CN1651918A - Method for making recombinant antigen pp 65 envelope enzyme-linked reaction plate and ELISA test kit - Google Patents
Method for making recombinant antigen pp 65 envelope enzyme-linked reaction plate and ELISA test kit Download PDFInfo
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- CN1651918A CN1651918A CN 200510037663 CN200510037663A CN1651918A CN 1651918 A CN1651918 A CN 1651918A CN 200510037663 CN200510037663 CN 200510037663 CN 200510037663 A CN200510037663 A CN 200510037663A CN 1651918 A CN1651918 A CN 1651918A
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Abstract
The present invention relates to a new method for making recombinant antigen PP65 coated enzyme labeled reaction plate and ELISA kit. Its key technique mainly liesin that using gene engineering method to prepare specific recombinant antigen-HCMV pp65, after the antigen is exracted and purified, using the coating plate as important component in the kit, besides, after the anti-human IgM monoclone antibody prepared by utilizing mouse-mouse hybridome technique is labeled by using horseradish peroxidase, it can be used for assembly of kit. Said method capable of creating sensitive quick ELISA antibody to trap HCMV specific IgM antibody in serum has high specificity and high stability, and said kit mainly can be used in laboratory research and clinical diagnosis.
Description
Technical field
The present invention relates to a kind of new biopreparate HCMVpp65 recombinant protein, is a kind of ELISA detection kit that is used to detect HCMV-IgM.
Background technology
(Human Cytomegalovirus HCMV) belongs to nerpes vinrus hominis β subfamily virus to human cytomegalovirus, and the infection in the crowd is very general, is worldwide distribution.Seroepidemiological survey shows that the 40-100% adult has HCMV antibody; Country variant, different economic situations, different crowds, different age brackets, HCMV infection rate difference, its popular no seasonal trend.Population in Asia and Africa 90% is infected, and wherein most of people promptly have antibody at adolescence, illustrate to infect the age early, and its infection of western countries takes place later; The low higher income crowd infection rate's height of income crowd.HCMV is one of modal pathogen of human congenital infection, and is huge to pregnant woman and fetus influence, often causes diseases such as miscarriage, stillborn foetus, fetal anomaly, hypoevolutism and tardy sexual centre nervous system defective.Can be asymptomatic during the baby due of congenital infection, but also have severe patient can involve many internal organs, in addition dead.The visible hepatosplenomegaly of symptom is arranged, jaundice, ecchymosis or purpura, choroidoretinitis, microcephalus etc.Hearing, hypopsia in various degree can appear in infant, consciousness, dyskinesia, baryencephalia etc.HCMV has become the important pathogen of infringement children's health, so the detection of HCMV becomes one of conventional project of pregnant woman and new born health.HCMV infects and can cause that body's immunological function reduces, and particularly cellular immune function decreased causes the generation of multiple disease, as hepatitis, organ transplant failure etc.In addition, HCMV can propagate through milk, uterine neck liquid, blood product, transplanted organ or cell etc.After the primary infection, hide in bone marrow cell etc.; When immune function depression, the virus of hiding easily is activated, and causes serious clinical symptoms, and especially in the crowd of fetus, neonate, immunosupress or the immunologic hypofunction of growing, HCMV infects and can cause or even the clinical symptoms of lethal.
In view of HCMV is bigger to the mankind's harmfulness, find that it is the key that ensures population quality and improve level of human health that the patient in time takes measures.The crowd that China need do the HCMV detection every year approximately has more than 1,000 ten thousand person-times at least.The various immunodiagnosis kit quality of HCMV are uneven in the market, both do not had unified quality standard, again in unordered production and application, cause serious hindrance for prevention and the diagnosis and treatment of HCMV.Though about the listing both at home and abroad of HCMV external diagnosis reagent, external product costs an arm and a leg, and is not suitable for China's national situation at present.And the domestic reagent box adopts specific IgM in the rough Detection of antigen serum of the totivirus of cellular incubation more, and viral yield is low, and gained viral antigen purity is lower, and its antigenicity may have with other herpesviral and intersect, and influences the accuracy of testing result; These kits have stability and repeatability is relatively poor, sensitivity and the not high shortcoming of specificity.In addition, the investigation contrast confirms, has bigger mass discrepancy between the product of each manufacturer production, and its minimum detectable activity can differ 15～30 times.Domestic detectable exists the main cause of mass discrepancy may be indifferent with the manufacturer quality mind, and it is relevant to lack quality control standard.
