CN103059135B - A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof - Google Patents
A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof Download PDFInfo
- Publication number
- CN103059135B CN103059135B CN201210576152.7A CN201210576152A CN103059135B CN 103059135 B CN103059135 B CN 103059135B CN 201210576152 A CN201210576152 A CN 201210576152A CN 103059135 B CN103059135 B CN 103059135B
- Authority
- CN
- China
- Prior art keywords
- mrjp1
- royal jelly
- antibody
- add
- mrjps
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229940109850 royal jelly Drugs 0.000 title claims abstract description 92
- 101001116000 Apis mellifera Major royal jelly protein 1 Proteins 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 title abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 63
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 54
- 239000000427 antigen Substances 0.000 claims abstract description 52
- 102000036639 antigens Human genes 0.000 claims abstract description 52
- 108091007433 antigens Proteins 0.000 claims abstract description 52
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 49
- 229920001184 polypeptide Polymers 0.000 claims abstract description 48
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 47
- 238000000034 method Methods 0.000 claims abstract description 33
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 28
- 238000004458 analytical method Methods 0.000 claims abstract description 17
- 238000011587 new zealand white rabbit Methods 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- 210000002966 serum Anatomy 0.000 claims abstract description 6
- 235000018102 proteins Nutrition 0.000 claims description 51
- 230000031700 light absorption Effects 0.000 claims description 23
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 18
- 239000008176 lyophilized powder Substances 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 12
- 125000000539 amino acid group Chemical group 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- 239000003593 chromogenic compound Substances 0.000 claims description 8
- 238000007789 sealing Methods 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 7
- 238000006467 substitution reaction Methods 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 238000000691 measurement method Methods 0.000 claims description 3
- 241000256844 Apis mellifera Species 0.000 abstract description 12
- 238000003556 assay Methods 0.000 abstract description 3
- 238000003908 quality control method Methods 0.000 abstract description 2
- 235000012907 honey Nutrition 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 27
- 238000001962 electrophoresis Methods 0.000 description 18
- 238000001262 western blot Methods 0.000 description 17
- 241000256836 Apis Species 0.000 description 15
- 239000006228 supernatant Substances 0.000 description 15
- 238000010790 dilution Methods 0.000 description 14
- 239000012895 dilution Substances 0.000 description 14
- 239000007788 liquid Substances 0.000 description 14
- 101100046775 Arabidopsis thaliana TPPA gene Proteins 0.000 description 12
- 239000000499 gel Substances 0.000 description 12
- XZZNDPSIHUTMOC-UHFFFAOYSA-N triphenyl phosphate Chemical compound C=1C=CC=CC=1OP(OC=1C=CC=CC=1)(=O)OC1=CC=CC=C1 XZZNDPSIHUTMOC-UHFFFAOYSA-N 0.000 description 12
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000003292 glue Substances 0.000 description 9
- 229920002401 polyacrylamide Polymers 0.000 description 9
- QHBZHVUGQROELI-UHFFFAOYSA-N Royal Jelly acid Natural products OCCCCCCCC=CC(O)=O QHBZHVUGQROELI-UHFFFAOYSA-N 0.000 description 8
- 229910021642 ultra pure water Inorganic materials 0.000 description 8
- 239000012498 ultrapure water Substances 0.000 description 8
- QHBZHVUGQROELI-SOFGYWHQSA-N (E)-10-hydroxydec-2-enoic acid Chemical compound OCCCCCCC\C=C\C(O)=O QHBZHVUGQROELI-SOFGYWHQSA-N 0.000 description 7
- 238000000502 dialysis Methods 0.000 description 7
- 238000004945 emulsification Methods 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- 230000008878 coupling Effects 0.000 description 6
- 238000010168 coupling process Methods 0.000 description 6
- 238000005859 coupling reaction Methods 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000009182 swimming Effects 0.000 description 6
- 238000005199 ultracentrifugation Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000013016 damping Methods 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 101150101800 MRJP1 gene Proteins 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000007822 coupling agent Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000012460 protein solution Substances 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000009010 Bradford assay Methods 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 101100077934 Apis mellifera MRJP2 gene Proteins 0.000 description 2
- 101100077935 Apis mellifera MRJP3 gene Proteins 0.000 description 2
- 101100077937 Apis mellifera MRJP5 gene Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 210000001715 carotid artery Anatomy 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000008073 immune recognition Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000005374 membrane filtration Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical group [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- KUPHXIFBKAORGY-UHFFFAOYSA-N 2-amino-3-iodo-4-methylbenzoic acid Chemical compound CC1=CC=C(C(O)=O)C(N)=C1I KUPHXIFBKAORGY-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 101100077936 Apis mellifera MRJP4 gene Proteins 0.000 description 1
- 101000693922 Bos taurus Albumin Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000012207 quantitative assay Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof. First, homology analysis is performed to amino acid sequences of proteins of all members of the Apismellifera major royal jelly protein MRJPs family (MRJP1-MRJP9) to select a specific polypeptide amino acid sequence unlike other MRJPs family members in MRJP1. The related specific polypeptide is synthesized by using a chemical method, and is used as an antigen to immunize New Zealand white rabbits; taking serum, and performing Elisa assay obtaining polyclonal antibody R2 with a relatively high titer, then purifying the antibody by using an affinity column prepared from the synthesized MRJP1 polypeptide. The titer of antibody R2 is detected via Elisa assay by using MRJP1 as the antigen, and the titer of the antibody is greater than 1:20000. The present invention provides a very reliable new rapid detection method for the qualitative and quantitative detection of MRJP1 in royal jelly, and also provides a very reliable technical means for quality control, freshness detection, and identification of genuine products of royal jelly and honey products for bee product quality supervision departments and processing and trading enterprises.
Description
Technical field
The present invention relates to the main albumen MRJP1 of a kind of royal jelly specific antibody and preparation method thereof and Elisa detection by quantitative.
Background technology
The physical and chemical index of existing " National Standard of the People's Republic of China-royal jelly " (GB 9697-2008) regulation is: (10-hydroxy-2-decenoic acid, is called for short 10-HDA for moisture (top grade product 67.5%, salable product 69.0%), 10-HAD; Top grade product content 1.8%, salable product content 1.4%), protein (11-16%), total reducing sugar (15%), ash content (1.5%), acidity and starch.But in these indexs, except 10-HDA, other index can only reflect the content of a certain class material of honeybee, can not reflect truly quality and the true and false of royal jelly.10-HDA is as the peculiar activeconstituents of royal jelly, the height of its content for a long time by global royal jelly trade as the most important index of weighing quality and telling truth from falsehood.But many research discoveries, 10-HDA stability is fine, even if at high temperature also only there is a small amount of degraded, is not suitable as the index of judging freshness of royal jelly and quality.
