CN101458256A - Hepatitis C virus envelope antigen ELISA kit and detecting method - Google Patents
Hepatitis C virus envelope antigen ELISA kit and detecting method Download PDFInfo
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Abstract
The invention discloses an ELISA kit for a hepatitis C virus envelope antigen and a detection method thereof. The ELISA kit comprises an ELISA plate, three polypeptides (12-peptide ZA, 12-peptide ZB and 12-peptide ZD) which are labeled by biotin and capable of being in specific binding with HCV E2 protein, a rabbit anti-E2 polyclonal antibody and streptavidin labeled by horseradish peroxidase. A method of the kit for detecting the hepatitis C virus envelope antigen comprises the following steps: firstly, coating the rabbit anti-E2 polyclonal antibody; then adding blood serum of a patient to be detected; thirdly, adding the polypeptides labeled by the biotin; fourth, adding the streptavidin labeled by the horseradish peroxidase; and fifth, adding o-phenylenediamine for color development and reading an OD value by an ELISA reader at OD450nm. The polypeptides and labels thereof are applied to the kit for detecting the hepatitis C virus envelope antigen. Meanwhile, the polypeptides can also be used for preparing a medicine for treating or preventing the hepatitis C virus infection. The ELISA kit can be widely used in medicine-related fields and the like.
Description
Technical field
The invention belongs to hepatitis C detection technique field.More specifically relate to a kind of hepatitis C virus envelope antigen and detect the ELISA kit, simultaneously, the invention still further relates to a kind of hepatitis C virus envelope antigen kit and detect hepatitis C virus envelope antigen and viral method, utilize the polypeptide and the E2 antibody of mark detecting (Enzyme-Linked ImmunosorbentAssay as the enzyme linked immunological absorption that detects hepatitis C virus surface-coating antigen, ELISA), can be widely used in association areas such as medical science and biology.
Background technology
Viral hepatitis type C (abbreviation hepatitis C) is by hepatitis C virus (Hepatitis C Virus, the liver diseases associated with inflammation that HCV) causes.HCV infects and is global distribution, mainly passes through blood propagation.According to World Health Organization's statistics, global HCV infection rate is about 3%, estimates at 1.7 hundred million people's HCV infection, annual New Development hepatitis C case about 3.5 ten thousand.China's seroepidemiological survey data shows that the general anti-HCV positive rate of crowd is 3.2%, and the each department infection rate has certain difference.Acute HCV infection person about 80% develops into chronic hepatitis C (CHC), and about 20% can develop into cirrhosis in the period of 20, has every year 1%～4% liver cirrhosis patient to develop into liver cancer approximately.CHC has become very important important diseases, finds and treats CHC patient early and come into one's own.The CHC therapeutic purposes comprise to be eradicated or the long term inhibition virus replication, alleviates inflammation and fiberization in the liver, and the final progress that stops is cirrhosis, liver cancer and liver failure, and improves patient's quality of life.
At present, the method that detects the HCV infection clinically roughly comprises: (1) ELISA method detection anti-HCV antibody comprises recombinant immune trace (RIBA); (2) PCR detects the RNA of HCV, comprises fluorescent PCR, immuno-PCR method (PCR-ELISA) and is used for quantitative bDNA and the NASBA technology; (3) genotype chip detection technology.The specificity that enzyme linked immunosorbent assay detects anti-HCV is good, highly sensitive, easy and simple to handle, has become the conventional method that anti-HCV detects in Blood Transfusion Services at present.Yet the concentration of anti-HCV sample is when the kit critical range, owing to influences such as the susceptibility, operating personnel's skills involved in the labour and the sense of responsibility thereof that are subjected to various uncertain factors such as kit, laboratory temperature, sample injectors, cause false positive or false-negative result probably.Document reported once that detecting anti-HCV ash zone blood sample with the ELISA method had 12.6% (11/87) false negative rate through duplicate detection.During the PCR that the random sampling sample that drops in the gray area scope is carried out detected, the positive rate of HCVRNA was 18% (3/17), showed that sample exists virus replication really in the gray area.If gray area is not set, be input in not guilty patient's body with regard to the sample that has 5 parts of anti-HCV positives within 1 year.For improving the recall rate of anti-HCV, prevent omission to greatest extent, improve the security of blood transfusion, the author thinks that the setting of gray area has great significance.Should much ability suitable as for the scope of gray area, also must do further research.Should carry out duplicate test for the sample that drops in the gray area.Unit with good conditionsi should detect by performing PCR, to improve the accuracy of check.PCR detects the RNA of HCV, and the maximum characteristics of particularly propping up chain DNA (bDNA) technology are without the amplification procedure that is exponential increase, and enlargement factor determines that unfavorable factor is few, and therefore stable, repeated high, the dynamic level result of study that is used for HCV RNA is accurate.But the limitation of bDNA technology is also apparent, and promptly enlargement factor is few, susceptibility is low, sensing range is narrow, be not suitable for the low level detection of HCVRNA.Biochip technology is applicable to epidemiology, variation trend, the route of transmission of research HCV, hepatitis C patients is judged that the state of an illness, guidance treatment, prediction curative effect and prognosis are significant, but its cost height is prone to false positive.
Because the various limitation of the method that existing clinical detection HCV infects, the clinical position person urgently wishes to occur the method for a kind of novel clinical detection HCV, and this method should have the specificity height, and is with low cost, easy and simple to handle, diagnoses characteristics rapidly.
Summary of the invention
The objective of the invention is to be to provide a kind of hepatitis C virus envelope antigen ELISA kit, can detect hepatitis C virus quick, sensitive, accurately.
Another object of the present invention is the detection method that has been to provide a kind of hepatitis C virus envelope antigen ELISA kit, this method specificity height, and with low cost, detection speed is fast, and is easy to operate, the efficient that saves time height.
But hepatitis C virus envelope antigen kit of the present invention widespread use in fast detecting HCV infects.
To achieve these goals, the present invention adopts following technical measures:
A kind of kit of fast detecting hepatitis C virus envelope antigen ELISA, this kit comprises:
A. 12 peptides that can combine of mark such as biotin with HCV E2 protein-specific;
B. polypeptide is the sequence shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3, the SEQIDNO:4;
C. the anti-E2 of rabbit polyclonal antibody;
D. the Streptavidin of horseradish peroxidase-labeled;
E. horseradish peroxidase substrate;
F. ELISA Plate (CellStar@ is available from Wuhan strong wind bio tech ltd);
G.E2 albumen is as standard positive control;
H, enzyme-linked immunosorbent assay instrument are (available from medical equipment company limited of Nanjing East China Electronics Co., Ltd group.
Ribosomes library screening technology is that DNA library with the peptide at random of encoding is in in-vitro transcription, translation, transcription product mRNA is owing to lack terminator, can not discharge from ribosomes, and with newborn polypeptide, ribosomes with covalent bonds, form mRNA one ribosomes one protein tripolymer structure, this trimeric form links together albumen and mRNA information, and it is unified that genotype and phenotype are obtained.Utilize the rondom polypeptide on the affine screening ribosomes of the specific monoclonal antibody surface of immobilization antigen or acceptor, separate mRNA one ribosomes one polypeptide complex of specificity combination, by reducing Mg
2+Concentration, ribosomes dissociates, and discharges mRNA, reclaims the mRNA in the ribosomes enrichment storehouse, and reverse transcription becomes cDNA, and pcr amplification, its product can be used as the external synthetic template of next round, pass through several screenings of taking turns, and can find the polypeptide that combines with antibody specificity.In recent years, use the ribosomal display technology and carried out many researchs, as screening part or acceptor, screening antibody or definite epitope are developed new protease inhibitor and new medicine.
