118098A; CN102199645A; CN102206693A).Along with the development of biotechnology, utilizing the protein engineering means to express recombinant protein becomes the first-selection that obtains a large amount of MT albumen.Escherichia expression system particularly, the maturation that possesses skills, expression amount be high, be easy to the many advantages such as cultivation and operation and production cost be low.But utilize this expression system, be difficult to obtain the MT of great amount of soluble.Mainly due to the MT molecular weight, and be rich in halfcystine, very easily form the inclusion body of lifeless matter activity.For acquisition has bioactive MT, must process through complicated change, renaturation, program is loaded down with trivial details, and loses a large amount of target proteins.Simultaneously MT is unstable in intestinal bacteria, and the demetalization sulfoprotein to problems such as the tolerance of metal ion are poor, is difficult to obtain higher productive rate to the toxicity of cell and cell always.For addressing this problem, also have the scholar utilize integration technology by the MT gene with have assist the correct folding fusion rotein of albumen as glutathione S-transferase (GST) or small molecules ubiquitin sample modified protein (SUMO) (referring to CN1348010A; CN101144093A) amalgamation and expression.Although this method has solved the problem of albumen solubility to a certain extent, has improved protein yield, because proteolytic enzyme is more expensive, enzyme is cut the problems such as consuming time, limited its use.
Simultaneously, due to the restriction of quantitative detecting method, MT also fails further to develop in the application of the aspects such as medical science, toxicology, environment remediation.The measuring method of MT content mainly contains plioweld, electrochemical process, red, orange, green, blue, yellow (ROGBY) etc. at present, and because of the singularity of MT albumen, the said determination method none be not subject to MT, in conjunction with the impact of the factors such as metal species, MT isomorph polymorphism, result is produced to 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, with other detection meanss, compare simultaneously, operate simple and easy, expense is cheap.The key of ELISA detection method is the preparation of antibody.Also there is at present ELISA to detect the relevant report of metallothionein(MT) both at home and abroad, but all adopt polyclonal antibody to realize.But polyclonal antibody poor specificity, output are limited, the physico-chemical property heterogeneity, are difficult to use in and set up quick, accurate, standardized enzyme-linked immune detection method.
Henan China river crab (Sinopotamon henanense) is transformed by the ocean crab is polynary, belongs to a special branch in Crustachia, Decapoda.As the unicellular lower eukaryote that moves in the 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 inventor is in early-stage Study, clone first Henan China river crab cDNA sequence, obtained its coding region gene (Ma, W.L., Yan, T., He, Y.J., Wang, L., 2009.Purification and cDNA cloning of a cadmium-binding metallothionein from the freshwater crab Sinopotamon henanense.Arch.Environ.Contam.Toxicol.56,747-753.).The result demonstration,, up to 30.5%, without die aromatischen Aminosaeuren, there is the characteristic sequence Cys-X-Cys of invertebrates MT in Henan China river crab MT cysteine content, 59 amino acid of encoding.As a kind of new MT gene that derives from Henan China river crab, than Mammals, MT compares, and river crab MT, in conjunction with the 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 the preparation method of a kind of easy and simple to handle, with low cost, purity is high, active good solubility is recombinated magnificent river crab metallothionein(MT) (recombinant crab metallothionein, rcMT).
Another object of the present invention is to utilize the magnificent river crab metallothionein(MT) of restructuring to prepare a kind of anti-magnificent river crab metallothionein(MT) monoclonal antibody.The anti-magnificent river crab metallothionein(MT) monoclonal antibody susceptibility of utilizing the inventive method to prepare is high, high specificity, can be applicable to the ELISA detection by quantitative of metallothionein(MT).
