CN101659975A - Preparation method of HRPII protein monoclonal antibody of plasmodium falciparum - Google Patents
Preparation method of HRPII protein monoclonal antibody of plasmodium falciparum Download PDFInfo
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Abstract
The invention relates to a preparation method of HRPII protein monoclonal antibody of plasmodium falciparum. The preparation method comprises the following steps of: adopting HRPII protein of plasmodium falciparum as target antigen and respectively analyzing and selecting two dominant antigen epitopes of A and B; respectively repeating the two dominant antigen epitopes of A and B, then continuously connecting four glycine and forming recombinant protein C; adopting most securest code of escherichia coli and converting the amino acid sequence of the recombinant protein C into corresponding nucleotide sequence; carrying out chemical synthesis to the former step to obtain the nucleotide sequence, and respectively adding enzyme cutting sites BamHI and EcoRI at the upstream and downstream thereof; inserting nucleotide fragment obtained by the former step into expression carrier PET-28a(+), constructing recombinant protein C expression carrier and inducing to express the recombinant proteinC in the escherichia coli BL21 (DE3); carrying out ultrasonic bacteria breaking and low-temperature centrifugation, then taking supernatant of the solution, affining a chromatographic column by nickel-agarose, eluting and obtaining purified recombinant protein C; after immunizing Balb/c mouse with the recombinant protein C for a plurality of times, taking and fusing spleen cells with sp2/0 myelomacells, and obtaining six hybridoma cell lines by multiple rounds of screening; and purifying monoclonal antibody, respectively marking horse radish peroxidase and prorating matching and combination of optimum monoclonal antibody by ELISA orthogonal experiment.
Description
Technical field
The present invention relates to the method, plasmodium falciparum HRPII albumen dominant antigen epi-position MONOCLONAL ANTIBODIES SPECIFIC FOR of plasmodium falciparum HRPII albumen dominant antigen epi-position tandem expression and detect a kind of preparation method of HRPII protein monoclonal antibody of plasmodium falciparum of the method for plasmodium falciparum with enzyme-linked immunosorbent assay, belong to biotechnology and make the field.
Background technology
At present, owing to be subjected to the influence of various factors, up to now, effectively malaria vaccine does not also drop into actual the use really, realizes that therefore the quick diagnosis of malaria and early stage treatment seem particularly important.Though traditional thick thin smear film smear for microscopic examination method is special, waste time and energy, and the operator that need be skilled in technique, so this method is not suitable for the quick diagnosis of malaria.Buffy coat quantitative method susceptibility is better than thick thin smear film smear for microscopic examination method greatly, but its employed consumption just costs an arm and a leg, and is not suitable for clinical large-scale promotion.In recent years, along with developing rapidly of molecular biology and Measurement for Biochemistry, zymetology method, quantitative fluorescent PCR and immunochromatographic method having occurred is the diagnostic techniques of representative.Wherein the zymetology method detects responsively, and accuracy is higher, but needs specific apparatus such as spectrophotometer, has hindered this method and has further popularized.Quantitative fluorescent PCR is according to plasmodial conserved sequence design primer, specific amplification fragment, mark fluorescent.This method is extremely sensitive, and realizes the difference of plasmodium kind.But this method is higher to technical requirements, needs professional's operation.At present, based on the serology detection of immunology, because it is easy and simple to handle, the result is easy to judge, has become the main flow direction of malaria diagnosis.This method mainly is to utilize monoclonal antibody to detect plasmodial specific antigens, and the tested antigen of report mainly is rich histidine protein (HRPII) at present, as ParaSight-F (dipstick) diagnostic kit of world health organisation recommendations.The conventional employed immunogen of HRPII Monoclonal Antibody is the HRPII intact proteins that genetic engineering technique is expressed, because the cause of base codon, this albumen is in the expression in escherichia coli difficulty, and expression product is based on inclusion body, and this makes that the preparation of monoclonal antibody is difficult.In addition, because the homology of epitope, the monoclonal antibody of using the HRPII intact proteins to prepare also may be discerned other albumen, as Rheumatoid factors, polyclonal, thereby causes the detected result false positive.