It is a kind of that to suppress the concrete preparation process of polypeptide that hepatitis C virus combines with human liver cell as follows:
One, the step of preparation E2-GST fusion is as follows:
A. picking contains pGEX-KG-E2 (document Li sees reference, et al., Engineering ofN-glycosylation of Hepatitis C Virus Envelope Protein E2 Enhances Tcell responses for DNA immunization, Vaccine.2007.25:1544-1551.) e. coli bl21 DE3 (available from U.S. invitrogen company) is inoculated in 3ml and contains in the LB nutrient culture media of ammonia joint penicillin (50 μ g/ml), the inoculation of contrast group contains the pGEX-KG (prokaryotic expression carrier of GST albumen, available from Amersham Biosciences company) e. coli bl21 DE3, cultivated 12～16 hours for 37 ℃.
B. the bacterium liquid in the small test tube being transferred to 200ml respectively contains the LB fluid nutrient medium of ampicillin (50 μ g/ml) (the LB fluid nutrient medium is: the 10g tryptone, the 5g yeast extract, 10g sodium chloride is dissolved to the 1000ml distilled water) in, 37 ℃ are cultivated OD600 is 1～2.
C. add 100mM isopropyl-(IPTG), making its final concentration is 0.1mM, induces 4～5 hours for 30 ℃.
D. will induce later bacterium liquid branch to install in the centrifuge tube of 250ml, 4 ℃, 7700g, 5min is centrifugal, abandons supernatant.
E. sediment washs 1 time with phosphate buffer PBS (140mM sodium chloride, 2.7mM potassium chloride, 10mM sodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate), and 4 ℃, 7700g, 5min is centrifugal, abandons supernatant.
F. precipitation is resuspended among the PBS that 8ml contains 1mM phenylmethylsulfonyl fluoride (PMSF:17.42mg PMSF is dissolved in the 1ml isopropyl alcohol ,-20 ℃ of preservations).
G. bacterium liquid is placed in the ice, ultrasonic broken bacterium adds 20% triton x-100 (Triton X-100:1mlTritonX-100 is dissolved in the 4ml aseptic double-distilled water), makes 1%, 4 ℃ of its final concentration, gently mixes 30 minutes.
H. the bacterium liquid that adds the 1ml ultrasonic degradation in every EP pipe, 4 ℃, 12000g, 10min is centrifugal, gets supernatant.
I.4 ℃, 12000g, centrifugal 10min gets supernatant again.
J. each EP pipe adds 20 μ l Ago-Gel 4B (Sepharose 4B: available from AmershamBiosciences company, the U.S.), mixing, and room temperature is hatched 30min for 20～25 ℃.4 ℃, 5000rpm, 5min, centrifugal, abandon supernatant.
K. respectively add 100 μ l 0.01M PBS washing in each EP pipe, the suspension that will contain Separose 4B behind the centrifuge washing is collected in a pipe, and 4 ℃, 5000rpm, 5min, centrifugal, abandon supernatant, wash altogether 3 times.
L. (Glutathione ElutionBuffer:0.0154g Agifutol is dissolved in the 1M filtration sterilization of 0.25ml pH 8.0 to the Agifutol elution buffer of adding and Sepharose 4B equivalent, packing, 4 ℃ of preservations), 20～25 ℃ of room temperatures are mixing 10min gently, 4 ℃, 5000rpm, 5min gets supernatant.
M. repeating step L is twice.The supernatant of collecting then is the E2-GST fusion.
Two, the step of the anti-E2 protein antibodies of preparation rabbit is as follows:
A. the E2-GST fusion with expression and purification is diluted to 1mg/ml with PBS, add complete Freund's adjuvant (CFA), be injected to 6 the subcutaneous and inguinal lymph nodes in new zealand rabbit (available from the Wuhan University Experimental Animal Center) vertebra both sides, every 0.1～0.2ml, every rabbit antigen immune dosage is 1mg.
B. after two weeks,, be adjuvant with incomplete Freund's adjuvant (IFA) with same antigen amount immunizing rabbit once more.Immunity site and quantity are selected same fundamental immunity.
C. altogether immunity 4 times, be 2 weeks interval time, twice immunizing dose in back be the first time 1.5 times of dosage.Heart blood sampling in the 10th day after room temperature leaves standstill and solidifies, changes 4 ℃ of refrigerator standing over night over to after the last immunity, and separation of serum, serum are the antibody of E2-GST fusion, and it is standby to place-80 ℃ of refrigerators to preserve.
Three, the step in preparation random dna library is as follows:
A. the former times acid of few nuclear is 3 '-CCT
CTA TAT AGG TAC CGA TCG (NNM)
12AGG CCG G
TAGTA GTA GTA GTA GTACCTAGG CCA-5 ' is (it is the BamHI restriction enzyme site that line part is respectively SD sequence, atg start codon, His Tag, italicized item) (N represents A, C, T or G, M represent A or C) (90bp)
B. the upstream primer U1 of first round PCR be 5 '-G CTC GTA TAA TGT GTG GCC ACAACG GAA GGA GAT ATA TCC ATG-3 ' (43bp) (the line part is that 5 ' end stem ring and italicized item are ribosome bind site: RBS), downstream primer D1 is 3 '-GTA GTA
CCT AGG CCA TCA CCT TCA CCA TCA CCG AGT GGA AGT TCA CAT ACG G-5 ' (52bp) (the line part is respectively BamHI restriction enzyme site, Gly-Ser sequence and 3 ' end stem ring)
C. the second upstream primer U2 that takes turns PCR be 5 '-GCG CTG CAG TTG ACA ATT AATCAT CGG CTC GTA TAA TGT GTG-3 ' (42bp), (the line part is respectively PstI restriction enzyme site and Tac promoter).Downstream primer is D1.SsDNA library and primer are synthetic by the rich inferior biological company limited in Shanghai at random.The structure synoptic diagram in random dna library is seen Fig. 1.
D. examining general acid with the widow is template, carries out first round PCR with primer U1 and D1, and reaction system is:
10 * Tag DNA Polymerase buffer (contains Mg
2+) 5 μ l
dNTP mix(10mM) 1μl
Primer U1 (20 μ M) 1 μ l
Primer D1 (20 μ M) 1 μ l
Oligonucleotides (0.05 μ g/ μ l) 1 μ l
Distilled water 40 μ l
Tag archaeal dna polymerase 1 μ l
Response procedures:
A.95 ℃, 2 minutes
B.95 ℃, 36 seconds; 63 ℃, 36 seconds; 72 ℃, 84 seconds; Totally 25 circulations.
C.72 ℃, 5 minutes
The E.PCR product reclaims with reclaiming kit (QIAEX II gel Extraction Kit, U.S. promega company) then through 2% Ago-Gel (0.4g agarose powder dissolution is in 20ml1 * TAE solution) electrophoresis.