And the main albumen 1-Major of royal jelly Royal Jelly Protein 1 (MRJP1) is topmost characteristic albumen in royal jelly, be the quality mark activeconstituents of the freshness of the represented royal jelly of generally acknowledging in the world, advise the detection index using MRJP1 content as royal jelly quality by many scholars in the world.Although passed through a lot of trials, still lacked MRJP1 in existing royal jelly national standard and international standard and detect index and detection method.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, the main albumen MRJP1 of a kind of royal jelly specific antibody and preparation method thereof and Elisa detection by quantitative are provided.
The object of the invention is to be achieved through the following technical solutions:
The preparation method of the main albumen MRJP1 of royal jelly specific antibody, adopts following steps preparation:
1) retrieve and download the aminoacid sequence of whole 9 the member MRJP1-MRJP9 coded by said gene of the main albumen MRJPs of royal jelly family from international GenBank, adopt bioinformatic analysis software to make homology analysis, select the distinctive polypeptide of MRJP1, the continuous homologous amino acid residue quantity that the distinctive polypeptide of described MRJP1 and other 8 MRJPs members share is no more than 3;
2) with the synthetic described distinctive polypeptide of MRJP1 of chemical method;
3) by the distinctive polypeptide immune New Zealand white rabbit of described MRJP1, gather serum from New Zealand white rabbit, obtain MRJP1 specific polyclonal antibody, frozen after purifying.
The distinctive polypeptide M4 of described MRJP1 is positioned at the 360-371 position of MRJP1 aminoacid sequence, and sequence is IKEALPHVPIFD.
The described MRJP1 specific polyclonal antibody > 1:20000 that tires.
MRJP1 specific antibody prepared by described method.
The Elisa fast quantitative measurement method for detecting of the main albumen MRJP1 of royal jelly, taking MRJP1 as antigen, process according to the following steps: envelope antigen, washing, sealing, wash, add primary antibodie, wash, add and two resist, wash, add chromogenic substrate, add colour developing stop buffer, with the light absorption value 450 nm-630 nm of microplate reader working sample, draw taking MRJP1 concentration as X-coordinate, with OD
450-OD
630for the typical curve of ordinate zou, set up regression equation;
Taking fresh royal jelly, Lac regis apis lyophilized powder or protein extract as antigen, process according to the following steps: envelope antigen, washing, sealing, wash, add primary antibodie, wash, add and two resist, wash, add chromogenic substrate, add colour developing stop buffer, then use the light absorption value 450 nm-630 nm of microplate reader working sample, by light absorption value substitution regression equation, obtain MRJP1 content in fresh royal jelly or Lac regis apis lyophilized powder.
The invention has the beneficial effects as follows:
(1) polypeptid specificity that the present invention filters out is strong, can solely identify the main albumen MRJP1 of royal jelly.Royal jelly protein family has 9 member MRJP1-MRJP9, from same ancestors, between each member, there is the amino acid sequence homology of height, and possesses following identical structure: all contain four conservative halfcystine sites, in sequence, have some identical amino acid regions, C-terminal all has highly hydrophobic characteristic structure.Just because of the conservative property of the main albumen MRJPs of royal jelly family, royal jelly MRJPs family protein can only be identified specifically with the polyclonal antibody that the MRJP1 complete sequence protein immunization rabbit of expression of recombinant e. coli obtains, and other non-royal jelly source protein can not be identified specifically.And the present invention screens the aminoacid sequence that the aminoacid sequence of polypeptide is different from other albumen in same family from MRJP1 complete sequence, chemical method synthesizes this polypeptide, for immune rabbit, can prepare specific polyclonal antibody, thereby realize can only the single albumen MRJP1 of specific recognition object.
(2) set up using MRJP1 as the New Set of differentiating the royal jelly true and false and quality, can fill up the deficiency of existing royal jelly national standard.Because the peculiar activeconstituents of existing royal jelly national standard (GB 9697-2008) taking Royaljelly acid 10-HDA as royal jelly is also the key index of weighing royal jelly quality and the true and false in international trade.But (the Antinelli such as Antinelli, et al. 2003, Food Chem, 80:85-89) research shows: 10-HDA preserves 12 months under-18 DEG C, 4 DEG C and room temperature condition, its content only reduces respectively 0.1%, 0.2% and 0.4%, highly stable, even if at high temperature also only there is a small amount of degraded.Therefore, be not suitable as the index of freshness of royal jelly and quality.And MRJP1 is that in royal jelly, content is the abundantest, determining Caste Differentiation in Honeybee, can make the increase of fruit bat individuality, fecundity enhancing, life, and the key protein matter of development time shortening, the leading indicator that sets it as reflection royal jelly quality product is very reasonable and science.
(3) the present invention provides a kind of simple and easy to do, Elisa rapid assay methods that accuracy is high for the mensuration of the real content of MRJP1 in royal jelly.Elisa method is as a kind of quantitative analysis method of antigen-antibody, because the catalytic efficiency of enzyme is very high, and greatly iodine effect, thus make measuring method reach very high sensitivity.Meanwhile, the method is simple to operate, rapidly convenient, with low cost, detects royal jelly quality very feasible by this method.
Brief description of the drawings
Fig. 1 is the amino acid sequence homology analysis of the full coded by said gene of 9 members (MRJP1-MRJP9) of the main albumen MRJPs of apis mellifera royal jelly family.Wherein the shared homology amino-acid residue of all MRJPs family members marks with " * " under the amino-acid residue of MRJP9 sequence.Homology amino-acid residue gray shade shared with it in the amino-acid residue of the MRJP1 specific polypeptide of selecting according to homology and other MRJPs family member marks.MRJP1 underscore part is signal peptide sequence, and the sequence that has housing is the N terminal amino acid sequence that mature peptide has checked order.
Fig. 2 is the SDS-PAGE electrophoretic analysis figure from the MRJPs of royal jelly centrifugation, the polyclonal antibody obtaining with the GST-MRJP1 fusion protein immunization rabbit of expression of recombinant e. coli that inserts MRJP1 gene with antibody 1() detect the Western blot engram analysis figure of MRJPs.Wherein, figure A is the SDS-PAGE electrophorogram of the main albumen MRJPs of royal jelly, and M is standard protein molecular weight Marker, and MRJPs is depicted as the main protein sample of royal jelly.The Western blot engram analysis result that figure B, C are MRJPs.MRJPs is depicted as apis mellifera royal jelly protein; Arrow is depicted as MRJPs member: the position of MRJP1, MRJP2, MRJP3 and MRJP5, apis mellifera royal jelly (Schmitzova et al 1998, Cell Mol Life Sci. 54 (9): 1020-1030) that the sign of position is delivered with reference to Schmitzova etc.