Summary of the invention
Purpose of design: avoid the weak point in the background technology, design a kind of can either specific recognition plasmodium falciparum HRPII albumen, can not cause the preparation method of a kind of HRPII protein monoclonal antibody of plasmodium falciparum of detected result distortion again.
Design: in order to realize above-mentioned purpose of design.The application: (1) is target antigen with plasmodium falciparum HRPII albumen, analyzes and selects A, two specificity dominant antigens of B epi-position, and the sequence comparative result shows that two epitopes of A, B and other protein sequence do not have obvious homology.(2) in order to strengthen of the stimulation of two dominant antigen epi-positions of A, B to Balb/c mouse immune system, promote two epitopes of A, B to produce corresponding monoclonal antibody, connect by soft segment (continuous four glycine) so respectively two dominant antigen epitope sequences of A, B are repeated the back, form the recombinant protein c aminoacid sequence.(3) adopt intestinal bacteria to have a liking for codon most, the recombinant protein c aminoacid sequence is converted to corresponding nucleotide sequences, be beneficial to the expression of target protein in intestinal bacteria.(4) the nucleotide sequence chemosynthesis that previous step is obtained is suddenly cut connection by enzyme, and the synthetic nucleotide fragments that obtains is inserted expression vector PET-28a (+), makes up the recombinant protein c expression vector.(5) recombinant protein c expression vector transformed into escherichia coli ER2566 competent cell, screening obtains the recombinant protein c expression strain.(6) after the recombinant protein c expression strain large scale culturing, behind carrying out ultrasonic bacteria breaking and the low-temperature centrifugation, get the solution supernatant by nickel agarose affinity chromatography post affinity chromatography, wash-out obtains purification of recombinant proteins C.(7) behind the repeatedly immune Balb/c mouse of the recombinant protein c behind the purifying, get its spleen cell and sp2/0 myeloma cell and merge, finally obtain 6 strain of hybridoma strains through multi-turns screen.(8) 6 strain of hybridoma strains are prepared the Balb/c mouse ascites respectively, use sad-ammonium sulfate method and Protein G monoclonal antibody purification in two steps, and difference mark horseradish peroxidase (HRP).(9) to obtain 2A2 monoclonal antibody bag be best of breed by detecting subtertian malaria with the 6E8-HRP pairing for ELISA orthogonal experiment screening.
The application compares with background technology, the one, pass through Protocols in Molecular Biology, realized repeating and tandem expression of plasmodium falciparum HRPII albumin A, B dominant antigen epi-position, strengthened of the stimulation of purpose epitope, got rid of the interference that irrelevant sequence may be brought the mouse immune system; The 2nd, only contain the distinctive A of plasmodium falciparum HRPII albumen, B dominant antigen epi-position as immunogenic recombinant protein c, guaranteed the monoclonal antibody specificity height that finally obtains; The 3rd, adopt intestinal bacteria to have a liking for codon optimized nucleotide sequence most, improved the expression level of recombinant protein c in intestinal bacteria greatly.
Embodiment
Embodiment 1: plasmodium falciparum HRPII dominant antigen epi-position is selected.
With plasmodium falciparum HRPII albumen is target antigen, utilizes biosoftware DNAssist2.0 to analyze the wetting ability and the antigenicity of its aminoacid sequence, selects two dominant antigen epi-positions of A, B.The sequence comparative result shows selected A, two dominant antigen epitope sequences of B specificity height, does not have obvious homology with other protein sequence.
A：LeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHis
B：AspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHis。
Embodiment 2: the series connection of plasmodium falciparum HRPII dominant antigen epi-position.
In order to strengthen of the stimulation of purpose epitope to the mouse immune system, connect by soft segment (continuous four glycine) again after plasmodium falciparum HRPII albumin A, two dominant antigen epitope sequences of B are repeated respectively, obtain the recombinant protein c aminoacid sequence, its concrete sequence is as follows:
LeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHisLeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHisGlyGlyGlyGlyAspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHisAspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHis。
Embodiment 3: the nucleotide sequence of optimizing the coding recombinant protein c.