The recombinate preparation method of magnificent river crab metallothionein(MT) (rcMT) of a kind of solubility comprises the following steps:
1, build the phoA-MT recombinant expression vector
The T carrier pGEM-MT that contains Henan China's river crab metallothionein(MT) coding region gene (SEQ ID NO:1) of take is template, utilizes Auele Specific Primer: Forward primer:5'-G
cCATGGCcCCTGATCCTTGCTGC-3'(is containing the NcoI restriction enzyme site) and Reverse primer:5'-A
gGATCCgGG GCAGCAGGAGCAAG-3'(is containing BamH I restriction enzyme site) pcr amplification; The phoA plasmid that amplified production and C end are introduced 6his tag is after NcoI and BamH I double digestion, and the T4DNA ligase enzyme connects rear Transformed E .coli DH5 α competent cell, and the picking positive colony shakes bacterium, carries out bacterium colony PCR and plasmid enzyme restriction and identifies, DNA sequencing;
2, the structure of engineering strain phoA-MT/BL21, induce and express
The phoA-MT plasmid of identifying through order-checking transforms e. coli bl21 according to standard method, builds colibacillus engineering strain phoA-MT/BL21; Engineering bacteria is inoculated in the LB substratum, and 37 ℃, 165r/min shaking culture 12h prepare seed; Induce according to 2.5% dilution bacterium to low-phosphorous substratum, add 1mM ZnSO simultaneously
4increase protein stability, 30 ℃, 165r/min inducing culture 22h;
Described low-phosphorous culture medium prescription is as follows: the 2.5g/L Tryptones, and the female extract of 0.25g/L enzyme, 3g/L (NH4) 2SO4,, 5g/LNacl,, 1g/LMgSO4,4g/L glucose;
3, rcMT protein purification and evaluation
Collect thalline, ice-bath ultrasonic smudge cells, centrifugal rear collection supernatant; Ni sepharose after 0.22 μ M membrane filtration
tM6Fast Flow affinitive layer purification; Target protein is through 3000MWCO super filter tube ultrafiltration and concentration; SDS-PAGE, Western blot and uv scan are identified.
Utilize above-mentioned recombinant protein to prepare a kind of anti-magnificent river crab metallothionein(MT) monoclonal antibody, can 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 are carried 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, with the myeloma cell, utilize PEG1500 reagent to carry out external fusion, using rcMT as the indirect non-competing ELISA method screening of envelope antigen utilization, through subclone repeatedly, obtain the hybridoma cell strain of the anti-rcMT of stably excreting;
3, ascites preparation
Using whiteruss as sensitiser, and choosing 14～22 week age BALB/c mouse is laboratory animal, with 0.5ml/ injecting fluid paraffin, after the hybridoma cell strain enlarged culturing that screening is obtained, with 1 * 10
5～1 * 10
7the inoculum density of individual/ml inoculation hybridoma cell strain is to production ascites in mouse peritoneal; Collect ascites after 10～12d, the centrifugal 15min of 3000rpm, results supernatant;
4, monoclonal antibody purifying, evaluation
The ascites supernatant of results, after MEP HyperCel hydrophobic chromatography post is slightly carried, then carry out purifying through Protein A-Sepharose CL-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) utilize alkaline phosphatase promoter (phoA) and signal sequence secreting, expressing China river crab metallothionein(MT) in intestinal bacteria, by phoA promoter regulation MT albumen soluble-expression between the cell pericentral siphon, overcome MT easily form inclusion body and with born of the same parents in the cytotoxicity defect that causes of melts combine.In the low-phosphorous abduction delivering of rcMT, add a certain amount of zine ion, improved stability and the purity of metallothionein(MT).The metallothionein(MT) that utilizes the present invention to prepare only, containing the 6his label, is cut processing without enzyme, utilizes 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.With polyclonal antibody, compare, its physicochemical character height homogeneous, biological activity is single, is convenient to artificial the processing and quality control; The high specificity of being combined with antigen, reduced possible cross reaction, makes test-results more credible.The monoclonal antibody made according to the inventive method, for developing a kind of quick, accurate, standardized MT enzyme-linked immunologic detecting kit.
The accompanying drawing explanation
Fig. 1 is phoA-MT recombinant plasmid bacterium colony PCR and double digestion qualification result.Mr is DNA Marker; 1 is that recombinant plasmid phoA-MT is through BamH I, Xho I double digestion; 2 is engineering bacteria phoA-MT/BL21PCR evaluation product.
Fig. 2 is phoA-MT recombinant plasmid partial sequence sequencing result.