Summary of the invention
Purpose of the present invention: a kind of new HCMVpp65-IgM type ELISA detection kit is provided.
Technical solution of the present invention is as follows:
Specificity recombinant antigen pp65 bag is prepared from through the following steps by the preparation method of reaction plate:
(1) acquisition of pp65 recombinant protein:
1. the prokaryotic expression of HCMV pp65: with HCMV AD169 DNA is template, and with Auele Specific Primer pcr amplification pp65 (1006-1521nt) genes of interest, genes of interest is cloned the prokaryotic expression carrier in pET-T0P0, transforms Top10 competence bacterium.Extract the clone according to the acillin resistance, the plasmid DNA of rapid extraction transformant is identified, obtains and the corresponding to segment of purpose fragmental molecule amount, is judged to positive findings.Extract plasmid with the PCR positive colony, carrier pET100-pp65-DNA order-checking, the result shows that the pp65 gene coded sequence correctly is connected into pET100, has an open reading frame (ORF) that 516bp is complete, with the homology of pp65 gene 98% among the GenBank.Calculate that its protein molecular weight is 22.8KD, pI is 4.18.
2. the pp65 recombinant protein is identified: with the recombinant plasmid PET/pp65 transformed into escherichia coli BL21 (DE3) of genes of interest, after IPTG induces, mycoprotein shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, compares with empty bacterium, at 20～30KD a dense newly-increased protein band that dyes is arranged.Identify through Western blotting, can with pp65 monoclonal antibody generation strong positive reaction, the pp65 recombinant protein has stronger immunogenicity.
3. the great expression of HCMV pp65 recombinant protein, purifying: through inducing in a large number, express, obtain a large amount of pp65 recombinant proteins behind the affinitive layer purification.
(2) with the pp65 recombinant protein as antigen, be diluted to behind the suitable concentration by 100 μ l/ holes bag by 96 hole ELISA Plate with the carbonate buffer solution of pH9.6, put in the wet box 4 ℃ 12～24 hours, PBST washes plate 3 times, each 3min pats dry then; Obtain antigen pp65 coated elisa plate.
(3) fill it up with hole (about 200 μ l/ holes) with the PBST that contains 10% calf serum, hatch for 37 ℃, sealing 1h washes plate 3 times, pats dry and seals, and can obtain the antigen coated enzyme reaction plate of pp65.
This new HCMVpp65 IgM type ELISA detection kit is characterized in that: described kit is made of by plate, sample diluent, cleansing solution, ELIAS secondary antibody, colour developing liquid, stop buffer above-mentioned specificity recombinant antigen pp65 bag.Specifically be divided into: (1) sample diluent: the phosphate buffer (PBS) that contains the 0.01M pH7.4 of 10% calf serum and 2% anti-human IgG; (2) cleansing solution: the phosphate buffer (PBST) that contains 0.05%Tween 20; (3) ELIAS secondary antibody: the mouse-anti people IgM monoclonal antibody of horseradish peroxidase-labeled; (4) colour developing liquid: TMB-H
2O
2Urea liquid; (4) stop buffer: 2mol/L H
2SO
4Solution.
HCMVpp65 participates in the regulation and control of HCMV viral gene expression, changes the host cell metabolism, helps the key protein of virus replication; Pp65 albumen mainly by phospholamban, is not only the major protein antigen in the viremia virusemia as HCMV, and is the main target molecule of HCMV specific CTL; It can produce by inducing specific antibody, also can induce the cellullar immunologic response of body; Be the main identification and the effect antigen of Th cell and T memory cell.In view of pp65 has these important function and good antigenicity, the present invention has chosen the partial sequence gene (1006～1521nt) of coding HCMV pp65 albumen, adopt gene recombination technology, prokaryotic expression obtains a kind of new pp65 recombinant protein after the affinity chromatography.Both keep its antigenicity through transformed pp65 recombinant protein, improved the prokaryotic expression amount of pp65 recombinant protein again.Adopt this recombinant protein, the present invention successfully uses indirect elisa method, detects specificity HCMV-IgM quickly and easily, and this result is compared with U.S. BioCheck kit, and consistance is 97.0%.
The detection positive rate of the testing result of kit of the present invention and homemade totivirus--ELISA method and U.S. BioCheck kit is respectively: this law is 44%; Totivirus--ELISA method 50%; U.S. BioCheck kit 45%.Specificity this law is 98.2%, is higher than totivirus--ELISA method 90.9%, correct index is 92.8%, and with U.S. BioCheck method higher consistance is arranged, and is 97.0%.