F. the PCR product that reclaims gained through step e carries out second and takes turns pcr amplification as template, and reaction system is:
10 * Tag DNA Polymerase buffer (contains Mg
2+) 5 μ l
dNTP mix(10mM) 1μl
Primer U1 (20 μ M) 1 μ l
Primer D1 (20 μ M) 1 μ l
First round PCR product 4 μ l
Distilled water 37 μ l
Tag archaeal dna polymerase 1 μ l
Response procedures:
A.95 ℃, 2 minutes
B.95 ℃, 36 seconds; 62 ℃, 36 seconds; 72 ℃, 84 seconds; Totally 25 circulations.
C.72 ℃, 5 minutes
G. the step F gained gets the PCR product through 2% agarose gel electrophoresis, reclaims with reclaiming kit then, reclaims the gained fragment and promptly can be used for the in-vitro transcription translation
Four, the step in preparation ribosomes library is as follows:
A. the preparation in ribosomes library (Peptides 2005,26 (11) for the document Wu that sees reference, et al.: 2057-2063.), make up and screening process as shown in Figure 2: will encode 10
14The dna library of individual 12 peptides at random carries out transcribing/translating of coupling external, and transcription product mRNA can not discharge from ribosomes, thereby form a stable mRNA-ribosomes-newborn complex of polypeptides structure owing to lack terminator.Utilization is connected with the rondom polypeptide on the affine screening ribosomes of E2-GST fusion (E2-GST-Sepharose 4B) surface of Sepharose 4B (sigma company), separates the mRNA-ribosomes-polypeptide complex of specificity combination, by reducing Mg
2+Concentration, ribosomes dissociates, and discharges mRNA, reclaims the mRNA in the ribosomes enrichment storehouse, and reverse transcription becomes the cDNA.PCR amplification, and its product can be used as the external synthetic template of next round, takes turns screening through six, can find the polypeptide that combines with the E2 protein-specific.
B. reaction system:
Dna profiling (0.3mg/ml) 10 μ l
S30 Premix without Amino Acids 20μl
Amino acid mixing liquid (no methionine) 2.5 μ l
Amino acid mixing liquid (no leucine) 2.5 μ l
S30 Extract 15μl
Each potpourri in the mixing EP pipe gently, 5000rpm centrifugal 30 seconds, allows potpourri be deposited on EP pipe bottom.
C.30 ℃ hatched 2.5 hours.
The D.EP pipe is placed 5 minutes stopped reactions on ice, and material is the ribosomal display library in the EP pipe.
Five, the step of the polypeptide that combines with hepatitis C virus E2 glycoprotein of ribosomal display library screening is as follows:
A. the DNase (Promega company, the U.S.) that in 100 μ l in-vitro transcription translation potpourri, adds the no RNA enzyme of 6 units, 10 * DNase I damping fluid (Promega company, the U.S.), 6 μ l, 37 ℃ of incubation 30min.Add 6 μ l then and end damping fluid, 65 ℃, 10min deactivation DNase.
B. get 60 μ l E2-GST-Sepharose 4B, 4 ℃, 5000rpm, 5min is centrifugal, abandons supernatant.
C. precipitate with 500 μ l elution buffers: (the Tris-acetic acid of washing buffer:50mM pH 7.5,150mM sodium chloride nacl, 0.1%Tween-20,50mM magnesium acetate) washing 3 times.
D. 100 μ l in-vitro transcription/translation potpourris are joined among the washed E2-GST-Sepharose 4B, 4 ℃ were shaken 1 hour.
E.4 ℃, 5000rpm, 5min is centrifugal, abandons supernatant, and precipitation is with washing buffer washing 3 times.
F. add 100 μ l elution buffer in the precipitation, 4 ℃ are shaken 10min mRNA and ribosomes are dissociated, and 4 ℃, 5000rpm, 5min is centrifugal, gets supernatant.
G. use RNaid Kit (available from bio101 company, the U.S.) to reclaim mRNA, concrete grammar is as follows:
A. RNABinding Salt (the RNaid Kit composition) mixing that adds 3 times of volumes in the supernatant.
B. add 10 μ l RNA MATRIX (RNaid Kit composition) again, room temperature is placed 5min.
C.13000rpm, 1min is centrifugal, abandons supernatant.
D. use RNA wash (RNaid Kit composition) washing 2 times, (RNAwash: ethanol=1:1).
E. add 10 μ l and do not have the H of RNase
2O, 45～55 ℃ of incubation 5min.
F.13000rpm, 2min is centrifugal, gets supernatant, and supernatant promptly is the RNA template, can be used for RT-PCR.
H.RT-PCR, its reaction system is
AMV/Tfl 5×Reaction buffer 10μl
Primer U1 (20 μ M) 1 μ l
Primer D1 (20 μ M) 1 μ l
MgSO
4(25mM) 2μl
AMV Reverse Transcription 1μl
Tfl DNA Polymerase 1μl
No enzyme water 29 μ l
Response procedures is: 48 ℃, and 45 minutes; 94 ℃, 2 minutes; 94 ℃, 30 seconds; 63 ℃, 1 minute, 72 ℃, 2 minutes; Totally 31 circulations; 72 ℃, 7 minutes
I.2% agarose agarose gel electrophoresis.
J. reclaim the molecular weight size and be the RT PCR product of 155bp.
K. be template to reclaim product, row second is taken turns pcr amplification again, and reclaiming the molecular weight size is the PCR product of 181bp.
That L. will reclaim second takes turns the PCR product and is connected on the carrier pUC19 (available from American I nvitrogen company), and concrete steps are:
A. PCR product and carrier pUC19 enzyme are cut, reaction system is
10×Y
+Buffer 2μl
BamHI 1μl
PstI 1μl
ddH
2O 17μl
37 ℃ of water-baths 2 hours
M. reclaim molecular weight size respectively and be the pUC19 purpose segment of 2.668kb, then with recovery kit (QIAEX II gel Extraction Kit, U.S. promega company) recovery for the dna fragmentation of 128bp and molecular weight size.
A. with dna ligase with step b DNA be connected with carrier pUC19, reaction system is:
DNA 4μl
pUC 19 2μl
10×Buffer 2μl
Hatched 20 hours for 16 ℃
B. the connection product with step c gained is converted into bacillus coli DH 5 alpha respectively.Get and connect product 10 μ l, add 100 μ l DH5 α and experience polypeptide cell, ice bath 30 minutes, 42 ℃ 90 seconds, ice bath is 5 minutes again, (the LB fluid nutrient medium is: the 10g tryptone to add 800 μ l LB fluid nutrient mediums, the 5g yeast extract, 10g sodium chloride is dissolved to the 1000ml distilled water), 37 ℃, 225rpm jolting 40～50 minutes.Get 200 μ l bacterium liquid and be evenly coated in ampicillin (Amp
r) the LB solid medium (the LB solid medium is: 10g tryptone, the 5g yeast extract, 10g sodium chloride, the 15g agar powder is dissolved to the 1000ml distilled water) of resistance goes up (ampicillin concentration is 50 μ g/ml), 37 ℃ of overnight incubation.
C. the single clone's bacterium colony of picking places 3ml LB fluid nutrient medium (containing ammonia benzyl mould 50 μ g/ml) overnight incubation.
D. the plasmid DNA of the bacterium liquid that obtains of extraction step e.
The DNA of e.PCR authentication step f gained, reaction system is:
10×Taq DNA polymerase buffer 5μl
DNTP mix potpourri (10mM) 1 μ l
Primer U2 (20mM) 1 μ l
Primer D1 (20mM) 1 μ l
Taq DNA Polymerase 1μl
Response procedures is: 95 ℃, and 2 minutes; 95 ℃, 30 seconds; 62 ℃, 1 minute, 72 ℃, 1 minute 24 seconds; Totally 25 circulations; 72 ℃, 5 minutes
F. the step g products therefrom is identified through BamHI and PstI that correct clone delivers to the order-checking of Shanghai Hua Nuo company.