The SDS-PAGE electrophoretic analysis that Fig. 3 is phoA-MT/BL21 expression product and purifying.Mr is the protein standard; 1 for not inducing full bacterium; 2 is the low-phosphorous full bacterium of inducing; 3 target proteins that are purifying.
The Western blot that Fig. 4 is recombinant protein rcMT identifies.
The ultraviolet spectrometry scanning spectra that Fig. 5 is recombinant protein rcMT.
The SDS-PAGE electrophoretic analysis that Fig. 6 is anti-rcMT monoclonal antibody purification of samples.Mr is the protein standard; 1,2 is HCLC column purification sample; 3 is the monoclonal antibody through Protein A-Sepharose CL-4B column purification;
Embodiment
Step 1: the selection of plasmid vector
Alkaline phosphate promotor (phoA) and signal sequence secreting, expressing foreign protein in intestinal bacteria has some advantages (referring to " molecular cloning experiment guide " third edition), the native conformation that can at utmost keep foreign protein, secretion is more stable to the protein of outer pericentral siphon simultaneously.In the phoA system, the alkaline phosphatase of phoA genes encoding intestinal bacteria phosphoric acid scavenge system, suppressed when excess phosphoric acid salt exists, under hungry state (bacterial growth enters the lag phase), the phoA gene is induced gradually.It is progressive in intracellular accumulation that this regulation and control make foreign protein, unlike the accumulation rapidly of other promoter regulations.This chronic accumulation has reduced the bacterial expression system overload and has formed the chance of inclusion body.When accumulation also makes to accumulate rapidly at a slow speed simultaneously, expression is alleviated, obtained to toxigenous foreign protein.The phoA plasmid has ampicillin resistance gene, through transformation C section, has introduced 6his tag.
Step 2: build the phoA-MT recombinant expression vector
The T carrier pGEM-MT that contains Henan China's river crab metallothionein(MT) coding region gene (SEQ ID NO:1) of take is template, utilizes Auele Specific Primer: Forward primer:5'-G
cCATGGCcCCTGATCCTTGCTGC-3'(is containing the NcoI restriction enzyme site) and Reverse primer:5'-A
gGATCCgGG GCAGCAGGAGCAAG-3'(is containing BamH I restriction enzyme site) pcr amplification, amplification condition is as follows: 94 ℃ of denaturation 5min, with 94 ℃ of sex change 30s, 65 ℃ of annealing 50s, 70 ℃ are extended 1min, then extend 10min, 3O circulation altogether, last 4 ℃ of preservations, product is through 15g/L agarose gel electrophoresis preliminary evaluation.Identify that correct PCR product reclaims test kit (love pursue progress Bioisystech Co., Ltd) through glue and reclaims the purpose fragment, utilize Nco I, BamH I double digestion, enzyme is cut product and is connected by the T4 ligase enzyme through the phoA plasmid of double digestion equally.Connect product and transform the bacillus coli DH 5 alpha competent cell, conversion fluid is coated (containing penbritin 50 μ g/ml) on the LB agar plate, selects positive colony, after bacterium colony PCR and enzyme are cut the (see figure 1) evaluation, and order-checking.Sequencing result shows (see figure 2), and magnificent river crab MT gene successfully is cloned into the 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 the LB substratum, and 37 ℃, 200r/min shaking culture 12h prepare 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) induce, add 1mM ZnSO simultaneously
4, 30 ℃, 180r/min inducing culture 22h.The molybdate method is surveyed phosphorus content, determines that phosphorus all runs out of, 8000r/min, and 25min collects bacterium.The wet bacterium of every 1g suspends with the broken liquid (20mM Tris, 500mM sodium-chlor, 1%Trionton, 10mM imidazoles pH7.8) of 5ml, ice bath stirs 30min, ice-water bath intermittently ultrasonic (360～400w) is processed to bacterium liquid inviscid, 4 ℃, the centrifugal 20min of 8000rpm, get full bacterium and carry out the 16.5%Tricine-SDS-PAGE analysis, and 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 soluble mode.
