CN103424546A - Specific identification method for biological active components in deer leather gel - Google Patents
Specific identification method for biological active components in deer leather gel Download PDFInfo
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- CN103424546A CN103424546A CN2012101544564A CN201210154456A CN103424546A CN 103424546 A CN103424546 A CN 103424546A CN 2012101544564 A CN2012101544564 A CN 2012101544564A CN 201210154456 A CN201210154456 A CN 201210154456A CN 103424546 A CN103424546 A CN 103424546A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a specific identification method for biological active components in deer leather gel. The identification comprises an enzyme linked immunosorbent assay (ELISA) for specific deer collagen in the deer leather gel, wherein the ELISA is used for determining whether the deer leather gel contains the specific deer collagen according to the color result and detecting the content of the specific deer collagen; adopts a ninhydrin post column derivation method, which is used for carrying out quantitative and qualitative analysis identifications for specific amino acid composition in deer leather gel; and carries out a PCR amplification test identification for deer leather gel specific DNA sequence in deer leather gel, and if stripes which represent the deer specific protein components can be amplified out, indicating that the sample to be test is a qualified product. The method mentioned above provides a scientific and reliable identification method for distinguishing true or false and high-quality or low-quality of deer leather gel products in the market, and detecting the content of effective biological components in deer leather gel.
Description
Technical field
The present invention relates to a kind of authentication method of pure deerskin glue bioactive ingredients, relate in particular to the specificity identification method of special collagen contained in a kind of deerskin glue, special amino acid and specific DNA.
Background technology
The effects such as qi-restoratives damage, benefiting essence-blood, strong waist kidney and tranquilizing mind to the deer goods in Chinese traditional medicine have a large amount of records.Then to the science data of deerskin glue, particularly to the contained bioactive ingredients in deerskin glue, as the specificity identification of collagen, amino acid and DNA still has no data available, adopt high-tech to be measured the bioactive ingredients of deerskin glue, contribute to produce and differentiate the quality of deerskin glue product.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the invention provides the specificity identification method of contained bioactive ingredients in a kind of deerskin glue, the true and false quality that the method is deerskin glue product on Vehicles Collected from Market provides science to identify reliably mode.
The present invention for the technical scheme that solves its technical matters and adopt is:
The specificity identification method of contained bioactive ingredients in a kind of deerskin glue, comprise the enzyme linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue; Adopt triketohydrindene hydrate post-column derivation method to identify contained special amino acid composition analysis in deerskin glue; With the pcr amplification experimental identification to contained deerskin glue specific DNA sequence in deerskin glue.
The described enzyme linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue, used the antibody immunoglobulin of the former albumen of special deer horn glue as coated antibody, adopts the former protein antibodies immunoglobulin (Ig) of the special deer horn glue of peroxidase labelling.
The described enzyme linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue comprises the following steps:
Be coated with the polypropylene reaction plate with the antibody immunoglobulin of the former albumen of special deer horn glue, and wash away the antibody immunoglobulin coating buffer of the former albumen of described special deer horn glue after being coated with; Testing sample and the antibody immunoglobulin that is coated in the former albumen of special deer horn glue on the polypropylene reaction plate are carried out to antigen-antibody reaction, and wash away described testing sample after above-mentioned antigen-antibody reaction finishes; Adopt the former protein antibodies immunoglobulin (Ig) of the described special deer horn glue of peroxidase labelling to form the former protein antibodies immunoglobulin (Ig) of special deer horn glue of peroxidase labelling; The former protein antibodies immunoglobulin (Ig) of the special deer horn glue of above-mentioned oxide enzyme labeling is added on above-mentioned polypropylene reaction plate, form the compound of the former protein antibodies immunoglobulin (Ig) of special deer horn glue of the former protein antibodies immunoglobulin (Ig) of special deer horn glue-former albumen-peroxidase labelling of special deer horn glue; Adopt the o-phenylenediamine substrate to be developed the color; Successfully show in testing sample to contain the former albumen of special deer horn glue if develop the color, adopt enzyme mark color comparator to measure the content of the former albumen of special deer horn glue in the quantitative sample of light absorption value of colour developing thing.
The described pcr amplification experimental identification to contained deerskin glue specific DNA sequence in deerskin glue comprises the following steps:
By traditional DNA purification method, the genomic DNA in deerskin glue is carried out to purification, obtain purified deerskin glue extracting genome DNA thing; Utilize primer pair CAGCTAGCTATAGTCT/GTATGGATAATGCAGTT to carry out the pcr amplification test to gained deerskin glue extracting genome DNA thing and increased, obtain amplified production one; Utilize primer pair CGTGGACTACGATGCTT/TAGTCCTTGGCTACGT to carry out the pcr amplification test to gained deerskin glue extracting genome DNA thing and increased, obtain amplified production two; Gained amplified production one and two is carried out respectively to the agarose gel electrophoresis detection, judge the true and false of deerskin glue according to the amplified production clip size.