In order to improve the expression amount of recombinant protein c, under the constant prerequisite of recombinant protein c aminoacid sequence, adopt intestinal bacteria to have a liking for will the encode nucleotide sequence optimization of recombinant protein c of codon most, optimized nucleotide sequence is as follows:
CTGAACCTGAACAAACGCCTGCTGCATGAAACCCAGGCCCATGTGGATGATGCCCATCATGCCCATCATGTGGCCGATGCCCATCATGCCCATCTGAACCTGAACAAACGCCTGCTGCATGAAACCCAGGCCCATGTGGATGATGCCCATCATGCCCATCATGTGGCCGATGCCCATCATGCCCATGGCGGCGGCGGCGATGCCCGCCATGCCACCGATGCCCATCATGCCGCCGATGCCCATCATGCCACCGATGCCCATGATGCCCGCCATGCCACCGATGCCCATCATGCCGCCGATGCCCATCATGCCACCGATGCCCAT
Embodiment 4: the nucleotide sequence of composite coding recombinant protein c.
The nucleotide sequence of the coding recombinant protein c that the trust Nanjing chemosynthesis embodiment of Jin Site company 3 obtains, and the downstream is added restriction enzyme site BamHI and EcoRI corresponding nucleotide sequences respectively thereon, concrete sequence is respectively: GGATCC and GAATTC.
Embodiment 5: make up the recombinant protein c expression vector establishment.
Use restriction enzyme BamHI and EcoRI in 37 ℃ of double digestion synthetic recombinant protein c nucleotide sequences and PET-28a (+) carrier 12s hour respectively, enzyme is cut product row 1% agarose gel electrophoresis respectively, and glue reclaims test kit (ancient cooking vessel Bioisystech Co., Ltd in the Ningbo) and reclaims recombinant protein c nucleotide fragments and PET-28a (+) carrier.Use the T4 ligase enzyme with the recombinant protein c nucleotide fragments that reclaims and PET-28a (+) carrier in 4 ℃ spend the night be connected after, connect product and transform DH5 α competent cell, and coat the LB flat board that contains that penicillin resistance of card (50 μ g/mL), in 37 ℃ of incubated overnight.Second day, the mono-clonal bacterial strain is to the LB liquid nutrient medium that contains that penicillin resistance of card (50 μ g/mL) on the picking flat board, and 37 ℃ of constant temperature shaking tables are cultivated after 12 hours the alkaline lysis method of extracting plasmid, after BamHI and the evaluation of EcoRI double digestion, obtain positive recombinant plasmid, called after PET-28a (+)-C.
Embodiment 6: make up the recombinant protein c expression strain.
With recombinant expression vector PET-28a (+)-C Transformed E .coli ER2566 competent cell that builds, and coat the LB flat board that contains that penicillin resistance of card (50 μ g/mL), in 37 ℃ of incubated overnight.Second day, the mono-clonal bacterial strain is to the LB liquid nutrient medium that contains that penicillin resistance of card (50 μ g/mL) on the picking flat board, 37 ℃ of constant temperature shaking tables were cultivated after 8 hours, added inductor isopropylthio-abduction delivering and prepared the protein electrophoresis sample after 4 hours.The polyacrylamide gel electrophoresis result shows the recombinant protein c successful expression, obtains the recombinant protein c expression strain.
Embodiment 7: purification of recombinant proteins C.
Inoculation recombinant protein c expression strain is to the LB liquid nutrient medium, Jia Kana penicillin to final concentration is 50 μ g/mL, 37 ℃ of constant temperature shaking tables were cultivated after 8 hours, with the LB liquid nutrient medium that contains 50 those penicillin of μ g/mL card with this bacterium by after 1: 100 dilution proportion, divide and be filled in the bacteria culture bottle, put 37 ℃ of constant temperature shaking tables and be cultured to OD600=0.8, adding inductor isopropylthio-to final concentration is 1.0mmol/L, continues to cultivate and induces 4 hours.Behind the centrifugal collection thalline, the low temperature carrying out ultrasonic bacteria breaking is got supernatant by nickel agarose affinity chromatography post behind the low-temperature centrifugation, finally obtain purification of recombinant proteins C through washing, wash-out.
Embodiment 8: the acquisition of hybridoma cell strain.