Kit of the present invention is chosen the partial sequence of the stronger pp65 albumen of HCMV antigenicity, by analyzing, select wherein epitope and hydrophilic region, adopt gene recombination technology, prokaryotic expression obtains a kind of new pp65 recombinant protein after the affinity chromatography, this antigen not only has high specific, and have high-purity and high stability, after extracting purifying, wrapper sheet is as the important component in the kit.Compare with totivirus antigen, but this recombinant antigen scale, standardized production is safer, economical.
Embodiment
1, reagent
(1) coating buffer (0.05mol/l, the sodium carbonate of pH9.6-sodium bicarbonate buffer liquid): Na
2CO
31.5g, NaHCO
32.9g, Na
2N
30.2g, add distilled water to 1000ml, transfer to pH9.6.
(2) sample diluent (the 0.01M PBS pH7.4 that contains 10% calf serum and 2% anti-human IgG): NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, thimerosal 0.1g adds distilled water to 1000ml, transfers to pH7.4, is PBS.
(3) confining liquid: (10% calf serum/PBS solution) calf serum 100ml, 1 * PBS (pH7.4) 900ml.
(4) cleansing solution (PBST, pH7.4): NaCl 8.0g, KH
2PO
40.2g, Na
2HPO
412H
2O 2.9g, KCl 0.2g, Tween 20 0.5ml, thimerosal 0.1g, add distilled water, transfer to pH7.4 to 1000ml.
(5) ELIAS secondary antibody (HRP
*Mouse-anti people IgM monoclonal antibody): the mouse-anti people IgM monoclonal antibody of horseradish peroxidase-labeled, produce voluntarily by this chamber.
(6) substrate solution (TMB-H
2O
2Urea liquid):
1. substrate solution A (3,3 ', 5,5 ' tetramethyl benzidine, TMB): TMB200mg, absolute ethyl alcohol 100ml adds distilled water to 1000ml.
2. substrate solution B damping fluid (0.1mol/L citric acid-0.2mol/L sodium hydrogen phosphate damping fluid, pH5.0～5.4): Na
2HPO
414.60g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml adds distilled water to 1000ml, transfers to pH5.0～5.4.
3. substrate solution A and B were mixed by 1: 1, TMB-H
2O
2Urea is used liquid.
(7) stop buffer: (2mol/L H
2SO
4Solution): distilled water 600ml, concentrated sulphuric acid 100ml (slowly drip and constantly stir) adds distilled water to 900ml.
2, key instrument
(1) microplate reader: BIO-RAD Model 550
(2) ELISA reaction plate: Corning Incorporated costar R 96 Well EIA/RIA plate
3, method
ELIAS secondary antibody is the selection of suitable titre
(1) wraps quilt with 100ng/ml people IgM, washing.
(2) enzyme being marked anti-people IgM monoclonal antibody does to add respectively after a series of dilutions with dilution and has wrapped in the hole of quilt insulation, washing.
(3) add the substrate colour developing.After adding sour cessation reaction, reading absorbance (A), get the A value at 1.0 o'clock enzyme labelled antibody dilutability, is 1: 1000 as the suitableeest titre of ELIAS secondary antibody.
The chessboard titrimetry is selected the suitableeest titre of envelope antigen
(1) with coating buffer antigen is done to wrap quilt after a series of dilutions washing.
(2) strong positive reference serum, weak positive reference serum and negative reference serum are diluted application of sample, insulation, washing by 10 times of ascending series with dilution.
(3) add the enzyme that dilutes by the suitableeest titre and mark anti-people IgM monoclonal antibody, insulation, washing.
(4) add substrate colour developing, read the A value after adding sour cessation reaction.
(5) to select the A value of strong positive reference serum be 0.8～1.0, the A value of negative reference serum less than the suitableeest titre of dilutability conduct of 0.1 envelope antigen is 20～40 μ g/ holes.
Specificity recombinant antigen pp65 bag is by the preparation method of reaction plate
(1) with the pp65 proteantigen with the carbonate buffer solution of pH9.6 dilution back bag by 96 orifice plates, 100 μ l/ holes, put in the wet box 4 ℃ 12～24 hours, PBST washes plate 3 times, each 3min pats dry then; Obtain ELISA Plate.
(2) fill it up with the hole with the PBS that contains 10% calf serum, hatch for 37 ℃, sealing 1h washes sealing behind the plate 3 times.This plate can obtain antigen pp 65 envelope enzyme-linked reaction plate.
The formation of kit
Described kit is made of specificity recombinant antigen pp 65 envelope enzyme-linked reaction plate, sample diluent, cleansing solution, colour developing liquid, stop buffer, and concrete component is:
(1) sample diluent: the 0.01M PBS (pH7.4) that contains 10% calf serum and 2% anti-human IgG;
(2) ELIAS secondary antibody: the mouse-anti people IgM monoclonal antibody of horseradish peroxidase-labeled;
(3) colour developing liquid: TMB-H
2O
2System;
(4) stop buffer: 2mol/L H
2SO
4
Utilize the mentioned reagent box to adopt the ELISA method to detect HCMV IgM in the serum:
(1) serum to be checked, the serum to be checked that adds dilution in 1: 100 is to wrapping by good enzyme reaction plate, and 1h is hatched for 37 ℃ in 100 μ l/ holes, washes plate 3 times;
(2) enzyme-added mark thing, the mouse-anti people IgM monoclonal antibody of adding HRP (horseradish peroxidase) mark, 40min is hatched for 37 ℃ in 100 μ l/ holes,
(3) colour developing washes that every hole adds TMB-H behind the plate
2O
2System's substrate 100 μ l, 37 ℃ or room temperature lucifuge colour developing 15 minutes;
(4) every hole adds 2mol/L H
2SO
450 μ l, cessation reaction;
(5) result judges, 450nm wavelength place absorbance is read in the blank well zeroing, i.e. OD 450 values.
(6) result of calculation divided by the average OD value of negative serum (N), is tried to achieve the P/N value with the average OD value of sample serum to be checked (P), and P/N 〉=2.1 are positive, and P/N＜2.1 are negative.
Claims (2)
1, the method for making of recombinant antigen pp 65 envelope enzyme-linked reaction plate is prepared from through the following steps:
(1), be template with HCMV AD169 DNA, amplification UL1006-1521nt gene, TOPO-pET is connected with clonal expression reagent, conversion BL21 (DE3) Star
TM, express large-scale purification pp65 albumen;
(2), with the pp65 proteantigen with the carbonate buffer solution of pH9.6 dilution back bag by 96 orifice plates, 100 μ l/ holes, put in the wet box 4 ℃ 12～24 hours, PBST washes plate 3 times, each 3min pats dry then;
(3), fill it up with the hole, hatch for 37 ℃, sealing 1h washes sealing behind the plate 3 times with the PBS that contains 10% calf serum; This plate is the pp65 antigen pp 65 envelope enzyme-linked reaction plate, and the specific detection that can be used for HCMV pp65-IgM is used.
2, use the ELISA detection kit of recombinant antigen pp 65 envelope enzyme-linked reaction plate, it is characterized in that: described kit is made of by reaction plate, sample diluent, cleansing solution, colour developing liquid, stop buffer specificity recombinant antigen pp65 bag, and concrete component is: (1), sample diluent: the 0.01M PBS (pH7.4) that contains 10% calf serum and 2% anti-human IgG; (2) ELIAS secondary antibody: HRP
*Mouse-anti people IgM monoclonal antibody; (3), colour developing liquid: TMB-H
2O
2System (4), stop buffer: 2mol/LH
2SO
4Solution.
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|---|---|---|---|
| CNB2005100376631A CN1307423C (en) | 2005-01-08 | 2005-01-08 | Method for making recombinant antigen pp 65 envelope enzyme-linked reaction plate and ELISA test kit |
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|---|---|
| CN1651918A true CN1651918A (en) | 2005-08-10 |
| CN1307423C CN1307423C (en) | 2007-03-28 |
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| US6133433A (en) * | 1984-07-27 | 2000-10-17 | City Of Hope | Method for detection and prevention of human cytomegalovirus infection |
| JP3241237B2 (en) * | 1995-05-26 | 2001-12-25 | 三菱化学メディカル株式会社 | Cytomegalovirus antigen test |
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| CN103901209B (en) * | 2014-02-18 | 2016-05-25 | 王明丽 | A kind of recombinant protein IE1 is coated with the preparation method of enzyme reaction plate and quantitatively detects human plasma HCMV neutralizing antibody kit |
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| CN1307423C (en) | 2007-03-28 |
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