CN104569387A - Kit for detecting human serum HCMV pp150 IgM antibody by using ELISA - Google Patents
Kit for detecting human serum HCMV pp150 IgM antibody by using ELISA Download PDFInfo
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Abstract
The invention discloses a kit for detecting a human serum HCMV IgM antibody by using a recombinant antigen HCMV pp150 by adopting an ELISA capturing method and relates to a preparation method of a novel horse radish peroxidase labeled recombinant HCMV pp150 antigen and an ELISA kit prepared on the basis of the preparation method and used for detecting the human serum HCMV IgM antibody. The method comprises the following steps: preparing a specific recombination HCMV pp150 antigen, a recombinant HCMV pp150 antigen horse radish peroxidase label and an anti-human IgM Mu chain antibody coating enzyme-labeled reaction plate by adopting a genetic engineering method and assembling the ELISA kit. Compared with other commercially-available ELISA kits, for the kit disclosed by the invention, the specificity of the kit is 100%; the sensitivity of the kit is 95.6%; the specificity and the sensitivity of the kit are superior to those of a HCMV IgM kit of a certain domestic company; the HCMV IgM kit has the advantages that the specificity is 95.6% and the sensitivity is 86.9%. The kit is mainly applied to laboratory study and clinically auxiliary diagnosis of HCMV active infection.
Description
Technical field
The invention belongs to technique for gene engineering, diagnostic reagent field, particularly relate to a kind of ELISA and detect human serum HCMV pp150 IgM antibody kit.
Background technology
Human cytomegalovirus (Human Cytomegalovirus, HCMV) belongs to nerpes vinrus hominis β subfamily virus, and the infection in crowd is very general, and in worldwide distribution, seroepidemiological survey shows, 40-100% adult has HCMV antibody.Current research shows that HCMV is that human congenital infects one of modal pathogen, affects huge on pregnant woman and fetus, and the HCMV primary infection harm in pregnancy period is particularly huge, and 2/3 congenital infection is caused by pregnancy period primary infection.In congenital infection, the infant of 90% does not show clinical symptoms, and 5%-10% can cause the diseases such as miscarriage, stillborn foetus, fetal anomaly, hypoevolutism and Delayed onset central nervous system deficit.In addition, HCMV infects also is one of organ transplant failure and AIDS patients accompanying infection main causes of death.
The method diagnosing and monitor HCMV Infection Status is fast and accurately be badly in need of clinically, particularly important in some specific crowds, the donor in such as organ transfer operation and acceptor, the women of child-bearing age and accept the crowd etc. of immunosuppressant treatment.HCMV usually presents symptomless infection in immunity normal population, and virus replication is suppressed, and has intermittent toxin expelling phenomenon to exist.And in HCMV Active infection, viral massive duplication, can at blood, urine, cerebrospinal fluid etc. detect live virus.Virus purification is one of most important index of diagnosis HCMV Active infection.But due to virus self copy feature, virus purification is longer for experimental period.And the complex operation of virus purification experiment own, to experiment condition, equipment has certain requirement, is not suitable for the clinical timely monitoring to this virus and diagnosis.The IgM antibody that in serum, HCMV is special diagnoses the important indicator recently infected, and IgM antibody can occur in the primary infection of body virus for 3-5 days, continues 12-16 time-of-week.Adopt ELISA kit to detect the special IgM antibody of HCMV, detection method is simple, and experimental period is short, repeatable high, is applicable to clinical diagnosis.
Prize law for the IgM antibody principle detecting HCMV in serum special is, adopts Anti-human IgMμ chain monoclonal antibody specific bag by enzyme reaction plate, can with the IgM antibody specific binding in serum.Add the antigen that horseradish peroxidase (HRP) marks again, add the substrate reactions regular hour and cessation reaction.In certain scope, in the size embodiment blood serum sample of absorbance (OD), the content of IgM antibody is how many.Relative to indirect method, prize law does not affect by the IgG antibody of titre high in serum, and specificity is better.