Fig. 3 is the SDS-PAGE electrophoretic analysis figure from the MRJPs of royal jelly centrifugation and the MRJP1 separating from royal jelly ultracentrifugation, the polyclonal antibody obtaining with the synthetic polypeptide immune rabbit of antibody 2(MRJP1 specificity) detect the Western blot engram analysis figure of MRJP1 and MRJPs.Wherein scheming A is the SDS-PAGE electrophoretic analysis figure of MRJPs and MRJP1, M is standard protein molecular weight Marker, MRJPs is for being depicted as from the main albumen of royal jelly, and MRJP1 is depicted as main albumen 1 standard model of royal jelly of purifying, and arrow is depicted as the protein band of MRJP1.Figure B is the Western blot engram analysis result of MRJP1 and MRJPs, and arrow is depicted as the Western blot of MRJP1.
Fig. 4, for taking restructuring MRJP1 whole protein antibody R1 as primary antibodie, taking from the purifying MRJP1 of royal jelly ultracentrifugation separation as standard antigen, through gradient dilution, adopts Elisa method to measure the survey light absorption value at 450 nm, 630nm place.With light absorption value OD
450– OD
630for ordinate zou, MRJP1 standard protein strength of solution is X-coordinate, the typical curve being depicted as: y=0.1100x+0.1239, R
2=0.998.
Fig. 5, for taking MRJP1 specific antibody R2 as primary antibodie, taking from the purifying MRJP1 of royal jelly ultracentrifugation separation as standard antigen, through gradient dilution, adopts Elisa method to measure the survey light absorption value at 450 nm, 630nm place.With light absorption value OD
450– OD
630for ordinate zou, MRJP1 standard protein strength of solution is X-coordinate, the typical curve being depicted as: y=0.1307x+0.0496, R
2=0.999.
Embodiment
The preparation method of the main albumen MRJP1 of royal jelly specific antibody, adopts following steps preparation:
1) retrieve and download the aminoacid sequence of whole 9 the member MRJP1-MRJP9 coded by said gene of the main albumen MRJPs of royal jelly family from international GenBank, adopt bioinformatic analysis software to make homology analysis, select the distinctive polypeptide of MRJP1, the continuous homologous amino acid residue quantity that the distinctive polypeptide of described MRJP1 and other 8 MRJPs members share is no more than 3;
2) with the synthetic described distinctive polypeptide of MRJP1 of chemical method;
3) by the distinctive polypeptide immune New Zealand white rabbit of described MRJP1, gather serum from New Zealand white rabbit, obtain MRJP1 specific polyclonal antibody, frozen after purifying.
The distinctive polypeptide M4 of described MRJP1 is positioned at the 360-371 position of MRJP1 aminoacid sequence, and sequence is IKEALPHVPIFD.
The described MRJP1 specific polyclonal antibody > 1:20000 that tires.
MRJP1 specific antibody prepared by described method.
The Elisa fast quantitative measurement method for detecting of the main albumen MRJP1 of royal jelly, taking MRJP1 as antigen, process according to the following steps: envelope antigen, washing, sealing, wash, add primary antibodie, wash, add and two resist, wash, add chromogenic substrate, add colour developing stop buffer, with the light absorption value 450 nm-630 nm of microplate reader working sample, draw taking MRJP1 concentration as X-coordinate, with OD
450-OD
630for the typical curve of ordinate zou, set up regression equation; Taking fresh royal jelly, Lac regis apis lyophilized powder or protein extract as antigen, process according to the following steps: envelope antigen, washing, sealing, wash, add primary antibodie, wash, add and two resist, wash, add chromogenic substrate, add colour developing stop buffer, then use the light absorption value 450 nm-630 nm of microplate reader working sample, by light absorption value substitution regression equation, obtain MRJP1 content in fresh royal jelly or Lac regis apis lyophilized powder.
Apis mellifera of the present invention (
apis mellifera) specific polypeptide of the main albumen MRJP1 of royal jelly derives from the aminoacid sequence of the MRJP1 coded by said gene of retrieving and downloading from international GenBank.By homology analysis, the continuously arranged homology amino-acid residue quantity that selected polypeptide and other 8 MRJPs members share is no more than 3.Using the polypeptide of chemosynthesis as antigen, immune New Zealand white rabbit, get serum and obtain the MRJP1 specific antibody R2 that tires higher.Analyzed and shown by Western blot, MRJP1 specific antibody R2 can identify the MRJP1 albumen in the main albumen MRJPs of royal jelly family in specific manner.Taking the pure MRJP1 albumen that separates from royal jelly as standard protein (antigen), make doubling dilution, adopt the absorbancy 450 nm-630 nm of microplate reader Elisa method detection different concns MRJP1 protein sample, set up the typical curve of absorbancy and MRJP1 protein concentration.By Lac regis apis lyophilized powder dilution, as antigen, adopt Elisa method to detect and obtain sample absorbancy, the typical curve that substitution is set up, calculates the content of the actual MRJP1 albumen in sample.
The preparation method of the main albumen MRJP1 of royal jelly specific antibody R2, comprises the steps:
(1) from international GenBank retrieve and download apis mellifera (
apis mellifera) aminoacid sequence of whole 9 members of the main albumen MRJPs of royal jelly family (MRJP1-MRJP9) coded by said gene, adopt bioinformatic analysis software GENTYX to make homology analysis, according to sequence information, select the peculiar polypeptide of one section of MRJP1.These polypeptide lay respectively at the 51-57 position (M1:QDAILSG) of MRJP1 aminoacid sequence, 73-80 position (M2:HDKIFVTM), 340-346 position (M3:NIRTVAQ), the polypeptide (M4:IKEALPHVPIFD) of 360-371 position.The common trait of selected polypeptide is: the continuously arranged homology amino-acid residue quantity that selected MRJP1 polypeptide and other 8 MRJPs members share is no more than 3.