The supernatant that upper step obtains utilizes Ni sepharose after 0.22 μ M membrane filtration
tM6Fast Flow affinity chromatography column separating purification.At first use level pad (20mM Tris, 500mM sodium-chlor, 20mM imidazoles pH7.8) balance Ni sepharose
tM6Fast Flow post, supernatant upper prop after then filtering.Be washed till baseline with level pad again after loading, then use elution buffer (20mM Tris, 500mM sodium-chlor, 50mM imidazoles pH7.8) wash-out foreign protein, finally use elution buffer (20mM Tris, 500mM sodium-chlor, 250mM imidazoles pH7.8) wash-out target protein peak.The elution peak component through 3000MWCO super filter tube ultrafiltration and concentration to storage liquid (0.01M Tris-HCl, 0.2mM PMSF, pH7.8).
Step 4: the evaluation of recombinant protein and concentration determination
Purified target protein is analyzed as the single band (see figure 3) in 8KD left and right through 16.5%Tricine-SDS-PAGE, shows and has obtained the rcMT that purity is higher.After the purifying protein electrophoresis, with electroporation (Bio-Rad), the albumen in SDS-PAGE glue is gone on pvdf membrane, with confining liquid (PBST+5% skim-milk) sealing, spend the night, PBST washes film 4 times, 10min/ time, add Anti-6His tag rabbit source how anti-as primary antibodie, 4 ℃ 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.The result demonstration, the rcMT of purifying is because being with the 6his label in 8KD place colour developing (see figure 4).
The rcMT ultrafiltration and concentration sample of purifying adopt UNICO UV-2102PC type ultraviolet-visible pectrophotometer under carry out full wavelength scanner (190nm-1100nm).Result shows, rcMT has an absorption peak because with zinc, being combined in the 225nm place, and at 280nm without conventional protein characteristic absorption peak, meet 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 1: mouse immune
The rcMT of purifying is usingd BSA as standard protein, utilizes the BCA method to measure protein concentration.
(1) fundamental immunity:
Get and be stored in damping fluid (0.01M Tris-HCl, 0.2mM PMSF, pH7.8) rcMT in is diluted to 120 μ g/ml, according to the volume ratio of 1: 1, with Quicklyantibody water-soluble adjuvant (purchased from the green spring bio tech ltd of Beijing health), fully mix, the intramuscular injection immune mouse, injection volume 0.1ml/ only.Immunity amount 0.1ml, 20 μ g rcMT/ only, with the cotton ball soaked in alcohol injection site of sterilizing, protect from infection after injection.(2) booster immunization: after 21d, the second immunisation mouse, immunity amount and immunization method are the same.(3) impact immunity: 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.After injection, with the cotton ball soaked in alcohol injection site of sterilizing, protect from infection.
Step 2: cytogamy and cloning
Immunity impact after the 3rd day, get the splenocyte of immune mouse, after carrying out cytogamy with the myeloma cell according to ordinary method, add the appropriate HAT containing 20% foetal calf serum minute to install to gently 96 orifice plates (100 μ l/ hole) of the feeder cell of preparation after suspendible.Put 37 ℃, cultivate in the CO2 case.Cultivate after 5 days, change liquid by HAT substratum half amount, within 7～10 days, change liquid with the HT substratum afterwards, within 14 days, with common perfect medium, cultivate afterwards, 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 three limited dilution clonings of positive aperture, be i.e. the magnificent river crab metallothionein(MT) in anti-Henan monoclonal antibody hybridoma strain.
Step 3: ascites preparation
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 that screening is obtained, with 1 * 10
5～1 * 10
7the inoculum density of individual/ml inoculation hybridoma cell strain is to production ascites in mouse peritoneal.Collect ascites after 10～12d, the centrifugal 15min of 3000rpm, results supernatant.
Step 4: monoclonal antibody purifying, evaluation
The ascites supernatant of results, carry out purifying (Zhao Fengmei, Qi Yanhong with reference to the method for the foundation such as Zhao Fengmei, what Yongji, Deng. a kind of research that utilizes the anti-rhTF243 monoclonal anti of HCIC fast purifying body method. Chinese Journal of Immunology, 2010,26(6): 548-551).The sample of purifying is analyzed the higher (see figure 6) of its purity through 12%SDS-PAGE.Conventional ELISA method detects antibodies specific, and result shows antibody and the coating protein rcMT specific effect of purifying, and the Recombulin somatomedin prepared with this laboratory of reference protein 6his-IGF() do not react.The rcMT of take measures and to utilize indirect non-competing method ELISA to measure antibody titer as envelope antigen, and result shows that this monoclonal antibody is tired and reaches 1 * 10
5.Utilize mouse monoclonal antibody hypotype to identify that it is IgG1 that ELISA test kit (cellway-lab company) is analyzed this monoclonal antibody.