Useful technique effect of the present invention is: the present invention is by the enzyme-linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue, judge in deerskin glue whether contain the former albumen of described special deer horn glue according to the colour developing result, and the content of the former albumen of contained special deer horn glue; Adopt triketohydrindene hydrate post-column derivation method to form and carry out the qualitative and quantitative Analysis and Identification contained special amino acid in deerskin glue; With the pcr amplification experimental identification to contained deerskin glue specific DNA sequence in deerskin glue, mean that if can amplify the band of deer differential protein composition shows that testing sample is certified products; The true and false quality that said method is deerskin glue product on Vehicles Collected from Market, and in deerskin glue, contained effective biotic component content provides science reliable authentication method.
The accompanying drawing explanation
Fig. 1 is the protein concentration typical curve of formulating while adopting enzyme mark color comparator to measure the former albumen of special deer horn glue in enzyme-linked immunosorbent assay;
Fig. 2 is amino acid contained in pure deerskin glue (except proline) HPLC analysis chart;
Fig. 3 is contained proline HPLC analysis chart in pure deerskin glue;
The agarose gel electrophoresis result that Fig. 4 is contained genomic DNA amplified production in pure deerskin glue, the primer pair that wherein A is used is EP-2/H1478 for DF/DR(), the primer pair that B is used is EP-1/H1478 for SF/SR().
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
(1) enzyme-linked immunosorbent assay (ELISA) is measured the contained former albumen of special deer horn glue in deerskin glue, the testing sample lysate that wherein adopted is respectively the deerskin glue solution of 50mg/ml and the deerskin glue dry powder solution of 50mg/ml, adopt two secondary holes in 96 hole polypropylene reaction plates, the method comprises the following steps:
(1) by the antibody immunoglobulin of the former albumen of special deer horn glue (purchased from the monoclonal anti-Collagen Type 1 of Sigma company, production number is C2456) coated 96 hole polypropylene reaction plates, after the reaction plate be coated with is placed in to 4 ℃ of lower 24h, adopt automatic plate washer (U.S. Biorad-680 model) to wash away the antibody immunoglobulin coating buffer of the former albumen of special deer horn glue;
(2) by each testing sample lysate respectively after diluting 20 times and 50 times, add respectively in two subsidiary reaction holes of the polypropylene reaction plate after process (1), hatch 1h under 37 ℃ after, the dilution of each testing sample lysate is discarded, and adopt automatic plate washer washing reaction plate;
(3) add the former protein antibodies immunoglobulin (Ig) of special deer horn glue through horseradish peroxidase (HRP) mark in two secondary holes of the polypropylene reaction plate after process (2), again under 37 ℃, hatched 1h, hatch and finish the rear automatic plate washer washing reaction plate that adopts;
(4) be added into o-phenylenediamine (OPD) substrate solution in two secondary holes of the polypropylene reaction plate after process (3), after waiting for colour developing, adopting enzyme mark color comparator (the Spectra Max M5 that Molecular Devices company produces) to be measured, is 350mg containing the former albumen of special deer horn glue in the pure deerskin glue of every gram of the present invention.
The detection principle of above-mentioned steps is as described below: be coated with the former protein antibodies immunoglobulin (Ig) of special deer horn glue on the polypropylene reaction plate, again to after adding the testing sample lysate in the reacting hole of reaction plate, if contain the former albumen of special deer horn glue in the testing sample lysate, the former albumen of special deer horn glue can form with the former protein antibodies immunoglobulin (Ig) of special deer horn glue the compound one of the former albumen of special deer horn glue-former protein antibodies immunoglobulin (Ig) of special deer horn glue, during then to the former protein antibodies immunoglobulin (Ig) of special deer horn glue that adds in above-mentioned compound through the HRP peroxidase labelling, can form the compound two of the former protein antibodies immunoglobulin (Ig) of special deer horn glue of the former protein antibodies immunoglobulin (Ig) of the special deer horn glue-former albumen of special deer horn glue-HRP peroxidase labelling.After in above-mentioned compound two systems, adding the OPD substrate solution, OPD can color reaction occur with peroxidase, the shade detected according to enzyme mark color comparator, compare with the typical curve of formulating on enzyme mark color comparator, draws the content of the former albumen of contained special deer horn glue in deerskin glue.
As shown in Figure 1, and to be contrasted in rear gained sample protein content as shown in table 1 with this typical curve for the protein concentration typical curve of formulating while adopting in above-mentioned steps (4) enzyme mark color comparator to measure:
In table 1 deerskin glue, contained special collagen concentration is measured
(2) triketohydrindene hydrate post-column derivation method is measured contained amino acid in deerskin glue, comprises the following steps:
(1) after adopting ultrapure water to be diluted to 1mg/ml the deerskin glue sample dissolution liquid to be measured of 50mg/ml, use miillpore filter to be filtered described dilution, using the gained filtered fluid as need testing solution;
(2) gained need testing solution in (1) is used microsyringe to the middle sample introduction 20.0 μ l of amino-acid analyzer (the full-automatic amino-acid analyzer of L-8800 type that HIT produces), the chromatographiccondition of described amino-acid analyzer is as described below: the cation exchange column of chromatographic column adopting 4.6mm * 60mm, model is the 2622SC of Hitachi, mobile phase is selected the citrate three sodium damping fluid, quaternary gradient eluent pH value is 3.2 ~ 4.9, the detection wavelength is 570nm, and eluent flow rate is 0.4ml/min; Column temperature is 50 ℃; The condition of post-column derivation is: reactant liquor A:L-propylene glycol monomethyl ether, 390g indenes diketone; Reactant liquor B propylene glycol monomethyl ether-sodium acetate, acetic acid (40:60, V/V); Temperature of reaction is 135 ℃, and the reactant liquor flow velocity is 0.35ml/min, and the scale factor adopted is 1:1000 and 1:2000.
The principle that above-mentioned automatic amino acid analyzer method is carried out amino acid whose component analysis is as described below: utilize not congruence property of various amino acid whose Acidity of Aikalinitys, polarity and molecular size range, use Zeo-karb to be separated on chromatographic column, after sample liquid adds the chromatographic column top, adopt the buffer solution of different pH values and ion concentration they can be eluted successively.The larger amino acid of acidic amino acid and polarity, be secondly nonpolar aromatic amino acid before this, is finally basic amino acid; Large first being eluted of ratio molal weight that molal weight is little, the amino acid eluted can develop the color with triketohydrindene hydrate, thus quantitative each seed amino acid.The amino acid whose foundation of quantitative measurement is that amino acid and ninhydrin reaction generate the bluish violet compound, and the shade of this compound amino acid whose content corresponding to each is directly proportional, and wherein proline and hydroxyproline generate the yellowish-brown compound with triketohydrindene hydrate.The shade detected according to color comparator, compare with the amino acid typical curve of formulating on color comparator, draws content amino acid contained in testing sample.In deerskin glue, the stratographic analysis figure of amino acid kind is shown in shown in Fig. 2 and Fig. 3, and wherein Fig. 3 is contained proline HPLC analysis chart in deerskin glue; The amino acid analysis result is as shown in table 2, and the computing method of wherein said amino acid content account for the mark of respective substance total amount for each amino acid content, wherein
※Mean that this seed amino acid is essential amino acid.
Contained amino acid content in table 2 deerskin glue
| The amino acid kind | Amino acid content (%) |
| The Asp(L-aminobutanedioic acid) | 6.42 |
| Thr ※(threonine) | 2.21 |
| The Ser(serine) | 3.38 |
| Glu(glutamic acid) | 11.17 |
| The Pro(proline) | 13.81 |
| The Gly(glycocoll) | 23.80 |
| The Ala(alanine) | 9.26 |
| The Cys(cystine) | 0.29 |
| Val ※(valine) | 2.64 |
| Met ※(methionine) | 0.59 |
| Ile ※(isoleucine) | 1.44 |
| Leu ※(leucine) | 3.51 |
| Tyr(tyrosine) | 1.04 |
| Phe ※(phenylalanine) | 2.02 |
| Lys ※(lysine) | 4.00 |
| The His(histidine) | 0.77 |
| The Arg(arginine) | 7.81 |
| NH3(ammonia) | (0.74) |
| Total amino acid content | 94.16 |
As shown in table 2, contain 17 kinds of different amino acid in pure deerskin glue, wherein threonine, valine, methionine, isoleucine, leucine, phenylalanine and lysine be the mankind can not self synthetic essential amino acid; The content of glycocoll is 23.8% in addition.
(3) the pcr amplification test for identification of specific DNA sequence in deerskin glue comprises the following steps:
(1) adopt the DNA that QLAGEN company produces to extract kit, carry out the extraction purifying of contained genomic dna sequence in deerskin glue according to the operation instruction on described kit, obtain purified deerskin glue extracting genome DNA thing;
(2) (1) gained extracting genome DNA thing is carried out to the PCR reaction under following reaction system and reaction conditions: reaction system is extracting genome DNA thing 1 μ g; 10 * PCR Buffer is 10 μ l; The Taq.DNA polymerase is 3U; Distilled water complements to 100 μ l; Each 80pmol of forward and reverse sequence that primer pair is every pair of primer pair.Mg wherein
2+Content is 1.5mmol/L; The dNTPs mixture content is every kind of each 200 μ mol/L; Wherein primer pair adopts EP-1/H1478(SF/SR) or primer pair EP-2/H1478(DF/DR), forward and reverse sequence of described SF/SR primer pair is: CGTGGACTACGAT GCTT; TAGTCCTTGGCTACGT; Forward and reverse sequence of described DF/DF primer pair is: CAGCTAGCTATAG TCT; GTATGGATAA TGCAGTT.Response procedures is 97 ℃ of sex change 7min; After annealing 30sec under 60 ℃, 68 ℃ of downward-extension 50sec carry out amplification cycles 30 times on PCR instrument (the MJ Mini model that Bio-Rad company produces) again;
(3) will carry out the agarose gel electrophoresis detection by the pcr amplification product of (2) gained: adopt the 1wt.% Ago-Gel, take DL200 DNA Maker(Takara company to produce) in DNA fragmentation calculate the size of pcr amplification product fragment as molecular weight standard (wherein DNA fragmentation length is 100bp, 00 bp, 300 bp, 400 bp, 500 bp and 1000bp).The agarose gel electrophoresis result of amplified production as shown in Figure 4.
Claims (4)
1. the specificity identification method of contained bioactive ingredients in a deerskin glue, is characterized in that: comprise the enzyme linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue; Adopt triketohydrindene hydrate post-column derivation method to identify contained special amino acid composition analysis in deerskin glue; With the pcr amplification experimental identification to contained deerskin glue specific DNA sequence in deerskin glue.
2. the specificity identification method of contained bioactive ingredients in deerskin glue according to claim 1, it is characterized in that: the described enzyme linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue, use the antibody immunoglobulin of the former albumen of special deer horn glue as coated antibody, adopt the former protein antibodies immunoglobulin (Ig) of the special deer horn glue of peroxidase labelling.
3. the specificity identification method of contained bioactive ingredients in deerskin glue according to claim 2, it is characterized in that: the described enzyme linked immunosorbent assay to the former albumen of contained special deer horn glue in deerskin glue comprises the following steps:
Be coated with the polypropylene reaction plate with the antibody immunoglobulin of the former albumen of special deer horn glue, and wash away the antibody immunoglobulin coating buffer of the former albumen of described special deer horn glue after being coated with; Testing sample and the antibody immunoglobulin that is coated in the former albumen of special deer horn glue on the polypropylene reaction plate are carried out to antigen-antibody reaction, and wash away described testing sample after above-mentioned antigen-antibody reaction finishes; Adopt the former protein antibodies immunoglobulin (Ig) of the described special deer horn glue of peroxidase labelling to form the former protein antibodies immunoglobulin (Ig) of special deer horn glue of peroxidase labelling; The former protein antibodies immunoglobulin (Ig) of the special deer horn glue of above-mentioned oxide enzyme labeling is added on above-mentioned polypropylene reaction plate, form the compound of the former protein antibodies immunoglobulin (Ig) of special deer horn glue of the former protein antibodies immunoglobulin (Ig) of special deer horn glue-former albumen-peroxidase labelling of special deer horn glue; Adopt the o-phenylenediamine substrate to be developed the color; Successfully show in testing sample to contain the former albumen of special deer horn glue if develop the color, adopt enzyme mark color comparator to measure the content of the former albumen of special deer horn glue in the quantitative sample of light absorption value of colour developing thing.
4. the specificity identification method of the contained bioactive ingredients of deerskin glue according to claim 1, it is characterized in that: the described pcr amplification experimental identification to contained deerskin glue specific DNA sequence in deerskin glue comprises the following steps:
By traditional DNA purification method, the genomic DNA in deerskin glue is carried out to purification, obtain purified deerskin glue extracting genome DNA thing; Utilize primer pair CAGCTAGCTATAGTCT/GTATGGATAATGCAGTT to carry out the pcr amplification test to gained deerskin glue extracting genome DNA thing and increased, obtain amplified production one; Utilize primer pair CGTGGACTACGATGCTT/TAGTCC TTGGCTACGT to carry out the pcr amplification test to gained deerskin glue extracting genome DNA thing and increased, obtain amplified production two; Gained amplified production one and two is carried out respectively to the agarose gel electrophoresis detection, judge the true and false of deerskin glue according to the amplified production clip size.
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