(2) adopt the synthetic MRJP1 specific polypeptide M4 immunity New Zealand white rabbit of chemical method, prepare polyclonal antibody, comprise following sub-step:
(2.1) adopt the synthetic MRJP1 specific polypeptide of chemical method, its aminoacid sequence is respectively: M1:C-QDAILSG-NH
2; M2:C-HDKIFVTM-NH
2; M3:C-NIRTVAQ-NH
2; M4:C-IKEALPHVPIFD-NH
2, the longest M4 of last Selective sequence is as experiment antigen;
(2.2) polypeptide and KLH coupling (coupling agent is Sulfo-SMCC) is as immunizing antigen, and polypeptide and BSA coupling (coupling agent is glutaraldehyde) are as detectable antigens;
(2.3) with PBS, antigen being diluted is respectively 1mg/ml, and packing is frozen in-20 DEG C of refrigerators;
PBS:NaCl 8.5g, KCl 0.2g, Na
2hPO
412H
2o 2.85g, KH
2pO
40.27g, ddH
2o 950 ml, regulate pH value to 7.2, add ddH
2o is settled to 1000 ml;
(2.4) the 1st days, every kind of antigen was got 1ml antigen and is added 1ml Freund's complete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection, 2 New Zealand white rabbit of every kind of antigen immune;
Inspection emulsification degree: an emulsification antigen liquid is splashed in physiological saline, if do not scatter, show to reach requirement;
(2.5) at the 15th, 29,43 days, every kind of antigen is got 1ml and is added 1ml freund 's incomplete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection, 2 New Zealand white rabbit of every kind of antigen immune;
(2.6) the 53rd days, carotid artery was got blood, and rabbit is put to death;
(2.7) rabbit blood spends the night 4 DEG C of placements, and 4 DEG C, the centrifugal 30min of 10000rpm collect supernatant; Supernatant is polyclonal antibody;
(2.8) specific polypeptide of chemosynthesis is connected on the Sulfolink Resin of activation, prepares antigen affinity column, 1ml Sulfolink Resin coupling 1mg polypeptide;
(2.9) 10 times of column volume PBS balances for affinity column, flow to end solution; Polyclonal antibody is through 0.45 μ m membrane filtration;
(2.10) serum is crossed antigen affinity column, flows to end solution, collects stream and wears;
(2.11) 10 times of column volume PBS balances, flow to end solution;
(2.12) add 5ml antibody elution liquid, be in charge of collection elutriant, every pipe 1ml;
Antibody elution liquid: glycine 5g, is dissolved in 100ml ddH
2o, with dense HCl adjusting pH value to 2.7,4 DEG C of preservations;
(2.13) detect the elutriant the collected absorbancy at 280nm place with spectrophotometer, absorbancy is greater than 1.0 component and merges, and is placed in dialysis tubing, ddH
2the 24h that dialyses in O, water is changed 3-4 time in centre, is specific antibody solution after dialysis; Dialysis tubing specification is molecular weight cut-off 14 kDa;
(2.14) taking the MRJP1 that synthesizes polypeptide and purifying as antigen, measure through Elisa method, this specific antibody R2 tires as > 1:20000.
(3) taking the GST-MRJP1 fusion rotein of expression of recombinant e. coli of the insertion MRJP1 gene that we build before as antigen, immune rabbit obtains polyclonal antibody R1, according to step (2.4)-(2.14), make Elisa method taking the MRJP1 of purifying as antigen and measure, R1 tires as > 1:10000.
(4) with Western blot detection method do MRJP1 antibody specificity differentiate, concrete steps are as follows:
(4.1) centrifugation of royal jelly protein MRJPs: take proper amount of fresh royal jelly, be dissolved in PBS with 1:1 ratio, 4 DEG C of extracting 24 h, then centrifugal 30 min at 12000g, 4 DEG C, get supernatant.Supernatant with the dialysis membrane of aperture 14 kDa in ultrapure water, 24 h that dialyse at 4 DEG C, water is changed 3-4 time in centre, then freeze-drying, obtained freeze-drying powder is royal jelly soluble proteins MRJPs ,-20 DEG C of preservations.
(4.2) centrifugation of royal jelly protein MRJP1: take proper amount of fresh royal jelly, the dilution of quality ultrapure water such as use, fully extracting 6 h.Mixed solution is centrifugal 5 h at 245000 g, 6 DEG C, occur after demixing phenomenon, and middle layer is taken out; Middle layer is dissolved with 2 times of quality ultrapure waters, under room temperature, extracting 1 h, mixes; By this liquid centrifugal 30 min at 30000g, 6 DEG C, get supernatant.Supernatant liquor is centrifugal 5 h at 245000 g, 6 DEG C, get precipitation.This precipitation is desirable proteins sample, i.e. MRJP-1 ,-20 DEG C of preservations.
(4.3) get above-mentioned MRJPs, MRJP1 protein sample, make denaturing polyacrylamide (SDS-PAGE) electrophoresis detection.Concrete grammar is, gets appropriate protein sample and is dissolved in ultrapure water, measures total protein content by Bradford method, is made into the solution that final concentration is 1mg/ml.Prepare denaturing polyacrylamide gel, concentrated gum concentration is 5%, and resolving gel concentration is 12%.Get MRJPs, MRJP1 protein solution, add sample-loading buffer, boil, centrifugal, get supernatant and add denaturing polyacrylamide gel sample hole by the applied sample amount in 15 μ l/ holes, on same glue, add protein standard substance simultaneously, after application of sample, glue is put to electrophoresis apparatus electrophoresis, get glue with examining the dyeing of Ma Shi light blue, through methyl alcohol decolouring, on gel, sample lane shows that respectively molecular weight ranges is at multiple bands of 25-87kDa and the single band (Fig. 2 A and Fig. 3 A) of molecular weight approximately 57 kDa.
(4.4) to the MRJP1 albumen seat-terminal amino acid sequencing separating from royal jelly, concrete steps are as follows: by the MRJP1 protein SDS-PAGE electrophoresis separating from royal jelly, turn again electrophoresis and print to nitrocellulose filter, with ABI PROCISETM492cLC (GC320078) instrument, press N-terminal sequencing standard method (SCI-S-006), measure the 1-5 aminoacid sequence of MRJP1 albumen-terminal amino acid sequence, result is NILRG, consistent with the MRJP1 albumen-terminal amino acid sequence of reporting.
(4.5) Western blot detects royal jelly MRJPs and MRJP1 albumen: taking MRJPs as antigen, taking antibody R1 as primary antibodie; Taking MRJPs and MRJP1 as antigen, taking antibody R2 as primary antibodie, carry out respectively Western blot analysis.Result as Fig. 2 B and Fig. 3 B and as shown in, from this two figure, antibody R1 can identify all albumen of MRJPs family, shows all Western blots (Fig. 2 B, 2C MRJPs swimming lane) of molecular weight at 25-87 kDa, does not possess narrow spectrum recognition capability.And R2 antibody can only immune response (Fig. 3 B MRJPs and MRJP1 swimming lane) occur with the MRJP1 that molecular weight is 57 kDa, there is the specificity of single-minded identification MRJP1.
(5) make the Elisa Quantitative detection of MRJP1 in royal jelly magma, lyophilized powder and protein extract with specific antibody R2.Process according to the following steps:
(5.1) taking MRJP1 as antigen, carbonate buffer solution (Carbonate Buffer solution, CBS) is coated, and coated concentration is 1-10 μ g/ml, and coated volume is 100 μ l/ holes, and 4 DEG C are spent the night;
Carbonate buffer solution: Na
2cO
31.59g, NaHCO
32.93g, ddH
2o 950 ml, regulate pH value to 9.6, add ddH
2o is settled to 1000 ml, 4 DEG C of preservations;
(5.2) taking MRJP1 specific antibody R2 as primary antibodie, with 10000-20000 times of PBS dilution, 100 μ l/ holes, 37 DEG C of standing 1-2 h; Taking the goat anti-rabbit igg of HRP mark as two anti-, dilution 5000-10000 doubly, 100 μ l/ holes, 37 DEG C of standing 0.5-1 h;
(5.3) be dissolved in PBS as confining liquid taking 5% skim-milk, 200 μ l/ holes, 37 DEG C of standing 1-2h; Taking PBST(PBS+0.05% Tween-20) be washings, every step washing 5-10 time, and pat dry;
(5.4), taking TMB as chromogenic substrate, 100 μ l/ holes, place 15min for 37 DEG C; With 2 M H
2sO
4for stop buffer, 50 μ l/ holes, add the light absorption value of measuring immediately wavelength 450 nm and 630nm place in microplate reader.450nm is the maximum absorption wavelength of this developer, disturbs and 630nm place light absorption value can reduce the light being caused by cut or finger mark etc. on container.