Get female Balb/c mouse in 6-8 age in week, the subcutaneous multi-point injection Fu Shi of every mouse of fundamental immunity Freund's complete adjuvant emulsive 50 μ g recombinant protein cs.Carry out booster immunization after 15 days, method is after getting the recombinant protein c usefulness freund 's incomplete adjuvant emulsification of same amount, subcutaneous multi-point injection.After 30 days, get 15 μ g recombinant protein c tail vein booster shots, and after after the booster shots of tail vein 72 hours, eye socket is got blood, and puts to death mouse, gets its spleen and prepares cell suspension, cell counting, get the good sp2/0 murine myeloma cell of growth conditions by 1/5 in the quantity of splenocyte, mixed centrifugal after, add polyoxyethylene glycol (PEG-4000) and make the two fusion.In addition, add isopyknic feeder cell again, the 96 porocyte plates that are placed in behind the mixing (200 μ L/ hole) are cultivated in 5% CO2gas incubator.After 5 days, half keeps and changes liquid, after 10 days, by microtiter plate (80ng/ hole), utilizes indirect enzyme-linked immunosorbent assay to detect Hybridoma Cell Culture supernatant in the 96 porocyte culture plates with recombinant protein c dilution back bag.For detecting the male hybridoma cell clone, re-use limiting dilution assay and carry out subclone.Through three subclones, screening obtains 6 strain of hybridoma strains (2A2,3E5,5B2,6D7,6E8,7A3) altogether.
Embodiment 9: a large amount of preparations and the purifying of monoclonal antibody.
Get the 8-10 healthy Balb/c mouse in age in week, abdominal injection pristane, every 200 μ L, 7 days pneumoretroperitoneum injection hybridomas (about 1 * 10
6Individual/only), after 15 days, mouse web portion is heaved, and collects ascites, and centrifugal 10 minutes of 3000rpm collects supernatant, adds 60mmol/L pH4.0 acetate buffer solution in 1: 4 ratio, transfers pH4.5 with 100mmol/L NaOH.Add the ratio of 25 μ L n-caprylic acid with every milliliter of ascites supernatant, slowly add n-caprylic acid under the magnetic agitation condition, after continuing at ambient temperature to stir 30min, centrifugal 30 minutes of 4 ℃ of 5000rpm abandon precipitation.With supernatant liquor precooling under 4 ℃ of conditions, the ratio that adds 0.227g ammonium sulfate with 1mL volume supernatant liquor, slowly add the ammonium sulfate powder, the limit edged stirs, room temperature condition continues down to stir after the 30min, centrifugal 15 minutes of 5000rpm, precipitation is dissolved in 10mmol/L PBS (pH7.4) damping fluid of 1/10 volume, and dialysis is to there not being ammonium sulfate.With Protein G adsorption and purification, elution peak is collected in the washing back with the monoclonal antibody after the dialysis, promptly obtains the monoclonal antibody behind the purifying.
The preparation of embodiment 10:HRP mark monoclonal antibody.
Get 10mg HRP and add 0.1mol/L sodium-acetate 2mL, fully mixing adds 0.08mol/L NaIO after about 5 minutes
4Solution 1mL, behind the mixing, room temperature reaction 20 minutes.Add 0.4mol/L ethylene glycol solution 0.5mL, after room temperature leaves standstill 30 minutes, add 21%NaCl solution 0.3mL, add the ice-cold dehydrated alcohol precipitation hydroformylation enzyme of 1.2mL again, after the centrifugal supernatant that goes, precipitation enzyme wash once with 6mL 80% ice-cold alcohol solution dipping again, the centrifugal removal ethanol that inclines).Precipitation is dissolved with 0.05mol/L carbonate buffer solution (pH9.6) 2mL, and monoclonal antibody 20mg stirs then, spends the night in 4 ℃.Add 10mg NaBH next day
4Mixing, react and add equivalent saturated ammonium sulphate enzyme conjugates after 3 hours, 4 ℃ of stirring reactions 30 minutes, centrifugal minute of 4 ℃ of 5000rpm, abandon supernatant, precipitation is put and is used 0.01mol/L PBS in 4 ℃ of dialyzed overnights in the dialysis tubing with 0.01mol/L PBS 3mL dissolving, add the aseptic glycerine of 3mL, behind the mixing in-20 ℃ of preservations.Respectively 2A2,3E5,5B2,6D7,6E8,7A3 monoclonal antibody are carried out the HRP mark with aforesaid method.
Embodiment 11: the screening of pairing monoclonal antibody.