According to sequencing result, ask Xi'an Mei Lian company to synthesize, obtained the polypeptide of 4 a kind of separation, its amino acid sequence is respectively:
PE2A(SEQ ID NO：1)：GFGRYRRHGSPW
PE2B(SEQ ID NO：2)：MAIGPYPACGSG
PE2C(SEQ ID NO：3)：LSVLVISMFNAV
PE2D(SEQ ID NO：4)：MARHRNWPLVMV
Six, it is as follows to utilize the hepatitis C virus envelope antigen kit to detect the step of the hepatitis C virus in the third hepatopathy human serum:
A.96 every hole adds 100 μ l (dilutability is 1:300～1:500) with the anti-E2-GST antibody of sodium bicarbonate buffer liquid (0.05M pH9.6) dilution in the orifice plate, or add the polypeptide (30～50ug/ hole) of 100 μ l with sodium bicarbonate buffer liquid (0.05M pH9.6) dilution, hatched 1 hour for 37 ℃.
B. cleansing solution (the PBS phosphate buffer of 0.1%Tween-20) washing is five times
C. seal quilt: every hole adds 100 μ l confining liquids (the PBS phosphate buffer of 1%BSA), 4 ℃ of overnight incubation.
D. cleansing solution (the PBS phosphate buffer of 0.1%Tween-20) washing is five times
E. every hole adds 100 μ l, third hepatopathy human serum or healthy human serum, and room temperature (26 ℃) was hatched 1 hour.
F. repeat the B step once.
G. biotin labeled polypeptide (the big genome company of Beijing China is synthetic for bio-ZD, the bio-ZA etc.) room temperature (26 ℃) that every hole adds 100 μ l10mg/L was hatched 1 hour.
H. repeat the B step once.
I. every hole adds the Streptavidin (dilutability is 1: 1000) of 100 horseradish peroxidase-labeled, and room temperature (26 ℃) was hatched 30 minutes.
Add the o-dihydroxy ammon colour developing, read the OD value under the microplate reader OD450nm.As a result the polypeptide that obtains of display separation can specificity in conjunction with hepatitis C virus E2 glycoprotein, and can directly combine with the virus in the third hepatopathy human serum, its OD value is greater than more than 2～3 times of the OD value that combines with normal human serum.Can reach the effect of viral level in the direct detection patient blood sample.
Seven, utilize the step of hepatitis C virus envelope antigen kit detection hepatitis C virus particle as follows:
A.96 every hole adds 18 μ l hepatitis C virus particles (3 * 10 in the orifice plate
5Individual virus) and 162 μ l sodium bicarbonate buffer liquid (0.05M pH9.6), mix, hatched 7 hours for 4 ℃.
B. cleansing solution (the PBS phosphate buffer of 0.1%Tween-20) washing is five times.
C. every hole adds 100 μ l confining liquids (the PBS phosphate buffer of 1%BSA), 4 ℃ of overnight incubation.
D. after cleansing solution washed five times times, the biotin labeled polypeptide that adds 2 μ g～8 μ g respectively was in 96 orifice plates, and every pore volume is 100 μ l, hatches 1h for 37 ℃.
E. after washing five times, the horseradish that adds 100 μ l dilutabilitys and be 1:1000 is crossed the Streptavidin (HRP-advin is available from crystalline substance U.S. company) of fosterization thing enzyme labeling.
F. add the o-dihydroxy ammon colour developing, read the OD value under the microplate reader OD450nm.Show that biotin labeled polypeptide can directly combine with the hepatitis C virus particle.
This experimental results show that separate the polypeptide obtain can specificity in conjunction with the E2 glycoprotein of hepatitis C virus particle (HCVcc), binding ability is dose dependent.
Advantage of the present invention
This experiment first Application ribosomes library screening the polypeptide that combines with hepatitis C virus E2 glycoprotein.Three peptide species (the ZD that screen, ZA, ZB) has high-affinity with hepatitis C virus surface-coating E2 glycoprotein albumen, polyclonal antibody and the biotin labeled polypeptide of anti-E2 used in this experiment first, the ELISA method detects the hepatitis C virus surface antigen in the third hepatopathy human serum, micromolecule polypeptide at hepatitis C virus E2 glycoprotein provided by the present invention (12 peptide) molecular weight is little, be easy to synthesize, anti-E2 polyclonal antibody preparation simple, highly sensitive in traditional detection HCV antibody method, near traditional HCVRNA detection method, cost is low than traditional detection HCVRNA detection method, does not need the PCR instrument.
Description of drawings
Fig. 1. the structure process flow diagram in random dna library.
RBS represents ribosome bind site, and N represents A, C, and T or G, M represent A or C
Fig. 2. the structure process flow diagram of the polypeptide that the ribosomal display library screening combines with hepatitis C virus E2 glycoprotein.
Fig. 3 .SPR detects the affinity of polypeptide PE2A, polypeptide PE2B, polypeptide PE2C, polypeptide PE2D and E2 albumen, and the affinity size of their combinations is: polypeptide PE2D〉polypeptide PE2B〉polypeptide PE2A〉polypeptide PE2C.
Fig. 4 A polypeptide suppresses HCVE2 albumen and combines with human liver cell Huh7.5 cell
Fig. 4 B polypeptide suppresses HCVE2 albumen and combines the ability (Fig. 4 A) that (A) flow cytometer detection polypeptide A, polypeptide B, polypeptide D suppress HCVE2 protein combination human liver cancer cell Huh7.5 respectively with the DC-SIGN+ target cell; Polypeptide suppresses the dose dependent (Fig. 4 A) of the ability of HCVE2 protein combination human liver cancer cell Huh7.5 respectively.(B) flow cytometer detects polypeptide A, polypeptide B, polypeptide D suppress E2 protein combination DC-SIGN respectively
+The ability of-NIH3T3 cell (Fig. 4 B); Polypeptide suppresses E2 protein combination human liver cancer cell DC-SIGN respectively
+The dose dependent of the ability of-NIH3T3 (Fig. 4 B).
Fig. 5. the competitive acceptor CD81 that suppresses E2 and HCV of polypeptide PE2D energy, the combination of heparin.
Fig. 6. polypeptide PE2D can specificity suppress third liver pseudovirus (HCVpp) target cell infection, and polypeptide PE2D inhibition ability is the dependence of dosage.Fig. 4 A is the construction step of pseudovirus, and Fig. 4 B is that pseudovirus is expressed E1, E2 albumen in 293 cells, and Fig. 4 C is that PE2D (SEQ ID NO:4) stops pseudovirus to infect the Huh7.5 cell, and is dose dependent.
Fig. 7. variable concentrations PE2D is dose dependent with the ability that HCV virion (HCVcc) combines.
It is different with the ability of Huh7.5 combination that the PE2D of Fig. 8 .ELISA detection variable concentrations can suppress HCVcc, and dosage is big more, and the inhibition ability is strong more.