(5.5) taking MRJP1 concentration as X-coordinate, with OD
450-OD
630for ordinate zou drawing standard curve, obtain regression equation.
(5.6) taking fresh royal jelly or Lac regis apis lyophilized powder as antigen, coated concentration is 100,200,300 ug/ml(or 50,100,200 μ g/ml), adopt the same step measurements fresh royal jelly in (5.1)-(5.4) or Lac regis apis lyophilized powder solution light absorption value, by light absorption value substitution regression equation, obtain the content of MRJP1 in fresh royal jelly or Lac regis apis lyophilized powder.
The present invention be set up in the world first fast, effectively, low cost detects the novel method of MRJP1 content, filled up the deficiency that lacks MRJP1 in existing royal jelly national standard and international standard and detect index and detection method.Of the present invention for royal jelly commercial quality control both at home and abroad and real and fake discrimination provide highly effective means, also learn fundamental research for the honeybee relevant to MRJP1 new means are provided.
Below in conjunction with specific embodiment and and accompanying drawing, further set forth the present invention.Should be understood that these embodiment are only not used in the restriction scope of the invention for the present invention is described.
the screening of embodiment 1:MRJP1 specific polypeptide
Login international gene information storehouse http://www.ncbi.nlm.nih.gov/, the aminoacid sequence of 9 members of MRJPs family (MRJP1-MRJP9) coded by said gene is downloaded in search, the sequence number of MRJP1 is NM_001011579, the sequence number of MRJP2 is NM_001011580, the sequence number of MRJP3 is NM_001011601, the sequence number of MRJP4 is NM_001011610, the sequence number of MRJP5 is NM_001011599, the sequence number of MRJP6 is NM_001011622, the sequence number of MRJP7 is NM_001014429, the sequence number of MRJP8 is NM_001011564, the sequence number of MRJP9 is NM_001024697.By the aminoacid sequence input GENTYX software of above-mentioned sequence MRJP1-MRJP9 coded by said gene, carry out homology analysis, complete and analyze rear output homology analysis figure (Fig. 1) as shown in Figure 1.
According to Fig. 1 homology analysis result, filter out the specific polypeptide of MRJP1, these polypeptide lay respectively at MRJP1 aminoacid sequence 51-57 position (M1:QDAILSG), 73-80 position (M2:HDKIFVTM), 340-346(M3:NIRTVAQ), 360-371 position (M4:IKEALPHVPIFD).The selection principle of these specific polypeptides of selecting from MRJP1 albumen is that the continuous arrangement homology amino-acid residue shared with other MRJPs protein sequence is no more than 3, is suitable as specific antigens (Fig. 1).
the preparation of embodiment 2:MRJP1 specific polyclonal antibody
1. antigen preparation
Select the M4 peptide sequence (IKEALPHVPIFD) in embodiment to carry out chemosynthesis.By synthetic 5 mg polypeptide and KLH coupling (coupling agent is Sulfo-SMCC), as immunizing antigen, another 5 mg are synthesized to polypeptide and BSA coupling (coupling agent is glutaraldehyde) as detectable antigens; Being diluted to concentration with PBS is respectively 1 mg/ml, and packing is frozen in-20 DEG C.
2. immune rabbit
Select 2 of healthy new zealand white rabbits, carried out immunity at the 1st, 15,29,43 days.Immune operation is as follows: get 1 ml immunizing antigen, add 1 ml Freund's complete adjuvant, emulsification, splashes into an emulsification antigen liquid in physiological saline, does multiple spot (at least 8 point) injection with syringe white rabbit nape portion is subcutaneous; At the 15th, 29,43 days, every kind of antigen was got 1ml and is added 1ml freund 's incomplete adjuvant, emulsification, the subcutaneous multiple spot of nape portion (at least 8 point) injection.
3. polyclonal antibody preparation
The 53rd day, get blood at rabbit carotid artery, rabbit blood is put to 4 DEG C and spend the night, centrifugal 30 min(4 DEG C, 10000 rpm), collect supernatant.Supernatant is polyclonal antibody.Detect by detectable antigens Elisa method the polyclonal antibody obtaining and tire, filter out the higher antibody of tiring.
embodiment 3: affinity chromatography purifying polyclonal antibody
1. affinity column preparation
With 1 ml Sulfolink Resin and the synthetic MRJP1 specific polypeptide coupling of 1 mg.Synthetic 5 mg MRJP1 polypeptide is connected on the Sulfolink Resin of activation, is prepared into antigen affinity column, 10 times of column volume PBS balances for affinity column, flow to end solution.
2. antibody purification
By 0.45 μ m membrane filtration for polyclonal antibody, then cross antigen affinity column, drain solution, collect stream and wear; The PBS damping fluid balance of using again 10 times of column volumes, drains solution; Add 5 ml antibody elution liquid (glycine 5g, is dissolved in 100 ml ultrapure waters, with dense HCl adjusting pH value to 2.7,4 DEG C of preservations), be in charge of collection elutriant by 1 ml/tube.
Detect the elutriant of collecting at 280 nm places with spectrophotometric, absorbancy is greater than to 1.0 component and merges, be placed in the dialysis tubing of molecular weight cut-off 14 kDa, 12 h dialyse in ultrapure water, water is changed once in centre, is the MRJP1 specific antibody solution of purifying after dialysis; Obtain altogether rabbit 1 antibody 2.1 ml, rabbit 2 antibody 2.3 ml, with 1 ml/tube packing, frozen in-20 DEG C.
embodiment 4:western blot detects the specificity of MRJP1 specific antibody R2
1. the centrifugation of MRJPs albumen and SDS-PAGE electrophoresis detection in royal jelly
Take proper amount of fresh royal jelly, be dissolved in PBS with 1:1 ratio, 4 DEG C of extracting 24 h, then centrifugal 30 min at 12000g, 4 DEG C, get supernatant.Supernatant with the dialysis membrane of aperture 14 k in ultrapure water, 24 h that dialyse at 4 DEG C, water is changed 3-4 time in centre, then freeze-drying, obtained freeze-drying powder is royal jelly soluble proteins MRJPs ,-20 DEG C of preservations.