The bag quilt:
2A2,3E5,5B2,6D7,6E8,7A3 use 50mmol/L carbonate buffer solution (pH9.6) dilution respectively, and final concentration is 0.8 μ g/mL, the every hole 100 μ l of 96 hole enzyme plates, and 4 ℃ are spent the night.
Sealing:
The PBST washings (0.01mol/mL PBS pH7.4,0.1%Tween20) after twice, every hole add 200 μ l confining liquids (0.01mol/mL PBS pH7.4,2%BSA), 37 ℃ of sealings 2 hours.
Application of sample:
Get positive clinical serum specimen of subtertian malaria and the negative clinical serum specimen of subtertian malaria, every hole adds 100 μ l, behind the vibration mixing, in 37 ℃ of reactions 1 hour.
Enzyme-added:
(0.01mol/mL PBS pH7.4,0.1%Tween20) after five times, every hole adds 100 μ l, and (0.01mol/mL PBS pH7.4 is 2%BSA) with the HRP mark monoclonal antibody of dilution in 1: 1000, in 37 ℃ of reactions 30 minutes with the enzyme diluent for the PBST washings.
Colour developing:
(0.01mol/mL PBS pH7.4,0.1%Tween20) after five times, every hole added each 50 μ l of A, B colour developing liquid to the PBST washings, in 37 ℃ of reactions 10 minutes.
Stop:
Every hole adds 2M H
2SO
450 μ l detect the A450/630 value in each hole in microplate reader behind the mixing.
Match with enzyme mark monoclonal antibody so that each Sheet of aforesaid method quadrature detection is anti-, ask P/N value (positive sample detects average and negative sample detects average ratio) value, see Table 1.
Each monoclonal antibody of table 1 and enzyme mark monoclonal antibody P/N primary system meter
By last table as can be known, 2A2 monoclonal antibody bag is a best of breed by detecting subtertian malaria with the 6E8-HRP pairing.
What need understand is: though the foregoing description is to the mentality of designing of the present invention detailed text description of contrasting; but these text descriptions; just the simple text of mentality of designing of the present invention is described; rather than to the restriction of mentality of designing of the present invention; any combination, increase or modification that does not exceed mentality of designing of the present invention all drops in protection scope of the present invention.
Claims (1)
1, a kind of preparation method of HRPII protein monoclonal antibody of plasmodium falciparum is characterized in that:
(1) be target antigen with plasmodium falciparum HRPII albumen, analyze and select A, two antigen advantages of B epi-position that the concrete sequence of its amino acid is as follows,
A：LeuAsnLeuAsnLysArgLeuLeuHisGluThrGlnAlaHisValAspAspAlaHisHisAlaHisHisValAlaAspAlaHisHisAlaHis、
B：AspAlaArgHisAlaThrAspAlaHisHisAlaAlaAspAlaHisHisAlaThrAspAlaHis；
(2) respectively two dominant antigen epitope sequences of A, B are repeated the back and connect, form the recombinant protein c aminoacid sequence by continuous four glycine;
(3) adopt intestinal bacteria to have a liking for codon most, the recombinant protein c aminoacid sequence is converted to corresponding nucleotide sequences, be beneficial to the expression of target protein in intestinal bacteria;
(4) nucleotide sequence that chemosynthesis previous step obtains is suddenly cut connection by enzyme, and the synthetic nucleotide fragments that obtains of previous step is inserted expression vector PET-28a (+), makes up the recombinant protein c expression vector;
(5) recombinant protein c expression vector transformed into escherichia coli ER2566 competent cell, screening obtains the recombinant protein c expression strain;
(6) after the recombinant protein c expression strain large scale culturing, carrying out ultrasonic bacteria breaking and low-temperature centrifugation are got the solution supernatant by nickel agarose affinity chromatography post, and wash-out obtains purification of recombinant proteins C;
(7) behind the repeatedly immune Balb/c mouse of recombinant protein c, get its spleen cell and sp2/0 myeloma cell and merge, finally obtain 6 strain of hybridoma strains through multi-turns screen;
(8) purified monoclonal antibody and mark horseradish peroxidase (HRP) respectively, the ELISA orthogonal experiment is determined the optimum monoclonal antibody combinations of pairs.
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