Claims (3)
1. the solubility preparation method of magnificent river crab metallothionein(MT) (rcMT) that recombinates, is characterized in that, comprises the following steps:
1) using and contain the phoA promotor and can secrete phoA plasmid to the signal sequence of cell pericentral siphon as expression vector, build the phoA-MT recombinant expression vector;
2) transform e. coli bl21 according to standard method, build colibacillus engineering strain phoA-MT/BL21; Engineering bacteria is inoculated in the LB substratum and prepares seed, then induce according to 2.5% dilution phoA-MT/BL21 bacterium to low-phosphorous substratum, add 1mM ZnSO simultaneously
4, 30 ℃, 165r/min inducing culture 22h;
Described low-phosphorous culture medium prescription is as follows: the 2.5g/L Tryptones, and the female extract of 0.25g/L enzyme, 3g/L (NH4) 2SO4,, 5g/LNacl,, 1g/LMgSO4,4g/L glucose;
3) collect thalline, ice-bath ultrasonic smudge cells, centrifugal rear collection supernatant; Ni sepharose
tM6Fast Flow affinitive layer purification.
2. the solubility that prepared by the method for claim 1 magnificent river crab metallothionein(MT) of recombinating.
3. the solubility anti-magnificent river crab metallothionein(MT) monoclonal antibody prepared by magnificent river crab metallothionein(MT) of recombinating as claimed in claim 2.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110438131A (en) * | 2019-07-23 | 2019-11-12 | 江西农业大学 | The prokaryotic expression carrier of cucumber metallothionein gene CsMT4 and its application |
| CN111363048A (en) * | 2020-04-01 | 2020-07-03 | 山西省农业科学院农产品加工研究所 | Soluble recombinant tartary buckwheat metallothionein FtMT with transmembrane activity and preparation method 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 |
| CN112546239A (en) * | 2020-11-19 | 2021-03-26 | 北京大学 | Use of protein nanoparticles for preparing antiviral products |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1549821A (en) * | 2001-08-27 | 2004-11-24 | A system for antibody expression and assembly |
-
2013
- 2013-09-18 CN CN201310430331.4A patent/CN103468735B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN1549821A (en) * | 2001-08-27 | 2004-11-24 | A system for antibody expression and assembly |
Non-Patent Citations (1)
| Title |
|---|
| WEN-LI MA，ET AL: "Purification and cDNA Cloning of a Cadmium-Binding Metallothionein from the Freshwater Crab Sinopotamon henanense", 《ARCH ENVIRON CONTAM TOXICOL》, no. 56, 10 October 2008 (2008-10-10), pages 747 - 753, XP019704686 * |
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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 |
| CN110438131A (en) * | 2019-07-23 | 2019-11-12 | 江西农业大学 | The prokaryotic expression carrier of cucumber metallothionein gene CsMT4 and its application |
| 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 |
| CN111363048A (en) * | 2020-04-01 | 2020-07-03 | 山西省农业科学院农产品加工研究所 | Soluble recombinant tartary buckwheat metallothionein FtMT with transmembrane activity and preparation method 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 |
| CN112546239A (en) * | 2020-11-19 | 2021-03-26 | 北京大学 | Use of protein nanoparticles for preparing antiviral products |
| CN112546239B (en) * | 2020-11-19 | 2022-09-02 | 北京大学 | Use of protein nanoparticles for preparing antiviral products |
| CN116121156A (en) * | 2021-11-12 | 2023-05-16 | 中国科学院青岛生物能源与过程研究所 | Genetically engineered bacterium for preparing metal quantum dots, application of genetically engineered bacterium and method for preparing CdSe quantum dots |
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