Get above-mentioned MRJPs protein sample, make denaturing polyacrylamide (SDS-PAGE) electrophoresis detection.Concrete grammar is, gets appropriate protein sample and is dissolved in ultrapure water, measures total protein content by Bradford method, is made into the solution that final concentration is 1 mg/ml.Prepare denaturing polyacrylamide gel, concentrated gum concentration is 5%, and resolving gel concentration is 12%.Get MRJPs protein solution, add sample-loading buffer, boil, centrifugal, get supernatant and add denaturing polyacrylamide gel sample hole by the applied sample amount in 15 μ l/ holes, on same glue, add protein standard substance simultaneously, after application of sample, glue is put to electrophoresis apparatus electrophoresis, got glue with examining the dyeing of Ma Shi light blue, decolour through methyl alcohol, on gel, sample lane shows the multiple bands of molecular weight ranges at 25-87 kDa, comprising MRJP1 albumen (Fig. 2 A and Fig. 3 A swimming lane MRJPs).
2. the ultracentrifugation separation of MRJP1 albumen, qualification and SDS-PAGE electrophoresis detection in royal jelly
Take proper amount of fresh royal jelly, the dilution of quality ultrapure water such as use, fully extracting 6 h.Mixed solution is centrifugal 5 h at 245000 g, 6 DEG C, occur after demixing phenomenon, and middle layer is taken out; Middle layer is dissolved with 2 times of quality ultrapure waters, under room temperature, extracting 1 h, mixes; By this liquid centrifugal 30 min at 30000g, 6 DEG C, get supernatant.Supernatant liquor is centrifugal 5 h at 245000 g, 6 DEG C, get precipitation.This precipitation is desirable proteins sample, i.e. MRJP-1 ,-20 DEG C of preservations.
Get the MRJP1 protein sample that ultracentrifugation obtains, make denaturing polyacrylamide (SDS-PAGE) electrophoresis detection.Concrete grammar is, gets appropriate protein sample and is dissolved in ultrapure water, measures total protein content by Bradford method, is made into the solution that final concentration is 1 mg/ml.Prepare denaturing polyacrylamide gel, concentrated gum concentration is 5%, and resolving gel concentration is 12%.Get MRJP1 protein solution, add sample-loading buffer, boil, centrifugal, get supernatant and add denaturing polyacrylamide gel sample hole by the applied sample amount in 15 μ l/ holes, on same glue, add protein standard substance simultaneously, after application of sample, glue is put to electrophoresis apparatus electrophoresis, got glue with examining the dyeing of Ma Shi light blue, decolour through methyl alcohol, on gel, sample lane shows the single band of molecular weight approximately 57 kDa, and this is pure MRJP1 albumen (Fig. 3 A MRJP1 swimming lane).
3. pair MRJP1 albumen that separation obtains from royal jelly is made-terminal amino acid sequencing
By the MRJP1 protein SDS-PAGE electrophoresis separating from royal jelly, turn again electrophoresis and print to nitrocellulose filter, with ABI PROCISETM492cLC (GC320078) instrument, press N-terminal sequencing standard method (SCI-S-006), measure the 1-5 aminoacid sequence of MRJP1 albumen-terminal amino acid sequence, result is NILRG, consistent with the MRJP1 albumen-terminal amino acid sequence of reporting.
4. Western blot detects royal jelly MRJPs and MRJP1 albumen
Taking MRJPs as antigen, the polyclonal antibody (antibody R1) that the GST-MRJP1 fusion protein immunization rabbit of the expression of recombinant e. coli of the insertion MRJP1 gene that before using, we build obtains is primary antibodie; Taking MRJPs and MRJP1 as antigen, with the polyclonal antibody (antibody R2) that the synthetic polypeptide immune rabbit of MRJP1 specificity obtains be primary antibodie, carry out respectively Western blot analysis.Result as Fig. 2 B and Fig. 3 B and as shown in, from this two figure, antibody R1 can identify all albumen of MRJPs family, shows all Western blots (Fig. 2 B, 2C MRJPs swimming lane) of molecular weight at 25-87 kDa, does not possess narrow spectrum recognition capability.And R2 antibody can only immune response (Fig. 3 B MRJPs and MRJP1 swimming lane) occur with the MRJP1 that molecular weight is 57 kDa, there is the specificity of single-minded identification MRJP1.
embodiment 5:Elisa method detects antibody titer
The polyclonal antibody of preparing with the expression of recombinant e. coli product GST-MRJP1 fusion rotein of antibody R1(MRJP1 gene described in embodiment 4 respectively), the polyclonal antibody of the synthetic polypeptide preparation of the MRJP1 specificity that filters out for antibody R2() be primary antibodie, operation according to the following steps:
(1) coated: use respectively MRJP1 standard protein, royal jelly as antigen, diluting with CBS is 1 μ g/ml, and 100 μ l/ holes add in 96 orifice plates, and 4 DEG C are spent the night;
(2) sealing: coating buffer is discarded, with 200 μ l/ holes, confining liquid is added in 96 orifice plates, place 1.5 h for 37 DEG C;
(3) add primary antibodie: discard confining liquid, add respectively the antibody R1 of different extension rates (5000,10000,20000 and 40000) or antibody R2(respectively from 2 rabbits), and contrast BSA, 100 μ l/well, place 1 h for 37 DEG C;
(4) washing: with tap water flushing 10 times, pat dry;
(5) add two to resist: 5000 times of enzyme mark goat-anti rabbit confining liquid dilutions, add in 96 orifice plates with 100 μ l/ holes, place 30 min for 37 DEG C;
(6) washing: with tap water flushing 10 times, pat dry;
(7) add TMB chromogenic substrate: 100 μ l/ holes add in 96 orifice plates, place 15 min for 37 DEG C;
(8) add stop buffer: with 50 μ l/ holes by 2M H
2sO
4add in 96 orifice plates;
(9) reading: be placed in immediately in microplate reader, 450n m place surveys light absorption value;
As shown in table 1 to the variance analysis of MRJP1 standard protein detected result.
Table 1 Elisa measures relatively MRJP1 whole protein polyclonal antibody (R1 antibody) and specific polyclonal antibody (R2 antibody) tiring to MRJP1 standard protein
Remarks: it is 450 nm that * light absorption value is measured wavelength; What * mean value institute marking-up parent phase was same indicates without significant difference, and significance level is p<0.01.
As known from Table 1, in 1% conspicuous level, the OD value utmost point of dilution 10000 antibody R1 is significantly higher than contrast BSA, dilutes the OD value utmost point of R2 antibody of 20000 times significantly higher than contrasting BSA; Therefore, the Elisa of the R1 antibody > 1:10000 that tires, the > 1:20000 and the Elisa of R2 antibody tires, is 2 times of R1 antibody; Simultaneously R2 antibody (being MRJP1 specific antibody) tire the utmost point significantly higher than R1 antibody (MRJP1 whole protein antibody).
As shown in table 2 to the variance analysis of royal jelly detected result.