Fig. 9 .HCV surface-coating ELISA kit test method: bag is by E2 antibody, add sample, the polypeptide that adds the biotin souvenir again, and the horseradish Streptavidin of crossing fosterization thing enzyme labeling detects HCV antibody positive or HCVRNA and detects virus in the third positive hepatopathy human serum.Normal human serum in contrast.
Embodiment
The configuration of hepatitis C virus envelope antigen kit:
A. 12 peptides that can combine of mark such as biotin with HCV E2 protein-specific;
B. polypeptide is the sequence shown in SEQIDNO:1, SEQIDNO:2, the SEQIDNO:4;
C. the anti-E2 of rabbit polyclonal antibody;
D. the Streptavidin of horseradish peroxidase-labeled;
E. horseradish peroxidase substrate;
F. ELISA Plate (CellStar@ is available from Wuhan strong wind bio tech ltd), microplate reader;
G.E2 albumen is as the standard positive hole;
H, enzyme-linked immunosorbent assay instrument (available from medical equipment company limited of Nanjing East China Electronics Co., Ltd group).
The step of one peptide species PF2A, PE2B, PE2C, PE2D is as follows:
One, the step of preparation E2-GST fusion is as follows:
A. picking contains pGEX-KG-E2 (document Li sees reference, et al., Engineering ofN-glycosylation of Hepatitis C Virus Envelope Protein E2 Enhances T cellresponses for DNA immunization, Vaccine.2007.25:1544-1551.) e. coli bl21 DE3 (available from U.S. invitrogen company) is incubated in the LB nutrient culture media that 3ml contains ampicillin (50g/ml), the inoculation of contrast group contains the pGEX-KG (prokaryotic expression carrier of GST albumen, available from Amersham Biosciences company), cultivated 12～16 hours for 37 ℃.
B. (the LB fluid nutrient medium is: the 10g tryptone bacterium liquid in the small test tube to be transferred to the LB fluid nutrient medium that 200ml contains ampicillin (50g/ml) respectively, the 5g yeast extract, 10g sodium chloride is dissolved to the 1000ml distilled water) in, 37 ℃ are cultivated OD600 is 1～2.
C. add 100mM isopropyl-(IPTG), making its final concentration is 0.1mM, induces 4～5 hours for 30 ℃.
D. will induce later bacterium liquid branch to install in the centrifuge tube of 250ml, 4 ℃, 7700g, 5min is centrifugal, abandons supernatant.
E. precipitation is washed 1 time with phosphate buffer PBS (140mM sodium chloride, 2.7mM potassium chloride, 10mM sodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate), and 4 ℃, 7700g, 5min is centrifugal, abandons supernatant.
F. precipitation is resuspended among the PBS that 8ml contains 1mM phenylmethylsulfonyl fluoride (PMSF:17.42mg PMSF is dissolved in the 1ml isopropyl alcohol ,-20 ℃ of preservations).
G. bacterium liquid is placed in the ice, added 20% triton x-100 (20%Triton X-100:1ml TritonX-100 is dissolved in the 4ml aseptic double-distilled water) behind the ultrasonic broken bacterium, make 1%, 4 ℃ of its final concentration, gently mixed 30 minutes.
H. the bacterium liquid that adds the 1ml ultrasonic degradation in every EP pipe, 4 ℃, 12000g, 10min is centrifugal, gets supernatant.
I. supernatant is centrifugal once more, and 4 ℃, 12000g, 10min gets supernatant again.
J. each EP pipe adds 20 μ l Ago-Gel 4B (Sepharose 4B: available from AmershamBiosciences company), and room temperature (20-25 ℃, below identical) is gently mixed 30min.4 ℃, 5000rpm, 5min, centrifugal, abandon supernatant.
K. each EP pipe respectively adds 100 μ l 0.01M PBS washing, and the suspension that will contain Separose 4B behind the centrifuge washing is collected in a pipe, and 4 ℃, 5000rpm, 5min, centrifugal, abandon supernatant, wash altogether 3 times.
L. add the Glutathione Elution Buffer (the 0.0154g Agifutol is dissolved in the 1M PBS filtration sterilization of 0.25ml pH 8.0, packing, 4 ℃ of preservations) with Sepharose 4B equivalent, room temperature is gently mixed 10min, and 4 ℃, 5000rpm, 5min gets supernatant.
M. repeating step L is twice.The supernatant of collecting then is required E2-GST fusion.
Two, the step of the anti-E2 protein antibodies of preparation rabbit is as follows:
A. the E2-GST fusion with expression and purification is diluted to 1m/ml with PBS, adds complete Freund's adjuvant (CFA), is injected to 6 the subcutaneous and inguinal lymph nodes in new zealand rabbit vertebra both sides, every 0.1～0.2ml, and every rabbit antigen immune dosage is 1mg.
B. after two weeks,, be adjuvant with incomplete Freund's adjuvant (IFA) with same antigen amount immunizing rabbit once more.Immunity site and quantity are selected same fundamental immunity.
C. altogether immunity 4 times, be 2 weeks interval time, twice immunizing dose in back be the first time 1.5 times of dosage.Heart blood sampling in the 10th day after room temperature leaves standstill and solidifies, changes 4 ℃ of refrigerator standing over night over to after the last immunity, and separation of serum, serum are E2-GST fusion antibody, and it is standby to place-80 ℃ of refrigerators to preserve.
Three, the following (see figure 1) of step in preparation random dna library:
A. the former times acid of few nuclear is 3 '-CCT
CTA TAT AGG TAC CGA TCG (NNM)
12AGG CCG G
TAGTA GTA GTA GTA GTACCTAGG CCA-5 ' is (it is the BamHI restriction enzyme site that line part is respectively SD sequence, atg start codon, His Tag, italicized item) (N represents A, C, T or G, M represent A or C) (90bp)
B. the upstream primer U1 of first round PCR be 5 '-G CTC GTA TAA TGT GTG GCC ACAACG GAA GGA GAT ATA TCC ATG-3 ' (43bp) (the line part is that 5 ' end stem ring and italicized item are ribosome bind site: RBS), downstream primer D1 is 3 '-GTA GTA
CCT AGG CCA TCA CCT TCA CCA TCA CCG AGT GGA AGT TCACAT ACG G-5 ' (52bp) (the line part is respectively BamHI restriction enzyme site, Gly-Ser sequence and 3 ' end stem ring)
C. the second upstream primer U2 that takes turns PCR be 5 '-GCG CTG CAG TTG ACA ATT AATCAT CGG CTC GTA TAA TGT GTG-3 ' (42bp), (the line part is respectively PstI restriction enzyme site and Tac promoter).Downstream primer is D1.The structure synoptic diagram in random dna library is seen Fig. 1.
D. examining general acid with the widow is template, carries out first round PCR with primer U1 and D1, and reaction system is:
10 * Tag DNA Polymerase buffer (contains Mg
2+) 5 μ l
dNTPmix(10mM) 1μl
Primer U1 (20 μ M) 1 μ l
Primer D1 (20 μ M) 1 μ l
Oligonucleotides (0.05 μ g/ μ l) 1 μ l
Tag archaeal dna polymerase 1 μ l
Response procedures:
A.95 ℃, 2 minutes
B.95 ℃, 36 seconds; 63 ℃, 36 seconds; 72 ℃, 84 seconds; Totally 25 circulations.
C.72 ℃, 5 minutes
The E.PCR product reclaims with reclaiming kit (QIAEX II gel Extraction Kit, U.S. promega company) then through 2% Ago-Gel (0.4g agarose powder dissolution is in 20ml1 * TAE solution) electrophoresis.
F. the PCR product that reclaims gained through step e carries out second and takes turns pcr amplification as template, and reaction system is:
10 * Tag DNA Polymerase buffer (contains Mg
2+) 5 μ l
dNTP mix(10mM) 1μl
Primer U1 (20 μ M) 1 μ l
Primer D1 (20 μ M) 1 μ l
First round PCR product 4 μ l
Distilled water 37 μ l
Tag archaeal dna polymerase 1 μ l
Response procedures:
A.95 ℃, 2 minutes
B.95 ℃, 36 seconds; 62 ℃, 36 seconds; 72 ℃, 84 seconds; Totally 25 circulations.
C.72 ℃, 5 minutes
G. the step F gained gets the PCR product through 2% agarose gel electrophoresis, reclaims with reclaiming kit then, reclaims the gained fragment and promptly can be used for in-vitro transcription translation dna profiling.
Four, the step in preparation ribosomal display library is as follows:
A. ribosomes library preparation and screening process (Peptides 2005,26 (11) for the document Wu that sees reference, et al.: 2057-2063.), make up and screening process as shown in Figure 2: will encode 10
14The dna library of individual 12 peptides at random carries out transcribing/translating of coupling external, and transcription product mRNA can not discharge from ribosomes owing to lack terminator, and with newborn polypeptide, ribosomes with covalent bonds, form a stable complex structure.Utilization is connected with the rondom polypeptide on the affine screening ribosomes of E2-GST albumen (E2-GST-Sepharose 4B) surface of Sepharose 4B, separates the mRNA-ribosomes-polypeptide complex of specificity combination, by reducing Mg
2+Concentration, ribosomes dissociates, discharge mRNA, reclaim the mRNA in the ribosomes enrichment storehouse, reverse transcription becomes the cDNA.PCR amplification, and its product can be used as the external synthetic template of next round, the anti-sieve of GST albumen through being connected with Sepharose 4B (GST-Sepharose 4B) is to remove the polypeptide of non-specific binding, E2-GST-Sepharose 4B just sieves, and takes turns screening through six, screens the polypeptide that combines with the E2 protein-specific.
B. reaction system:
Dna profiling (0.3mg/ml) 10 μ l
S30 Premix without Amino Acids 20μl
Amino acid mixing liquid (no methionine) 2.5 μ l
Amino acid mixing liquid (no leucine) 2.5 μ l
S30 Extract，linear 15μl
Each potpourri among the mixing step B gently, 5000rpm centrifugal 30 seconds, allows potpourri be deposited on EP pipe bottom.
C.30 ℃ hatched 2.5 hours.
The D.EP pipe is placed 5 minutes stopped reactions on ice.Promptly obtain the ribosomal display library.
Five, the following (see figure 2) of step of the polypeptide that combines with hepatitis C virus E2 glycoprotein of ribosomal display library screening:
A. the DNase (Promega company, the U.S.) that in 100 μ l in-vitro transcription translation potpourri, adds the no RNA enzyme of 6 units, 10 * DNase I damping fluid (Promega company, the U.S.), 6 μ l, 37 ℃ of incubation 30min.Add 6 μ l then and end damping fluid, 65 ℃, 10min deactivation DNase.
B. get 60 μ l E2-GST-Sepharose 4B, 4 ℃, 5000rpm, 5min is centrifugal, abandons supernatant.
C. precipitation is washed 3 times with 500 μ l washing buffer (the Tris-acetic acid of 50mM pH 7.5,150mMNaCl, 0.1%Tween-20,50mM magnesium acetate).
D. 100 μ l in-vitro transcription/translation potpourris are joined among the washed E2-GST-Sepharose 4B, 4 ℃ were shaken 1 hour.
E.4 ℃, 5000rpm, 5min is centrifugal, abandons supernatant, and precipitation is with elution buffer washing 3 times.
F. add 100 μ l elution buffer in the precipitation, 4 ℃ are shaken 10min mRNA and ribosomes are dissociated, and 4 ℃, 5000rpm, 5min is centrifugal, gets supernatant.
G. use RNaid Kit (available from bio101 company, the U.S.) to reclaim mRNA, concrete grammar is as follows:
G. RNABinding Salt (the RNaid Kit composition) mixing that adds 3 times of volumes in the supernatant.
H. add 10 μ l RNA MATRIX (RNaid Kit composition) again, room temperature is placed 5min.
I.13000rpm, 1min is centrifugal, abandons supernatant.
J. use RNA wash (RNaid Kit composition) washing 2 times, (RNAwash: ethanol=1:1).
K. add 10 μ l and do not have the H of RNase
2O, 55 ℃ of incubation 5min.
L.13000rpm, 2min is centrifugal, gets supernatant, and supernatant promptly is the RNA template, can be used for RT-PCR.
H.RT-PCR, its reaction system is
AMV/Tfl 5×Reaction buffer 10μl
Primer U1 (20mM) 1 μ l
Primer D1 (20mM) 1 μ l
Magnesium sulphate (25mM) 2 μ l
AMV Reverse Transcription 1μl
Tfl archaeal dna polymerase 1 μ l
No enzyme water 29 μ l
Response procedures is: 48 ℃, and 45 minutes; 94 ℃, 2 minutes; 94 ℃, 30 seconds; 63 ℃, 1 minute, 72 ℃, 2 minutes; Totally 31 circulations; 72 ℃, 7 minutes
I.2% agarose agarose gel electrophoresis.
J. reclaim the molecular weight size and be the RTPCR product of 155bp.
K. be template to reclaim product, row second is taken turns pcr amplification again, and reclaiming the molecular weight size is the PCR product of 181bp.
That L. will reclaim second takes turns the PCR product and is connected on the carrier pUC19 (available from U.S. invitrogen company), and concrete steps are:
A. PCR product and carrier pUC19 enzyme are cut, reaction system is
10×Y
+Buffer 2μl
BamHI 1μl
PstI 1μl
ddH
2O 17μl
37 ℃ of water-baths 2 hours
B. reclaim molecular weight size respectively and be the pUC 19 purpose segments (recovery method is the same) of 2.668kb for the dna fragmentation of 128bp and molecular weight are big or small.
C. with dna ligase with step b DNA be connected with carrier pUC19, reaction system is:
DNA 4μl
pUC 19 2μl
10×Buffer 2μl
Hatched 20 hours for 16 ℃
D. the connection product with step c gained is converted into bacillus coli DH 5 alpha respectively.Get and connect product 10 μ l, add 100 μ l DH5 α and experience polypeptide cell, ice bath 30 minutes, 42 ℃ 90 seconds, ice bath is 5 minutes again, (the LB fluid nutrient medium is: the 10g tryptone to add 800 μ l LB fluid nutrient mediums, the 5g yeast extract, 10g sodium chloride is dissolved to the 1000ml distilled water), 37 ℃, 225rpm jolting 40～50 minutes.Get 200 μ l bacterium liquid and be evenly coated in ampicillin (Amp
r) the LB solid medium (the LB solid medium is: 10g tryptone, the 5g yeast extract, 10g sodium chloride, the 15g agar powder is dissolved to the 1000ml distilled water) of resistance goes up (ampicillin concentration is 50 μ g/ml), 37 ℃ of overnight incubation.
E. the single clone's bacterium colony of picking places 3ml LB fluid nutrient medium (containing ammonia benzyl mould 50 μ g/ml) overnight incubation.
F. the plasmid DNA of the bacterium liquid that obtains of extraction step e.
The DNA of g.PCR authentication step f gained, reaction system is:
10×Taq DNA polymerase buffer 5μl
DNTP mix potpourri (10mM) 1 μ l
Primer U2 (20 μ M) 1 μ l
Primer D1 (20 μ M) 1 μ l
Taq DNA Polymerase 1μl
Response procedures is: 95 ℃, and 2 minutes; 95 ℃, 30 seconds; 62 ℃, 1 minute, 72 ℃, 1 minute 24 seconds; Totally 25 circulations; 72 ℃, 5 minutes
H. the step g products therefrom is identified through BamHI and PstI that correct clone delivers to the order-checking of Shanghai Hua Nuo company.
M. according to sequencing result, please Xi'an Mei Lian company synthesize 12 peptides, see Table 1.
Table 1 filter out with the protein bound peptide sequence of E2
| The peptide that separates | Amino acid sequence | |
| PE2A | GFGRYRRHGSPW | SEQIDNO：1 |
| PE2B | MAIGPYPACGSG | SEQIDNO：2 |
| PE2C | LSVLVISMFNAV | SEQIDNO：3 |
| PE2D | MARHRNWPLVMV | SEQIDNO：4 |
The step of the affinity of surface plasma resonance technology (SPR) detection polypeptide and E2 albumen is as follows:
A. allow testing tool Biocore (U.S.) pass through one section HBS damping fluid until baseline stability.
B. inject 40 μ l amino coupled reagent (containing 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide (EDC) of 0.02M and the N-hydroxy-succinamide (NHS) of 0.05M), the carboxyl on the activation CM5 vane.
C. be that 5.0 acetate buffer solution is diluted to 5mg/ml with E2 albumen with the pH value, inject this solution 40 μ l.
D. go up machine testing, flow velocity is 20 μ l/min.
E. be variable concentrations with polypeptide PE2A, PE2B, PE2C, PE2D dilution, with the E2 albumen effect on machine and the CM5 vane on the 2 μ l/min 7 minutes.
F. the HCl regenerated liquid of injecting 10 μ l with the flow velocity of 2 μ l/min recovers baseline, gathers response signal in real time.
G. the special software with BIACORE carries out data analysis, and the affinity that we find to analyze PE2D and E2 albumen is the highest, the results are shown in Figure 3, and its affinity has related parameter to see Table 2.
The dynamics data of table 2 polypeptide and the E2 albumen effect of mutually combining
| Polypeptide | ka/M -1s -1(×10 4) | kd/s -1(×10 -3) | k A/M -1(×10 7) |
| PE2A | 3.22 | 1.39 | 2.32 |
| PE2B | 2.56 | 1.22 | 2.10 |
| PE2C | no | no | no |
| PE2D | 7.51 | 1.45 | 5.18 |
It is as follows with the step that the target molecule cell combines that flow cytometer detects polypeptide:
A. cultivate human liver cancer cell Huh7.5 (the document Zhong that sees reference, et al., Robust hepatitis C virusinfection in vitro, 2005, PNAS, 102:9294-9299) and DC-SIGN
+(-NIH3T3 (there is the fibroblast of the mouse of DC-Sign molecule on the surface) cell sees reference document Wu, et al., Functional evaluation of DC-SIGN monoclonal antibodies reveals DC-SIGNinteractions with ICAM-3 do not promote human immunodeficiency virustype I transmission.J.virol.2002,76:5905-5914).Condition of culture is the DMEM nutrient culture media (purchase of Gibco company) of 10% hyclone, is incubated at 37 ℃, contains in the incubator of 5% carbon dioxide.
B. polypeptide PE2A, PE2B, PE2D are mixed (control tube adds GST albumen) respectively with E2-GST albumen, cumulative volume is 50 μ l, and wherein the concentration of polypeptide is 500 μ M, and the concentration of E2-GST albumen and GST albumen is 20 μ g/ml, hatches 30 minutes for 37 ℃.
C. with the potpourri of the polypeptide of step B gained and albumen respectively with the Huh7.5 and the DC-SIGN of results
+-NIH3T3 mixing with cells, cumulative volume are 100 μ l, and wherein number of cells is 2 * 10
6Individual.Hatched 30 minutes for 37 ℃.
D. every pipe adds 1ml PBS washing, 1000rpm, and 5min abandons supernatant, with 80 μ l re-suspended cells.
E. the E2-GST antibody that adds 20 μ l 1:500 was hatched 30 minutes for 37 ℃.
F. repeating step D.
G. add goat-anti rabbit two anti-of 1 μ l PE mark, hatched 30 minutes for 37 ℃.
H. every pipe adds 1ml PBS washing, 1000rpm, and 5min abandons supernatant, with 500 μ l re-suspended cells.
I. through last machine testing, the applicant finds PE2A, peace PE2B, PE2D respectively with the effect of E2-GST albumen after, can significantly suppress the E2 protein combination to Huh7.5 cell (Fig. 4) and DC-SIGN
+-NIH3T3 cell (Fig. 5), and become dose-dependence, polypeptide dosage is bigger, and its affinity with E2 is stronger, suppresses HCVE2 invasion and attack target cell abilities (Fig. 4 A and Fig. 4 B) more by force.
Embodiment 4
Polypeptide D can part competitive inhibition E2 and the acceptor CD81 of HCV and the combination (Fig. 5) of heparin.
PE2D can suppress HCVppv pseudovirus (Fig. 6 A, 6B) structure of invasion and attack people target cell (Fig. 6 C) .HCVppv pseudovirus is seen figure Fig. 6 A, and the ability that 6B, PE2D suppress HCVppv pseudovirus invasion and attack people target cell is dose dependent, polypeptide D concentration is bigger, its inhibition ability stronger (Fig. 6 C).
Embodiment 6
It is as follows with the step that hepatitis C virus particle (HCVcc) combines to detect polypeptide PE2D:
A.96 every hole adds 18 μ l hepatitis C virus particles (3 * 10 in the orifice plate
5Individual virus) and 162 μ l sodium bicarbonate buffer liquid (0.05M pH9.6), mix, hatched 7 hours for 4 ℃.
B. cleansing solution (the PBS phosphate buffer of 0.1%Tween-20) washing is five times.
C. every hole adds 100 μ l confining liquids (the PBS phosphate buffer of 1%BSA), 4 ℃ of overnight incubation.
D. after cleansing solution washs five times times, add 0 μ g respectively, 2 μ g, 4 μ g, the bio-PE2D of 8 μ g are in 96 orifice plates, and every pore volume is 100 μ l, hatches 1h for 37 ℃.
E. after washing five times, the horseradish that adds 100 μ l dilutabilitys and be 1:1000 is crossed the Streptavidin (HRP-advin is available from crystalline substance U.S. company) of fosterization thing enzyme labeling.
F. add the o-dihydroxy ammon colour developing, read the OD value under the microplate reader OD492nm.Show that ZD can directly combine with the hepatitis C virus particle.
This experimental results show that separate the polypeptide obtain can specificity in conjunction with hepatitis C virus particle (HCVcc), binding ability is dose dependent (Fig. 7).
Embodiment 7
The step that detects polypeptide PE2D inhibition hepatitis C virus particle (HCVcc) infected person liver cell Huh7.5 cell is as follows
A. cultivate human liver cancer cell Huh7.5 in 24 orifice plates, place 37 ℃, contain in the incubator of 5% carbon dioxide, condition of culture is the DMEM nutrient culture media of 10% hyclone.
B. every hole adds PE2D and 3 * 10
5Individual hepatitis C virus particle, the concentration of PE2D are divided into 200 μ M, 400 μ M.
C.37 ℃ hatched 5 hours, behind the washed cell, added medium culture 3 days.
D. adding fluorescently-labeled HCV-E2 antibody (available from the Shanghai Institute Pasteur) hatched 30 minutes with cell
E. fluorescent microscope down the every hole of counting red fluorescence count (ffu/well, 58nm).
This experimental results show that separating the polypeptide PE2D obtain can specificity suppress the hepatitis C virus particle (HCVcc infected person liver cell, and dosage is big more, the inhibition ability is strong more, is dose dependent (Fig. 8).
Embodiment 8
Utilize the hepatitis C virus envelope antigen kit detect hepatitis C virus in the 150 routine hepatitis C patients serums (this virus is from Hubei. the Wuhan related hospitals is collected the hepatitis C virus in the hepatitis C virus human serum), 150 routine normal human serums are (it is reference substance that normal human serum is collected in health check-up) in contrast, and step is as follows:
A.96 every hole adds the anti-E2 antibody (dilutability be 1:400) of 100 μ l with sodium bicarbonate buffer liquid (0.05M pH9.6) dilution in the ELISA Plate of hole, hatches 1-2 hour (or 4 ℃ spend the night) for 37 ℃.
B. cleansing solution (the PBS phosphate buffer of 0.1%Tween-20) washing is five times.
C. seal quilt: every hole adds 100 μ l confining liquids (the PBS phosphate buffer of 1%BSA), 4 ℃ of overnight incubation.
D. repeat the B step once.
E. every hole adds 100 μ l, third hepatopathy human serum or healthy human serum, hatches 1～2 hour for 26 ℃-37 ℃.
F. repeat the B step once.
G. the biotin labeled polypeptide (Beijing China big genome company synthetic) that every hole adds 100 μ l 10mg/L was hatched 1～2 hour for 26 ℃-37 ℃.
H. repeat the B step once.
I. every hole adds the Streptavidin (dilutability is 1:1000) of 100 μ l, 1 horseradish peroxidase-labeled, hatches 30 minutes for 26 ℃-37 ℃.
J. add the o-dihydroxy ammon colour developing, read OD value (see figure 9) under the microplate reader OD492nm.The result show biotin labeled polypeptide can specificity with the third hepatopathy human serum in virally directly combine, its OD value is greater than more than 2～3 times of the OD value that combines with normal human serum.Can reach the effect of viral level in the direct detection patient blood sample.
SEQUENCE LISTING
＜110〉Wuhan University
＜120〉hepatitis C virus envelope antigen ELISA kit and detection method
＜130〉hepatitis C virus envelope antigen ELISA kit and detection method
<160>4
<170>PatentIn version 3.1
<210>1
<211>12
<212>PRT
＜213〉synthetic
<400>1
<210>2
<211>12
<212>PRT
＜213〉synthetic
<400>2
<210>3
<211>12
<212>PRT
＜213〉synthetic
<400>3
<210>4
<211>12
<212>PRT
＜213〉synthetic
<400>4
Claims (2)
1, a kind of hepatitis C virus envelope antigen ELISA kit is characterized in that this kit comprises:
A. 12 peptides that can combine of mark such as biotin with HCV E2 protein-specific;
B. polypeptide is the sequence shown in SEQIDNO:1, SEQIDNO:2, SEQIDNO:3, the SEQIDNO:4;
C. the anti-E2 of rabbit polyclonal antibody;
D. the Streptavidin of horseradish peroxidase-labeled;
E. horseradish peroxidase substrate;
F. ELISA Plate, microplate reader;
G.E2 albumen is as the standard positive hole.
2, utilize the described a kind of kit of claim 1 preparing the method that detects hepatitis C virus envelope antigen ELISA kit, this detection method comprises the following steps:
A, every hole adds the rabbit anti-E2 antibody of 100 μ l with the dilution of sodium bicarbonate buffer liquid in 96 hole ELISA Plate, and dilutability is 1: 400, hatches for 37 ℃ to spend the night in 1-2 hour or 4 ℃, and PBS washing 3 times dries, and sodium bicarbonate buffer liquid is 0.05M, pH9.6;
B, cleansing solution washing five times, cleansing solution is the PBS phosphate buffer of 0.1%Tween-20;
C, envelope quilt: every hole adds 100 μ l confining liquids, 4 ℃ of overnight incubation, and confining liquid is the PBS phosphate buffer of 1%BSA;
D, repetition B step are once;
E, every hole add 100 μ l, third hepatopathy human serum or healthy human serum, hatch 1～2 hour for 26 ℃-37 ℃;
F, repetition B step are once;
The biotin labeled polypeptide room temperature that G, every hole add 100 μ l 10mg/L was hatched 1～2 hour for 26 ℃-37 ℃;
H, repetition B step are once;
I, every hole add the Streptavidin of 100 μ l horseradish peroxidase-labeled, and dilutability is 1:1000, hatch 30 minutes for 26 ℃-37 ℃, add the colour developing of horseradish peroxidase substrate o-dihydroxy ammon, read the OD value under the microplate reader OD492nm.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102043048A (en) * | 2010-09-29 | 2011-05-04 | 中国医学科学院医学生物学研究所 | HCV antigen detection board and method for detecting HCV antigen by using same |
| CN102081093A (en) * | 2009-11-30 | 2011-06-01 | 武汉大学 | Hepatitis C virus as well as kit of surface antigen and detection method thereof |
| CN102175872A (en) * | 2010-12-31 | 2011-09-07 | 石药集团中奇制药技术（石家庄）有限公司 | Enzyme immunoassay for quantitatively detecting Exendin-4 |
| CN103649316A (en) * | 2011-05-02 | 2014-03-19 | 约翰霍普金斯大学 | A synthetic hepatitis c genome and methods of making and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102081093A (en) * | 2009-11-30 | 2011-06-01 | 武汉大学 | Hepatitis C virus as well as kit of surface antigen and detection method thereof |
| CN102043048A (en) * | 2010-09-29 | 2011-05-04 | 中国医学科学院医学生物学研究所 | HCV antigen detection board and method for detecting HCV antigen by using same |
| CN102175872A (en) * | 2010-12-31 | 2011-09-07 | 石药集团中奇制药技术（石家庄）有限公司 | Enzyme immunoassay for quantitatively detecting Exendin-4 |
| CN102175872B (en) * | 2010-12-31 | 2014-03-05 | 石药集团中奇制药技术（石家庄）有限公司 | Enzyme immunoassay for quantitatively detecting Exendin-4 |
| CN103649316A (en) * | 2011-05-02 | 2014-03-19 | 约翰霍普金斯大学 | A synthetic hepatitis c genome and methods of making and use |
| CN111122880A (en) * | 2020-02-14 | 2020-05-08 | 烟台大学 | Competitive ELISA detection method for dolafectin and kit thereof |
| CN111122880B (en) * | 2020-02-14 | 2023-08-15 | 烟台大学 | Detection method of Du-raglutide competition ELISA and kit thereof |
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