HCMV pp150 is the peculiar gene of herpesviral β subfamily, in the numerous structural protein of HCMV, pp150 is that immunogenicity is the strongest, being the main target antigen stimulating body to produce humoral immunity, is also that cytotoxic T lymphocyte (CTL) mainly identifies antigen.In the convalescence of virus infections, long compared with the antibody life period for other antigens for the antibody life period of pp150 in serum.HCMV pp150 still can stimulate body to produce one of IgM CMV antigen few in number.Therefore adopt this albumen not only can ensure that the reliability of detection method can also reduce the cross reaction between herpesviral as prize law enzyme-labelled antigen, thus improve the specificity of detection method.Confirm that pp150 is most important to the packaging maturation of virus in tenuigenin by the structure of deletion mutation strain, pp150 deletion mutation pnca gene copies and can normally carry out, but virus can not complete final packaging, can not infect contiguous cell.Complete pp150 is made up of, containing multiple epitope 1048 amino acid.But found by shorten expression or these epitopes of single expression, they can both react with CMV positive serum.Existing research also shows, the same epitope of simple expressing in series can not strengthen immunogenicity and the immunoreactivity of this antigen, and different epitope expressing in series can well be strengthened its immunogenicity and immunoreactivity.Given this, we adopt the method for fusion DNA vaccine, by two of HCMVpp150 dominant antigen epi-position (peptide section aa495-691, aa862-1048) coding region directly couples together and inserts pET32 (b) expression vector, imports colon bacillus BL21 (DE3).Brachymemma pp150 albumen is obtained through abduction delivering.Detect the enzyme-labelled antigen of the ELISA kit of IgM antibody as prize law with this albumen, the kit of packaging compares with the HCMV IgM kit of the HCMV IgM detection kit of German DiaSorin company and certain company domestic.With the HCMV totivirus of German DiaSorin company packaging IgM detection kit as standard, result shows: this project invention kit specificity is 100%, susceptibility is 95.6%, is all better than the HCMV IgM kit (being respectively 95.7% and 86.9%) of certain company domestic.Though susceptibility is a little less than HCMV totivirus packaging IgM detection kit (100%) of German DiaSorin company, but specificity is higher than HCMV totivirus packaging IgM detection kit (98.9%) of German DiaSorin company.
Summary of the invention
The object of the invention is the deficiency of existing clinical detection technique, a kind of kit being detected HCMV IgM antibody in sample by ELISA method is fast provided.
The present invention adopts following technical scheme to achieve these goals:
A kind of ELISA detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the enzyme-labelled antigen of use is the recombinant protein HCMV pp150 of horseradish peroxidase-labeled.
Described a kind of ELISA detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the preparation method of the recombinant protein HCMV pp150 of described horseradish peroxidase-labeled is:
A, HCMV pp150 recombinant protein obtains
(1) according to the HCMV gene order announced in Genebank, determine HCMV pp150 gene coded sequence, be designed for the fusion DNA vaccine primer of amplification aa495-691 and aa862-1048, with HCMV AD169 strain DNA for template, the object clip size amplified is 1155bp, be cloned on pET32b carrier, construction recombination plasmid pET32b/pp150, the correct recombinant vector of HCMV pp150 gene nucleic acid sequence is being obtained by DNA sequencing, transformation of E. coli BL21 (DE3), after IPTG induction, mycoprotein is identified through SDS-PAGE protein electrophoresis and Western blotting,
(2) by express HCMV pp150 recombinant protein Escherichia coli through mass propgation to logarithmic phase, add IPTG and induce 5 hours, collected by centrifugation thalline, after broken, 12000rpm ultracentrifugation 30min collects supernatant, obtains a large amount of HCMV pp150 recombinant protein after affinitive layer purification;
B, horseradish peroxidase-labeled antigen recombinant protein HCMVpp150
(1) taking 4.8-5.2mgHRP is dissolved in 0.8-1.2ml distilled water;
(2) in step (1) gained solution, add the NaIO of the 0.05-0.15M that 0.15-0.25ml newly joins
4solution, under room temperature, lucifuge stirs 15-25 minute;
(3) step (2) gained Solutions Solution is loaded in bag filter, the sodium-acetate buffer of 0.5-1.5mM PH4.3-4.5 is dialysed, places 6-8 hour at 3-5 DEG C, obtain hydroformylation HRP;
(4) 20 μ l 0.2M PH9.5 carbonate buffer solutions are added, the PH of above hydroformylation HRP is made to be elevated to 9.0-9.5, then add the HCMV pp150 recombinant protein after 10mg purifying immediately in 1ml 0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently;
(5) the 4mg/ml NaBH that 0.1ml newly joins is added
4liquid, mixing, then place 2 hours at 4 DEG C;
(6) loaded in bag filter by above-mentioned solution, dialyse with 0.15M PH7.4 PBS, 4 DEG C are spent the night;
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, place 1 hour at 4 DEG C;
(8) 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of 5ml 0.15M PH7.4;
(9) above-mentioned solution is loaded in bag filter, dialyse by the PBS buffer saline of 0.15M PH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, supernatant is horseradish peroxidase-labeled antigen recombinant protein HCMVpp150, after packing, and about-20 DEG C preservations.
Described a kind of ELISA detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: this kit is made up of by enzyme reaction plate, sample diluent, cleansing solution, enzyme-labelled antigen, nitrite ion, stop buffer, positive control, negative control anti-IgMμchainantibody bag.
Described a kind of ELISA detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the serum of described positive control to be clinical definite the be HCMV IgM positive; The serum of negative control to be clinical definite be HCMV IgM feminine gender; Sample diluent refers to the phosphate buffer of the 0.01M pH7.4 containing 10% calf serum and blood plasma developer; Cleansing solution refers to the phosphate buffer containing 0.05%Tween 20; Enzyme-labelled antigen is the recombinant protein HCMV pp150 of horseradish peroxidase-labeled; Nitrite ion is TMB-H
2o
2urea liquid; Stop buffer is 2mol/L H
2sO
4solution.
Described a kind of ELISA detects human serum HCMV pp150 IgM antibody kit, it is characterized in that:
Anti-human IgMμ chain monoclonal antibody bag by the acquisition methods of enzyme reaction plate is:
(1) be diluted to by the carbonate buffer solution of anti-IgMμchainantibody pH9.6 by Bao Bei96 hole, 100ng/ hole ELISA Plate after 1mg/ml, put in wet box and place 24 hours at 4 DEG C, PBST washes plate 3 times, and each 3min, then pats dry;
(2) by Bao Bei96 hole, the PBS solution 100 μ l/ hole ELISA Plate containing 1%BSA, 10% calf serum, hatch for 37 DEG C, close 1h, seal after washing plate 3 times; This plate is anti-IgMμchainantibody bag by enzyme reaction plate.
Described a kind of ELISA detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the amino acid residue sequence of described recombinant protein pp150 is as shown in SEQ No.1.
Described a kind of ELISA detects human serum HCMV pp150 IgM antibody kit, and the DNA nucleotide sequence of described recombinant protein pp150 is as shown in SEQ No.2.
Adopt Over-voltage protection mark recombinant antigen HCMV pp150.
(1) take 5mgHRP to be dissolved in 1ml distilled water.
(2) in upper liquid, add the 0.1M NaIO that 0.2ml newly joins
4solution, under room temperature, lucifuge stirs 20 minutes.
(3) loaded in bag filter by above-mentioned solution, dialyse to the sodium-acetate buffer of 1mM PH4.4,4 DEG C are spent the night.
(4) add 20 μ l 0.2M PH9.5 carbonate buffer solutions, make the PH of above Quanization Bedside-stand RP be elevated to 9.0-9.5, then add the HCMV pp150 after 10mg purifying immediately in 1ml 0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently.
(5) add the 4mg/ml NaBH4 liquid that 0.1ml newly joins, mixing, then put 4 DEG C 2 hours.
(6) loaded in bag filter by above-mentioned liquid, dialyse to 0.15M PH7.4 PBS, 4 DEG C are spent the night.
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour.
(8) 3000rpm centrifugal half an hour, supernatant is abandoned.Sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of a small amount of 0.15M PH7.4.
Loaded in bag filter by above-mentioned solution, dialyse to the PBS buffer saline of 0.15M PH7.4, after removing ammonium ion, 10,000rpm removes precipitation in centrifugal 30 minutes, and supernatant is enzyme conjugates, after packing, and-20 DEG C of preservations.
Compared with existing product, the present invention has following beneficial effect:
1. the present invention can accurately, whether be HCMV pp150 IgM positive to rapid screening if going out sample.
2. this law compares with domestic and international similar kit, and specificity is 100%, and susceptibility is 95.6%, is all better than the HCMV IgM kit (being respectively 95.7% and 86.9%) of certain company domestic.Susceptibility is a little less than HCMV totivirus packaging IgM detection kit (100%) of German DiaSorin company, but specificity is higher than HCMV totivirus packaging IgM detection kit (98.9) of German DiaSorin company.
Accompanying drawing explanation
Escherichia coli mycoprotein after IPTG induction that Fig. 1 expresses HCMV pp150 recombinant protein is identified through SDS-PAGE protein electrophoresis; Swimming lane 1 is contrast containing the BL21 (DE3) of empty expression vector pET32b; Swimming lane 2-7 is the BL21 (DE3) containing pET32b-pp150 carrier;
The western blot of Fig. 2 HCMV pp150 recombinant protein identifies.Swimming lane 1 is the BL21 (DE3) containing pET32b-pp150 carrier; Swimming lane 2 is contrast containing the BL21 (DE3) of empty expression vector pET32b;
Fig. 3 HCMV pp150 recombinant protein prokaryotic expression plasmid sequencing result.
Embodiment
One, HCMV pp150 recombinant protein preparation
1. prepare before restructuring
According to the HCMV gene order announced in Genebank, determine HCMV pp150 gene coded sequence, be designed for the fusion DNA vaccine primer of amplification HCMV pp150 gene 1483-2073nt (corresponding amino acid 495-691aa) and 2584-3144nt (corresponding amino acid 862-1048aa).
2. target DNA fragment PCR obtains
From HCMV AD169 strain virus culture, extract DNA as template, amplify object fragment by fusion DNA vaccine
3. recombinant dna fragment inserts expression vector
By pET32 (b) carrier and target DNA fragment through double digestion, connect through T4 DNA ligase after digestion products purifying, connect product conversion e. coli bl21 (DE3) competence, dull and stereotyped upper 37 DEG C of the LB coated containing Amp is inverted overnight incubation, and the single bacterium colony on next day picking flat board carries out bacterium colony PCR and order-checking qualification.
4. to recombinate the abduction delivering of HCMV pp150 albumen and purifying
By the bacterial strain filtered out, be inoculated in the LB liquid medium containing Amp, being cultured to OD value is about 0.5, adds IPTG abduction delivering 5h, collects thalline and obtains the restructuring HCMV pp150 albumen after purifying through ultrasonication, histidine affinity column and ion-exchange chromatography.
5. the Western blot of restructuring HCMV pp150 albumen verifies
After HCMV pp150 albumen above-mentioned steps prepared mixes by one to one with 2 X SDS-PAGE sample-loading buffers, boil 5min.Sample is transferred to pvdf membrane after SDS-PAGE is separated, closes 1h, pp150 monoclonal antibody overnight incubation, wash film 3 times with the TBST solution containing 5% skimmed milk power, each 5min, HRP mark sheep anti mouse two is anti-hatches 1h.Wash film 3 times, each 5min.The immunoreactivity of ECL method exposure tests HCMV pp150 albumen.
Two, horseradish peroxidase-labeled antigen recombinant protein HCMVpp150
(1) taking 4.8-5.2mgHRP is dissolved in 0.8-1.2ml distilled water;
(2) in step (1) gained solution, add the NaIO of the 0.05-0.15M that 0.15-0.25ml newly joins
4solution, under room temperature, lucifuge stirs 15-25 minute;
(3) step (2) gained Solutions Solution is loaded in bag filter, the sodium-acetate buffer of 0.5-1.5mM PH4.3-4.5 is dialysed, at 3-5 DEG C, places 6-8 hour;
(4) 20 μ l 0.2M PH9.5 carbonate buffer solutions are added, the PH of above hydroformylation HRP is made to be elevated to 9.0-9.5, then add the HCMV pp150 recombinant protein after 10mg purifying immediately in 1ml 0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently;
(5) the 4mg/ml NaBH that 0.1ml newly joins is added
4liquid, mixing, then place 2 hours at 4 DEG C;
(6) loaded in bag filter by above-mentioned solution, dialyse with 0.15M PH7.4PBS, 4 DEG C are spent the night;
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, place 1 hour at 4 DEG C;
(8) 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of 5ml 0.15M PH7.4;
(10) above-mentioned solution is loaded in bag filter, dialyse by the PBS buffer saline of 0.15M PH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, supernatant is horseradish peroxidase-labeled antigen recombinant protein HCMVpp150, after packing, and about-20 DEG C preservations.
Three, the suitableeest wrapper sheet concentration determination of anti-IgMμchainantibody
Checkerboard titration method selects coated antibody optimum content
(1) with coating buffer by antibody (anti-IgMμchainantibody) 0.01mg/ml stoste, be diluted to 0 respectively, after 9 μ g/ml, 0.8 μ g/ml, 0.7 μ g/ml, 0.6 μ g/ml, 0.5 μ g/ml, 0.4 μ g/ml, 0.3 μ g/ml, 0.2 μ g/ml, 0.1 μ g/ml, every hole adds 100 μ l and carries out bag quilt, washing.
(2) strong positive reference serum, weak positive reference serum and negative reference serum dilution are diluted by 10 times of ascending series, application of sample, insulation, washing.
(3) the enzyme mark HCMV pp150 recombinant protein antigen by optimum concentration dilution is added, insulation, washing.
(4) add substrate colour developing, after acid adding cessation reaction, read OD
450value.
(5) OD of strong positive reference serum is selected
450the dilutability that value is between 0.8 ~ 1.0, the A value of negative reference serum is less than the coated antibody of 0.1 is 100ng/ hole as the suitableeest titre.
Four, anti-IgMμchainantibody enzyme reaction plate bag quilt
(1) by anti-IgMμchainantibody, to be diluted to after suitable concentration by Bao Bei96 hole, 100ng/ hole ELISA Plate with the carbonate buffer solution of pH9.6, to put in wet box 4 DEG C 24 hours, PBST washes plate 3 times, and each 3min, then pats dry.
(2) with the PBS 100 μ l/ hole containing 1%BSA and 10% calf serum, hatch for 37 DEG C, close 1h, seal after washing plate 3 times; This plate is anti-IgMμchainantibody bag by enzyme reaction plate.
Five, the assembling of ELISA kit and application
1. the selection of enzyme mark pp150 antigen optimum concentration
(1) bag quilt is carried out with 100ng/ml anti-IgMμchainantibody, washing.
(2) add respectively after pp150-HRP dilution being done a series of dilution and wrapped in the hole of quilt, insulation, washing.
(3) substrate colour developing is added.After acid adding cessation reaction, read absorbance OD450 value, get the enzyme-labelled antigen dilutability of OD450 value 1.0 time, the most dilutability obtained as enzyme-labelled antigen is 1:1000.
2. the assembling of kit
Described kit is by anti-IgMμchainantibody bag by enzyme reaction plate, positive control serum, negative control sera, and sample diluent, cleansing solution, nitrite ion, stop buffer are formed, and concrete component is:
(1) clinical diagnosis is HCMV IgM positive serum and negative serum;
(2) sample diluent: containing the phosphate buffer (PBS) of the 0.01M pH7.4 of 10% calf serum and blood plasma developer;
(3) cleansing solution: the phosphate buffer (PBST) containing 0.05%Tween 20;
(4) enzyme-labelled antigen: the recombinant protein pp150 of horseradish peroxidase-labeled;
(5) nitrite ion: TMB-H
2o
2urea liquid;
(6) stop buffer: 2mol/L H
2sO
4solution.
Six, the HCMV IgM kit of mentioned reagent box and German DiaSorin company HCMV totivirus packaging IgM kit and certain company domestic compares:
(1) serum to be checked, add the serum to be checked of 1:100 dilution to wrapping by good enzyme reaction plate, 100 μ l/ holes, hatch 1h for 37 DEG C, wash plate 3 times;
(2) add enzyme-labelled antigen, HRP-pp150 is diluted to working concentration, and 100 μ l/ holes, hatch 30min for 37 DEG C,
(3) develop the color, after washing plate, every hole adds TMB-H
2o
2system substrate 100 μ l, 37 DEG C or room temperature lucifuge develop the color 15 minutes;
(4) every hole adds 2mol/L H
2sO
450 μ l, cessation reaction;
(5) result judges, blank well returns to zero, and read 450nm wavelength place absorbance, namely OD 450 is worth.
(6) judged result, detects same batch sample with three kinds of kits, calculates specificity and the susceptibility of kit of the present invention and German DiaSorin company HCMV totivirus packaging IgM kit and domestic certain company HCMV IgM kit.
Table 1 packs comparing of IgM detection kit and domestic certain company HCMV IgM kit for kit of the present invention and the HCMV totivirus of German DiaSorin company.
Table 1
The amino acid sequence (SEQ No.1) of the pp150 recombinant protein described in the present invention:
LVSPQVTKASPGRVRRDSAWDVRPLTETRGDLFSGDEDSDSSDGYPPNRQDPRFTDTLVDITDTETSAKPPVTTAYKFEQPTLTFGAGVNVPAGAGAAILTPTPVNPSTAPAPAPTPTFAGTQTPVNGNSPWAPTAPLPGDMNPANWPRERAWALKNPHLAYNPFRMPTTSTASQNTVSTTPRRPSTPRAAVTQTASDVVSPATSPLSMLSSASPSPAKSAPPSPVKGRGSRVGVPSLKPTLGGKAVVGRPPSVPVSGSAPGRLSGSSRAASTTPTYPAVTTVYPPSSTAKSSVSNAPPVASPSILKPGASAALQSRRSTGTAAVGSPVKSTTGMKTVAFDLSSPQKSGTGPQPGSAGMGGAKTPSDAVQNILQKIEKIKNTEE
The DNA nucleotide sequence (SEQ No.2) of the pp150 recombinant protein described in the present invention:
CTCGTCTCCCCGCAGGTGACCAAGGCCAGCCCGGGAAGGGTCCGTCGGGACAGCGCGTGGGACGTGAGGCCGCTCACGGAGACCAGAGGGGATCTTTTCTCGGGCGACGAGGATTCCGACAGCTCGGATGGCTATCCCCCCAACCGTCAAGATCCGCGTTTCACCGACACGCTGGTGGACATCACGGATACCGAGACGAGCGCCAAACCGCCCGTCACCACCGCGTACAAGTTCGAGCAACCGACGTTGACGTTCGGCGCCGGAGTTAACGTTCCTGCTGGCGCCGGCGCTGCCATCCTCACGCCGACGCCTGTCAATCCTTCCACGGCCCCCGCTCCGGCCCCGACACCTACCTTCGCGGGTACCCAAACCCCGGTCAACGGTAACTCGCCCTGGGCTCCGACGGCGCCGTTGCCCGGGGATATGAACCCCGCCAACTGGCCGCGCGAACGCGCGTGGGCCCTCAAGAATCCTCACCTGGCTTACAATCCCTTCAGGATGCCTACGACTTCCACGGCTTCTCAAAACACCGTGTCCACCACCCCTCGGAGGCCGTCGACTCCACGCGCCGCGGTGACACAAACAGCGTCTGATGTCGTGTCCCCCGCCACATCGCCGCTGTCCATGCTTTCGTCAGCCTCTCCGTCCCCGGCCAAGAGTGCCCCCCCGTCTCCGGTGAAAGGCCGGGGCAGCCGCGTCGGTGTTCCTTCCTTGAAACCTACTTTGGGCGGCAAGGCGGTGGTAGGTCGACCGCCCTCGGTCCCCGTGAGCGGTAGCGCGCCGGGTCGCCTGTCCGGCAGCAGCCGGGCCGCCTCGACCACGCCGACGTATCCCGCGGTAACCACCGTTTACCCACCGTCGTCTACGGCCAAAAGCAGCGTATCGAATGCGCCGCCTGTGGCCTCCCCCTCCATCCTGAAACCGGGGGCGAGCGCGGCTTTGCAATCACGCCGCTCGACGGGGACCGCCGCCGTAGGTTCCCCCGTCAAGAGCACGACGGGCATGAAAACGGTGGCTTTCGACCTATCGTCGCCCCAGAAGAGCGGTACGGGGCCGCAACCGGGTTCTGCCGGCATGGGGGGCGCCAAAACGCCGTCGGACGCCGTGCAGAACATCCTCCAAAAGATCGAGAAGATTAAGAACACGGAGGAATAG
Claims (7)
1. ELISA detects a human serum HCMV pp150 IgM antibody kit, it is characterized in that: the enzyme-labelled antigen of use is the recombinant protein HCMV pp150 of horseradish peroxidase-labeled.
2. a kind of ELISA according to claim 1 detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the preparation method of the recombinant protein HCMV pp150 of described horseradish peroxidase-labeled is:
A, HCMV pp150 recombinant protein obtains
(1) according to the HCMV gene order announced in Genebank, determine HCMV pp150 gene coded sequence, be designed for the fusion DNA vaccine primer of amplification aa495-691 and aa862-1048, with HCMV AD169 strain DNA for template, the object clip size amplified is 1155bp, be cloned on pET32b carrier, construction recombination plasmid pET32b/pp150, the correct recombinant vector of HCMV pp150 gene nucleic acid sequence is being obtained by DNA sequencing, transformation of E. coli BL21 (DE3), after IPTG induction, mycoprotein is identified through SDS-PAGE protein electrophoresis and Western blotting,
(2) by express HCMV pp150 recombinant protein Escherichia coli through mass propgation to logarithmic phase, add IPTG and induce 5 hours, collected by centrifugation thalline, after broken, 12000rpm ultracentrifugation 30min collects supernatant, obtains a large amount of HCMV pp150 recombinant protein after affinitive layer purification;
B, horseradish peroxidase-labeled antigen recombinant protein HCMVpp150
(1) taking 4.8-5.2mgHRP is dissolved in 0.8-1.2ml distilled water;
(2) in step (1) gained solution, add the NaIO of the 0.05-0.15M that 0.15-0.25ml newly joins
4solution, under room temperature, lucifuge stirs 15-25 minute;
(3) step (2) gained Solutions Solution is loaded in bag filter, the sodium-acetate buffer of 0.5-1.5mM PH4.3-4.5 is dialysed, places 6-8 hour at 3-5 DEG C, obtain hydroformylation HRP;
(4) add 20 μ l 0.2M PH9.5 carbonate buffer solutions, make the PH of above hydroformylation HRP be elevated to 9.0-9.5, then add the HCMV pp150 recombinant protein after 10mg purifying immediately in 1ml 0.01M carbonate buffer solution, room temperature lucifuge stirs 2 hours gently;
(5) the 4mg/ml NaBH that 0.1ml newly joins is added
4liquid, mixing, then place 2 hours at 4 DEG C;
(6) loaded in bag filter by above-mentioned solution, dialyse with 0.15M PH7.4 PBS, 4 DEG C are spent the night;
(7) under agitation dropwise add equal-volume saturated ammonium sulfate, place 1 hour at 4 DEG C;
(8) 3000rpm centrifugal half an hour, abandon supernatant, sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of 5ml 0.15M PH7.4;
Above-mentioned solution is loaded in bag filter, dialyses by the PBS buffer saline of 0.15M PH7.4, after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, supernatant is horseradish peroxidase-labeled antigen recombinant protein HCMVpp150, after packing, and about-20 DEG C preservations.
3. a kind of ELISA according to claim 1 detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: this kit is made up of by enzyme reaction plate, sample diluent, cleansing solution, enzyme-labelled antigen, nitrite ion, stop buffer, positive control, negative control anti-human IgM μ chain antibody bag.
4. a kind of ELISA according to claim 3 detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the serum of described positive control to be clinical definite the be HCMV IgM positive; The serum of negative control to be clinical definite be HCMV IgM feminine gender; Sample diluent refers to the phosphate buffer of the 0.01M pH7.4 containing 10% calf serum and blood plasma developer; Cleansing solution refers to the phosphate buffer containing 0.05%Tween 20; Enzyme-labelled antigen is the recombinant protein HCMV pp150 of horseradish peroxidase-labeled; Nitrite ion is TMB-H
2o
2urea liquid; Stop buffer is 2 mol/ L H
2sO
4solution.
5. a kind of ELISA according to claim 3 detects human serum HCMV pp150 IgM antibody kit, it is characterized in that:
Anti-Human IgM μ chain monoclonal antibody bag by the acquisition methods of enzyme reaction plate is:
(1) be diluted to by the carbonate buffer solution of anti-human IgM μ chain antibody pH9.6 by 100 Bao Bei96 hole, ng/ hole ELISA Plate after 1mg/ml, put in wet box and place 24 hours at 4 DEG C, PBST washes plate 3 times, and each 3 min, then pat dry;
(2) by Bao Bei96 hole, the PBS solution 100 μ l/ hole ELISA Plate containing 1% BSA, 10% calf serum, hatch for 37 DEG C, close 1 h, seal after washing plate 3 times; This plate is anti-human IgM μ chain antibody bag by enzyme reaction plate.
6. a kind of ELISA according to claim 1 detects human serum HCMV pp150 IgM antibody kit, it is characterized in that: the amino acid residue sequence of described recombinant protein pp150 is as shown in SEQ No.1.
7. a kind of ELISA according to claim 1 detects human serum HCMV pp150 IgM antibody kit, and the DNA nucleotide sequence of described recombinant protein pp150 is as shown in SEQ No.2.
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