Table 2 Elisa measures relatively MRJP1 whole protein polyclonal antibody (R1 antibody) and specific polyclonal antibody (R2 antibody) tiring to royal jelly
Remarks: it is 450 nm that * light absorption value is measured wavelength; What * mean value institute marking-up parent phase was same indicates without significant difference, and significance level is
p<0.01.
As known from Table 2, in 1% conspicuous level, it is also > 1:10000 that the Elisa of R1 antibody tires, and it is also > 1:20000 that the Elisa of R2 antibody tires, and is 2 times of R1 antibody; Simultaneously R2 antibody (being MRJP1 specific antibody) tire the utmost point significantly higher than antibody 1(MRJP1 whole protein antibody).This is very consistent with the trend of table 1.
According to Western blot detected result, R1 antibody is except producing immune recognition reaction with MRJP1, can also with MRJPs family in other albumen produce immune recognition reaction.And R2 antibody can only be identified MRJP1.Therefore, tiring of antibody 2 matches higher than antibody 1 and Western blot detected result.
the content of MRJP1 in embodiment 6:Elisa indirect method quantitative assay royal jelly
1. the preparation of antigen, antibody-solutions
The MRJP1 standard protein that ultracentrifugation is obtained is dissolved in CBS, is made into the solution of 10 μ g/ml.Be primary antibodie with MRJP1 whole protein polyclonal antibody (R1 antibody, 1:5000 dilution) and specific polyclonal antibody (R2 antibody, 1:10000 dilution) respectively; Taking the goat anti-rabbit igg of HRP mark as ELIAS secondary antibody, add in PBS by 1:5000, be made into two anti-solution.
2. the foundation of MRJP1 typical curve
By the MRJP1 standard protein solution of 10 μ g/ml, press respectively multiple proportions gradient dilution method application of sample in 96 orifice plates, every hole adds 100 μ l/ holes, 4 DEG C of coated spending the night.Abandon liquid in hole, wash 5 times with PBST damping fluid, add skimmed milk confining liquid with the amount in 200 μ l/ holes, 37 DEG C of sealing 1.5 h.Abandon skimmed milk confining liquid, wash 5 times with PBST damping fluid, with the amount in 100 μ l/ holes, add respectively primary antibodie R1 antibody, R2 antibody-solutions, 37 DEG C of reaction 1 h.Abandon primary antibodie solution, with PBST damping fluid washing 5 times, add two anti-solution with the amount in 100 μ l/ holes, 37 DEG C of reaction 0.5 h.Abandon two anti-solution, with PBST damping fluid washing 5 times, add nitrite ion TMB with the amount in 100 μ l/ holes, 37 DEG C of reaction 15 min, add TMB stop buffer 2M H with the amount in 50 μ l/ holes
2sO
4.Be placed in immediately in microplate reader, measure the survey light absorption value at 450 nm, 630 nm places.
(1) obtain table 3 with above-mentioned R1 antibody test.
The absorbancy that table 3 obtains with the MRJP1 of R1 TPPA gradient dilution
With OD
450-OD
630light absorption value mean value (n=3) is ordinate zou, and MRJP1 standard protein strength of solution is X-coordinate, obtains typical curve (Fig. 4): (R1) y=0.1100x+0.1239, R
2=0.998;
(2) obtain table 4 with above-mentioned R2 antibody.With OD
450-OD
630light absorption value mean value (n=3) is ordinate zou, and MRJP1 standard protein strength of solution is X-coordinate, obtains typical curve (Fig. 5): (R2) y=0.1307x+0.0496, R
2=0.999.
The absorbancy that table 4 obtains with the MRJP1 of R2 TPPA gradient dilution
3. measure the effect comparison of MRJP1 content in royal jelly with antibody R1 and R2
Accurately weigh respectively Lac regis apis lyophilized powder 0.1 g, be dissolved in PBS, be made into the solution that concentration is 100 μ g/ml, press respectively multiple proportions gradient dilution method application of sample in 96 orifice plates, every hole adds 100 μ l/ holes.By above method, measure respectively the OD of Lac regis apis lyophilized powder as primary antibodie with R1 and R2
450-OD
630value.Result is as follows:
(1) R1 TPPA result
The light absorption value mean value obtaining by R1 TPPA is 1.306 ± 0.110(table 5); The X item of substitution equation y=0.1100x+0.1239, the MRJP1 content that obtains Lac regis apis lyophilized powder solution is 10.75 ± 1.00 μ g/ml.
(2) R2 TPPA result
The light absorption value mean value obtaining by R2 TPPA is 0.822 ± 0.024(table 5); The Y item of substitution equation y=0.1307x+0.0496, the MRJP1 content (X) that obtains Lac regis apis lyophilized powder solution is 5.91 ± 0.19 μ g/ml(%).
R1 antibody and the comparison of R2 TPPA Lac regis apis lyophilized powder MRJP of the same race content results for table 5
Remarks:
*: t
=0.0012, * *: represent that two measurement results exist utmost point significant difference.
(3) R1 antibody and R2 TPPA result and analysis
Above-mentioned two measurement results are carried out to t inspection, find that result exists utmost point significant difference (t<0.001), in the Lac regis apis lyophilized powder obtaining by R1 TPPA, MRJP1 content is 10.75 ± 1.00 μ g/ml (%), higher than MRJP1 content in the Lac regis apis lyophilized powder obtaining by R2 TPPA (5.91 ± 0.19 % μ g/ml) 4.84 μ g/ml (%), R1 is higher by 81.90% than the detected result of R1.The MRJP1 content obtaining according to R2 TPPA is 55% (100 × 5.91/10.75) of R1 TPPA result.
According to Western blot detected result, R1 can identify all albumen of MRJPs family, and R2 antibody can only be identified MRJP1 albumen.Therefore, the measurement result of R1 is MRJPs, and the measurement result of R1 is MRJP1.
According to report, MRJP1 accounts for the more than 48% of water-soluble protein (MRJPs family) (Hanes and Simuth., J. 1992, J. Apicult. Res. 31:22-26).Therefore, to account for 55% the result of MRJPs be consistent with bibliographical information to this MRJP1.
Claims (3)
1. a preparation method for the main albumen MRJP1 of royal jelly specific antibody, is characterized in that, adopts following steps preparation:
1) retrieve and download the aminoacid sequence of whole 9 the member MRJP1-MRJP9 coded by said gene of the main albumen MRJPs of royal jelly family from international GenBank, adopt bioinformatic analysis software to make homology analysis, select the distinctive polypeptide of MRJP1, the continuous homologous amino acid residue quantity that the distinctive polypeptide of described MRJP1 and other 8 MRJPs members share is no more than 3;
2) with the synthetic described distinctive polypeptide of MRJP1 of chemical method;
3) by the distinctive polypeptide immune New Zealand white rabbit of described MRJP1, gather serum from New Zealand white rabbit, obtain MRJP1 specific polyclonal antibody, frozen after purifying;
The distinctive polypeptide M4 of described MRJP1 is positioned at the 360-371 position of MRJP1 aminoacid sequence, and sequence is IKEALPHVPIFD;
The described MRJP1 specific polyclonal antibody > 1:20000 that tires.
2. a MRJP1 specific antibody prepared by method according to claim 1.
3. the Elisa fast quantitative measurement method for detecting of a MRJP1 specific antibody as claimed in claim 2, it is characterized in that, taking MRJP1 as antigen, process according to the following steps: envelope antigen, washing, sealing, wash, add primary antibodie, wash, add and two resist, wash, add chromogenic substrate, add colour developing stop buffer, with the light absorption value 450 nm-630 nm of microplate reader working sample, draw taking MRJP1 concentration as X-coordinate, with OD
450-OD
630for the typical curve of ordinate zou, set up regression equation; Taking fresh royal jelly, Lac regis apis lyophilized powder or protein extract as antigen, process according to the following steps: envelope antigen, washing, sealing, wash, add primary antibodie, wash, add and two resist, wash, add chromogenic substrate, add colour developing stop buffer, then use the light absorption value 450 nm-630 nm of microplate reader working sample, by light absorption value substitution regression equation, obtain MRJP1 content in fresh royal jelly or Lac regis apis lyophilized powder; Described primary antibodie is described MRJP1 specific antibody, and described two resist for enzyme mark goat-anti rabbit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210576152.7A CN103059135B (en) | 2012-12-26 | 2012-12-26 | A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210576152.7A CN103059135B (en) | 2012-12-26 | 2012-12-26 | A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103059135A CN103059135A (en) | 2013-04-24 |
| CN103059135B true CN103059135B (en) | 2014-07-30 |
Family
ID=48102058
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210576152.7A Active CN103059135B (en) | 2012-12-26 | 2012-12-26 | A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103059135B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104316587A (en) * | 2014-10-20 | 2015-01-28 | 浙江大学 | Establishment method and application for detecting freshness of royal jelly by adopting running gel protein band |
| CN105218654A (en) * | 2015-05-08 | 2016-01-06 | 西北大学 | A kind of characteristic protein differentiating Mel Jujubae flower source |
| CN105693858A (en) * | 2016-03-15 | 2016-06-22 | 浙江大学 | Preparing method and detecting method for MRJR1 antibody pairs in honey and kit |
| CN106699899B (en) * | 2016-12-27 | 2020-11-24 | 重庆大学 | Immunogen for obtaining Nrf1D protein antibody, Nrf1D protein antibody and Elisa detection kit |
| KR20190139913A (en) * | 2017-04-13 | 2019-12-18 | 더블유와이엘 사이언스 인크. | Antibodies to royal lactin and uses thereof |
| CN107462713A (en) * | 2017-07-05 | 2017-12-12 | 浙江大学 | The preparation method and its test strips of honey Rapid detection test strip and application |
| CN107892713B (en) * | 2017-09-14 | 2020-04-28 | 中国农业科学院蜜蜂研究所 | Monoclonal antibody for resisting royal jelly major protein 3 and enzyme linked immunosorbent assay kit for detecting royal jelly major protein 3 |
| CN110208539A (en) * | 2019-03-15 | 2019-09-06 | 中国农业科学院蜜蜂研究所 | The pairing monoclonal antibody of anti-MRJP4, the ELISA kit and colloid gold immune test paper for detecting MRJP4 |
| CN109884290A (en) * | 2019-03-27 | 2019-06-14 | 扬州大学 | A kind of method of Rapid identification royal jelly effect |
| CN111751364B (en) * | 2020-06-28 | 2023-05-23 | 浙江省农业科学院 | Method for rapidly determining water-soluble protein and total sugar of royal jelly |
| CN111735966B (en) * | 2020-07-23 | 2021-01-15 | 中国农业科学院蜜蜂研究所 | Method for detecting royal jelly major protein 5 in honey and Chinese honey by liquid chromatography-tandem mass spectrometry |
| CN111733142B (en) * | 2020-08-14 | 2020-12-25 | 中国农业科学院蜜蜂研究所 | Monoclonal antibody and colloidal gold detection card of Apis mellifera MRJP1 |
| CN113176348A (en) * | 2021-03-07 | 2021-07-27 | 杭州艾蜂健康科技有限公司 | Biological marker for freshness of royal jelly and screening method thereof |
| CN118146339B (en) * | 2024-05-13 | 2024-08-23 | 中国农业科学院蜜蜂研究所 | Italian bee MRJP1 mutant and application thereof as internal standard protein in bee product analysis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101289664A (en) * | 2008-05-23 | 2008-10-22 | 浙江大学 | Method for preparing MRJP1 eucaryon expression products of China honey bee for promoting growth of cells |
-
2012
- 2012-12-26 CN CN201210576152.7A patent/CN103059135B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103059135A (en) | 2013-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103059135B (en) | A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof | |
| CN102747042B (en) | Hybridoma cell line 10G4 and anti-alfatoxin total (B1, B2, G1 and G2) monoclonal antibody produced by the same | |
| CN102628867B (en) | Double antibody latex intensified Retinal-binding protein detection kit | |
| CN101993855B (en) | Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof | |
| CN102279269A (en) | Preparation method of cystatin C detection kit | |
| Söulling | Free light chains of immunoglobulins in normal serum and urine determined by radioimmunoassay | |
| CN102759623A (en) | Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof | |
| CN101592661B (en) | Brucellosis antibody competitive enzyme-linked immunosorbent assay reagent kit | |
| CN105021830A (en) | Double-antibody sandwich colloidal gold detection test strip for human lactoferrin | |
| CN102746403A (en) | Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin | |
| CN106432440A (en) | Brucella melitensis antibody PPA-ELISA detection kit and preparation method thereof | |
| CN101592660B (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit | |
| CN101799470A (en) | Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit | |
| CN103215230A (en) | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit | |
| CN105693858A (en) | Preparing method and detecting method for MRJR1 antibody pairs in honey and kit | |
| CN107144686B (en) | A kind of accurate fluorescence quantitative detecting method | |
| NO303852B1 (en) | Procedure for detecting analyte variants, as well as kits for performing the method | |
| CN108570104A (en) | Recombinating adiponectin antigen, antibody and adiponectin nano rubber latex enhances immunoturbidimetry kit | |
| CN105651996A (en) | Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof | |
| CN107478848B (en) | The kit and preparation method thereof of quantitative detection people NT-proBNP | |
| CN101294956B (en) | Application of human retinol conjugated protein-4 in metabolism exception detection | |
| CN104965086A (en) | Dog toxoplasma gondii antibody indirect ELISA detection kit | |
| Burnie | Developments in the serological diagnosis of opportunistic fungal infections | |
| CN102336814B (en) | Polypeptide sequence for preparing anti-liver-neoplasm marker CK-19 antibody, polyclonal antibody and application thereof | |
| CA2259413A1 (en) | Methods for determining the presence of brain protein s-100 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |