CN105944093B - The purposes of Periodic Brugia M29 epitope gene DNA vaccination - Google Patents

The purposes of Periodic Brugia M29 epitope gene DNA vaccination Download PDF

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CN105944093B
CN105944093B CN201610398014.2A CN201610398014A CN105944093B CN 105944093 B CN105944093 B CN 105944093B CN 201610398014 A CN201610398014 A CN 201610398014A CN 105944093 B CN105944093 B CN 105944093B
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方政
吴佳倩
徐倩
方浩
张波
陆施娟
陶然
刘芹
谢东方
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Hai'an Baiyou Matrix Bioengineering Co ltd
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Abstract

The invention discloses a kind of purposes of Periodic Brugia M29 epitope gene DNA vaccination, using the Periodic Brugia of China's prevalence as research object, Wuchereria malayi myosin genetic fragment is expanded, Periodic Brugia myosin Eukaryotic expression recombinant plasmid is constructed.It is analyzed according to sequencing result, thus it is speculated that then its amino acid sequence utilizes relevant software prediction their B cell epitope and t cell epitope, prepares Periodic Brugia myosin epitope gene vaccine.By the vaccine immunization BALB/c mouse, and detect the humoral immunity of its vaccine and the response effect of cellular immunity.The experiment results proved vaccine can induce the immune response that mouse generates specificity, obtain promising result, and manufacturing cycle type Wuchereria malayi myosin epitope gene vaccine succeeds.

Description

The purposes of Periodic Brugia M29 epitope gene DNA vaccination
The application is application number: 201410564361.9, applying date 2014.10.21, title " Periodic Brugia M29 The divisional application of the preparation method of epitope gene DNA vaccination ".
Technical field
The present invention relates to a kind of preparation methods of Periodic Brugia M29 epitope gene DNA vaccination.
Background technique
Filariasis is most ancient one of the disease in the whole world.According to the Gu Ai before 4000 of Cairo, EGY Museum Exhibit And Pharaoh's statue, display suffer from right lower extremity elephant hide swell, as one of the disease typical clinical manifestations.According to WHO report, the whole world has 83 Countries and regions share lymph filarial infection person about 1.2 hundred million, wherein bancroftosis about 1.07 hundred million, wuchereria malayi filariasis and Timor's silk Parasitosis total about 13,000,000.The clinical manifestation of about 4400 universal Filariasis in these the infecteds, about 76,000,000 be microfilaremia Disease person.It is more than 1,100,000,000 that, which there is compromised population in the estimation whole world, accounts for about the 20% of total world population.China was once global Filariasis Popular one of the countries with the most serious ..., compromised population is up to 3.3 hundred million.Although the prevention and treatment of filariasis achieves huge in the world Achievement, but since climate warming etc. is natural, the nocturnal periodicity and microfilaremia of biology and society and lymph filaria There is epidemic situation re-ignition in some countries and regions in recent years and becomes with what is spread in mostly being widely present without factors such as clinical symptoms The prevalence of gesture, filariasis is not only seriously endangered to the health care belt of the people, also becomes the one of the major reasons driven into poverty by medical crises.It is newest Research report shows that intermediate density and higher density are micro- the microfilaraemia of the infected's Residual source of infection sustainable 20 years or more Silk larva of a tapeworm or the cercaria of a schistosome mass formed by blood stasis person has the function of propagating filariasis.So the monitoring in later period and prevention and treatment task are still very arduous.
In mainly bancroft's filaria and the Wuchereria malayi that China is popular.The latter's history of life is more complex, mainly includes that larva exists In mosquito body and adult is in the intracorporal growth course of people, can additionally reach maturity in a variety of vertebrate bodies other than human body. Filariasis can lead to patient permanently or disable for a long time, be classified as the second-biggest-in-the-world disease act of violence that disables by WHO.Find control filariasis The research of vaccine is increasingly paid attention to by people.It screens being effectively protected property antigen and organically combines to obtain best protection effect It is an important content of vaccine research.Myosin (myosin) is a kind of function egg being widely present in eucaryote It is white, be not only present in myocyte, and in non-myocyte there is also.Its contractile protein important as one kind of organism Matter plays an important role in terms of adjusting contraction of muscle and cell.Molecular biology in relation to helminth myosin With immunology aspect research shows that it is a kind of dominant molecule of early immune, there is more significant immunogenicity, animal is immunized It can produce stronger immanoprotection action.This is also the reason of researcher is dedicated to myosin molecular vaccine and drug research.
Summary of the invention
Easy, easy to operate, the good Periodic Brugia M29 epitope of effect that the purpose of the present invention is to provide a kind of methods The preparation method of gene DNA vaccine.
The technical solution of the invention is as follows:
A kind of preparation method of Periodic Brugia M29 epitope gene DNA vaccination, it is characterized in that: including
The following steps:
(1) design of primers
According to Periodic Brugia myosin gene (highly conserved sequence area, using Primer in GenBank Premier5.0 software Design primers, and I/BamH of Xho, I restriction enzyme site is introduced respectively, originate termination codon and protectiveness Base；
P1: 5 '-CC of upstreamGGA TCC ATG AAA TGC AAA AAA T‐3’
Underlined sequences are I restriction enzyme site Tm=56.84 of BamH
P2: 5 '-CC of downstreamCTC GAG ATG GTG AAC GGA GTA CG‐3’
Underlined sequences are I restriction enzyme site Tm=66.90 of Xho
(2) collection of Periodic Brugia polypide and microfilaria and the extraction of total serum IgE and measurement
(1) adult for being derived from meriones unguiculatus peritoneal fluid and microfilaria are moved into 1.5ml homogenizer, 1mlTrizol is added Reagent grinding, is stored at room temperature 5min；
(2) 0.2ml chloroform is added, stands 2min after shaking 15s, 4 DEG C, 12000rpm centrifugation 15min takes supernatant；
(3) 0.5ml isopropanol is added to mix, is stored at room temperature 10min, 4 DEG C, 12000rpm is centrifuged 10min and abandons supernatant；
(4) 75% ethyl alcohol of 1ml is added to mix, 4 DEG C, 9000rpm is centrifuged 5min and abandons supernatant；
(5) DEPC processing water 30ul, 60 DEG C of 10min are dissolved in after dry 5min；
(6) it takes 2ul total serum IgE to dilute 200 times, OD260 and OD280 value is surveyed on ultraviolet specrophotometer, determines its concentration And purity, and carry out formaldehyde electrophoresis detection；
(3) synthesis of cDNA
(1) total serum IgE 5ul is taken, it is rear that 10mM dNTPmix 1ul, 0.5ug/ul oligo (dT) 12-18ul, DEPC is added Handle water 3ul, 65 DEG C of heating 5min, rear ice bath 5min；
(2) 10 × Buffer 2ul is separately taken, 25mM MgCl2 4ul, 0.1M DTT 2ul, RNase inhibitor 1ul are mixed, 42 DEG C of incubation 2min；
(3) step (2) mixed liquor is added in step (1) substance and is mixed, add 1ul reverse transcriptase Superscript II mixes；42 DEG C of incubations 50min, 70 DEG C of 15min terminate reaction, and -70 DEG C save backup；
(4) PCR amplification Bm-myosin gene
P1=56.84, P2=66.90, reaction system cDNA 1.5ul, 10 × PCR Buffer 2.5ul, 2.5mM DNTPmix 4.0ul, P1 P2 each 0.7ul, 5U/ul TaqDNA polymeric protease 0.2ul, ddH2O 15.4ul；It mixes in DNA PCR is carried out on amplification instrument PTC-100；Loop parameter are as follows: 94 DEG C of 3min, 94 DEG C of 50s, 51 DEG C of 45s, 72 DEG C of 90s, totally 33 are followed Ring, last time recycle 72 DEG C of extension times as 7min, and 4 DEG C of coolings terminate reaction；Negative control is set up simultaneously.PCR is produced Object is identified through 1.0% agarose gel electrophoresis；
(5) connection and conversion of Bm-myosin genetic fragment purifying and carrier T
(1) it purifies Bm-myosin genetic fragment: taking 50ulPCR product through 1.0% agarose gel electrophoresis, in ultraviolet lamp Lower gel of the cutting containing target gene is illustrated, purification and recovery product by plastic recovery kit；
(2) preparation of competent E.coli: bacillus coli DH 5 alpha 37 DEG C of overnight incubations on fresh LB agar plate；It chooses Single bacterium is taken to fall in 3ml LB culture medium, 37 DEG C of shaken overnights；300ul bacterium solution is taken to be added in 30ml LB culture medium, 37 DEG C 220rpm vibrate 4h, ice bath 1h, after be dispensed into 4 DEG C of 1.5ml Eppendorf pipe from 8000rpm 5min；Supernatant is abandoned, ice is added It bathes 0.1mol/L MgCl2 100ul to suspend, 4 DEG C of centrifugations, 8000rpm 5min abandons supernatant, and ice bath 0.1mol/L is added CaCl2- 10% glycerite 300ul suspends, and -70 DEG C save backup；
(3) connection of Bm-myosin gene and pGEM-T carrier: taking the PCR product 3ul of purifying, and 2 × quick connect buffers Liquid 5ul, pGEM-T carrier 1ul, T4DNA ligase 3weiss/ul 1ul, total 10ul mixing, 4 DEG C of connection 20h；
(4) is converted: taking ligation reaction 10ul that freshly prepared bacillus coli DH 5 alpha competent cell 300ul is added mixed Even, ice bath 30min, 42 DEG C of heat shock 90s are quickly moved to ice bath 3min, and 200ul LB culture medium, 37 DEG C of incubation 45min are added. Culture 100ul is coated in containing X-gal, IPTG, on 1.5% agar plate of Amp, is inverted plate, 37 DEG C are incubated overnight；
(6) screening and identification of Positive recombinant clones
(1) screening of Positive recombinant clones: the good white bacterial plaque of picking growth conditions, and extract plasmid；
(2) PCR is identified: using recombinant plasmid Bm-myosin/pGEM-T as template, with primer P1, P2 of Bm-myosin into Row PCR reaction, reaction system 25ul, Bm-myosin/pGEM-T recombinant plasmid 1.5ul, 10 × PCR Buffer 2.5ul, 2.5mM dNTPmix4.0ul, P1, P2 each 0.7ul, 5U/ul TaqDNA polymeric protease 0.2ul, ddH2O 15.4ul；Circulation Parameter are as follows: 94 DEG C of 3min, 94 DEG C of 50s, 51 DEG C of 45s, 72 DEG C of 90s, totally 33 recycle, when last time recycles 72 DEG C of extensions Between be 7min, 4 DEG C cooling to terminate reaction；PCR product is identified through 1.0% agarose gel electrophoresis；
(3) digestion is identified: the PCR that learns from else's experience identifies correct recombinant plasmid, with BamH I and I double digestion of Xho, with 1.0% fine jade Sepharose electrophoresis detection endonuclease bamhi；
(7) Bm-MYOSIN secondary structure prediction
Respectively using SOPMA, GOR, nnPredic, HNN method and the analysis of EMBOSS program on EXPASY server Predict the secondary structure of Bm-myosin；
(8) Bm-MYOSIN hydrophily, surface accessibility, antigenicity, polarity and flexibility parameter prediction
Using Hopp&Woods hydrophilic parameter, the surface Emini accessibility parameter, Jameson-Wolf antigenicity parameter, Zimmerman polarity parameters and flexibility parameter prediction；
(9) Bm-MYOSIN t cell epitope is predicted
(1) prediction of source of people HLA class Ⅰmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server selects AA length for 9-mers, genotype HLA-A0201, HLA-A1101, predicts HLA class Ⅰmolecule Binding peptide retains the result of output；
(2) prediction of source of mouse MHC class Ⅰmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server selects AA length for 9-mers, genotype H2-d, including H2-Kd, H2-Ld and H2-Dd, is H2-d type The MHC class Ⅰmolecule of mouse expression；It predicts MHC class Ⅰmolecule binding peptide, retains the result of output；
(3) prediction of source of people HLA class Ⅱmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server, selects genotype for HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1*0401, HLA-DRB1* 0701, HLA-DRB1*0801, HLA-DRB1*0901, HLA-DRB1*1101, HLA-DRB1*1301, HLA-DRB1*1501, in advance HLA class Ⅱmolecule binding peptide is surveyed, the result of output is retained；
(4) prediction of source of mouse MHC class Ⅱmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server selects genotype for I-Ad, I-Ed, predicts source of mouse MHC class Ⅱmolecule binding peptide, retains output As a result；
(10) selection of epitope gene segment
According to B cell Antigen Epitope Prediction and t cell epitope prediction result, selection is rich in the genetic fragment 562bp- of epitope The segment using plasmid pcDNA3.1 (+)-BmM55 as template, and is cloned into prokaryotic expression carrier pET- by 1269bp design primer In 28a (+), pET-BmM29 is constructed.Using 5 software Design primers of Primer；
Upstream P1:5 '-GCGGATCCGCTAATGAATTGAATATGC-3 '
Wherein GGATCC is I restriction enzyme site of BamH；
Downstream P2:5 '-CGGAATTCCTATGGTGAACGGAGTACGGT
Wherein GAATTC is I restriction enzyme site of EcoR；
Primer is dissolved using preceding with ddH2O, and concentration is 10 μm of ol/L；
(11) epitope gene segment PCR amplification
(1) using pcDNA3.1 (+)-BmM55 as template, P1, P2 are respectively upstream and downstream primer, reaction system:
Pcr amplification reaction system
Amplified reaction parameter are as follows:
5 μ l samples are taken to detect PCR amplification target gene with 1% agarose gel electrophoresis；
(12) target gene is connect and identification with carrier
A, PCR product and pET-28a (+) vector plasmid double digestion system:
Double enzyme digestion reaction system
Target gene is connect with carrier, specific reaction system:
Target gene and carrier coupled reaction system
It mixes, reacts at room temperature 30min or more, product is for converting；
B, the preparation of E.coli DH5 α competent cell
(1) it is gone bail for oese and is stored in -80 DEG C of DH5 α bacterium solutions, draw bacterium on not antibiotic LB plate, 37 DEG C are inverted About 14-16h is cultivated to single colonie appearance；
(2) the LB liquid that single bacterium drops down onto 5ml non-resistant, 37 DEG C of shaken cultivation about 10-12h are chosen；
(3) for the bacterium solution for taking 1ml to be incubated overnight to containing in 100ml nonreactive LB liquid, 37 DEG C of shaken cultivation about 2-2.5h arrive OD value Up to 0.5, ice bath 5-10min；
(4) 2 50ml are dispensed into, sterile centrifuge tube is pre-chilled, 4 DEG C, 1600rpm is centrifuged 7min；
(5) cell is resuspended with 10ml ice-cold 0.1M CaCl2 solution, 4 DEG C, 1100rpm is centrifuged 5min；
(6) cell is resuspended with 10ml ice-cold 0.1M CaCl2 solution, after placing 30min on ice, 4 DEG C, 1100rpm centrifugation 5min；
(7) the 0.1M CaCl ice-cold with 2ml2Cell is resuspended in solution, and 15% glycerol freezes in -80 DEG C after packing；
C, conversion and identification
(1) 100 μ l are taken to be stored in -80 DEG C of calcification bacterium, ice bath melts；10 μ l connection products are added, mix gently, ice bath 30min；
(2) heat shock, 42 DEG C of water-bath 30-90s, is not moved；
(3) it is quickly transferred in ice bath, cooling 1-2min；
(4) plus 2.25ml LB liquid is to test tube, then plus 0.25ml mixture, slant setting, 37 DEG C, 100rpm shaken cultivation 45min；
(5) 5000rpm is centrifuged 1min；
(6) part supernatant is abandoned, is poured on after mixing on the LB plate containing Amp, is applied uniformly, is stored at room temperature with spreader, until Liquid is absorbed, and 37 DEG C of incubators are inverted culture 12-16h；
(7) LB liquid of the monoclonal to 5ml containing Amp is chosen with white pipette tips, 37 DEG C, 220rpm shaken cultivation 12-16h, until OD value Between 0.6-0.8；
(8) positive control is done by above-mentioned steps simultaneously, negative control transformation reacts；
(9) it extracts plasmid and carries out plasmid electroresis appraisal and PCR identification；
(10) it chooses PCR and identifies positive bacterium solution, be sequenced after being expanded in LB liquid medium；
(13) BmM29 epitope gene construction of eukaryotic expression vector
It purifies purpose epitope gene and is subcloned to gram of the BamH I and EcoR I of eukaryotic expression vector pcDNA3.1 (+) Grand site.The molar ratio of target gene and carrier is 5:1, and reaction system 25ul, 16 DEG C of water-bath connections are overnight；Take connection liquid conversion Competent E.coli DH5 α, is coated on LB-Amp plate, wherein containing Amp100mg in every mlLB, 37 DEG C are incubated overnight；From Random picking single bacterium colony, is inoculated in respectively in LB-Amp culture medium on LB-Amp plate, 37 DEG C of shaken overnight cultures, extracts weight Group plasmid, through I digestion of BamH I and EcoR, rear 1.0% agarose gel electrophoresis identification；
The test of (14) BmM29 epitope gene vaccine immune response
A.pcDNA3.1 (+) and pcDNA3.1 (+)-BmM29 plasmid largely extract
(1) 50ml centrifuge tube is added in the bacterium solution for taking 100ml to be incubated overnight, and 12000rpm is centrifuged 3min and collects bacterium, as far as possible Abandon supernatant to the greatest extent；
(2) 12000rpm is centrifuged 3min, and remaining a small amount of liquid is sucked out with pipettor；
(3) solution 1 of a large amount of extraction agent boxes of 10ml plasmid is added, it is outstanding by cell using turbula shaker and pipettor It is floating, no small bacteria block；
(4) solution 2 of a large amount of extraction agent boxes of 10ml plasmid is added, leniently spins upside down 6-8 times, is stored at room temperature immediately 5min；
(5) solution 4 of a large amount of extraction agent boxes of 10ml plasmid is added, leniently spins upside down 6-8 times, is stored at room temperature immediately 10min, 12000rpm are centrifuged 20min, and solution is all poured into Filter column, slowly push push handle, filtrate collect 50ml from In heart pipe；
(6) add 8ml isopropanol into filtrate, adsorption column is transferred to after mixing, 12000rpm is centrifuged 2min, abandons in collecting pipe Waste liquid；
(7) add 3ml to remove endotoxin buffer into adsorption column, be stored at room temperature 2min, 12000rpm is centrifuged 2min, abandons and collects Waste liquid in pipe；
(8) rinsing liquid W2 is added into adsorption column, 12000rpm is centrifuged 2min, abandons the waste liquid in collecting pipe.It is repeated once；
(9) adsorption column is placed back in into collecting pipe, 12000rpm is centrifuged 5min, removes liquid remaining in adsorption column.
(10) adsorption column is placed in clean 50ml collecting pipe, adds 1ml eluent TE, is stored at room temperature 5min, 12000rpm from Heart 2min；
(11) plasmid of purifying is diluted to final concentration of 1mg/ml with physiological saline, -20 DEG C save backup.
In step (12) C (7), OD value is 0.7.
A kind of immunologic adjuvant, it is characterized in that: sequence is as follows: 1,826 5 '-TCCATGACGTTCCTGACGTT- of CpG ODN 3 ', and full chain phosphorothioate backbone modification is to enhance its nuclease resistant.
The method of the present invention is easy, easy to operate, and effect is good.The present invention is research pair with the Periodic Brugia of China's prevalence As expanding Wuchereria malayi myosin genetic fragment, constructing Periodic Brugia myosin Eukaryotic expression recombinant plasmid (pcDNA3.1(+)‐Bm‐myosin).It is analyzed according to sequencing result, thus it is speculated that then its amino acid sequence utilizes relevant software The B cell epitope and t cell epitope for predicting them prepare Periodic Brugia myosin epitope gene vaccine.It should Vaccine immunization BALB/c mouse, and detect the humoral immunity of its vaccine and the response effect of cellular immunity.Test result card The vaccine, which is illustrated, can induce the immune response that mouse generates specificity, obtain promising result, manufacturing cycle type Wuchereria malayi Myosin epitope gene vaccine succeeds.
Below with reference to embodiment, the invention will be further described.
Specific embodiment
One, design of primers
According to the highly conserved sequence area of Periodic Brugia myosin gene (Bm-myosin) in GenBank, answer With Primer Premier5.0 software Design primers, and introduce I/BamH of Xho, I restriction enzyme site respectively, originate termination codon with And protectiveness base, primer are synthesized by Shanghai Bio-engineering Corporation.
P1: 5 '-CC of upstreamGGA TCC ATG AAA TGC AAA AAA T‐3’
Underlined sequences are I restriction enzyme site Tm=56.84 of BamH
P2: 5 '-CC of downstreamCTC GAG ATG GTG AAC GGA GTA CG‐3’
Underlined sequences are I restriction enzyme site Tm=66.90 of Xho
Two, the collection of Periodic Brugia polypide and microfilaria and the extraction of total serum IgE and measurement
1. the adult for being derived from meriones unguiculatus peritoneal fluid and microfilaria are moved into 1.5ml homogenizer, 1mlTrizol examination is added Agent is fully ground, and is stored at room temperature 5min.
2. 0.2ml chloroform is added, 2min is stood after shaking 15s, 4 DEG C, 12000rpm centrifugation 15min takes supernatant.
3. 0.5ml isopropanol is added to mix, it is stored at room temperature 10min, 4 DEG C, 12000rpm is centrifuged 10min and abandons supernatant.
4. 1ml75% ethyl alcohol is added to mix, 4 DEG C, 9000rpm is centrifuged 5min and abandons supernatant.
5. being dissolved in DEPC processing water 30ul, 60 DEG C of 10min after dry 5min.
6. take 2ul total serum IgE dilute 200 times, on ultraviolet specrophotometer survey OD260 and OD280 value, determine its concentration and Purity, and carry out formaldehyde electrophoresis detection.
Three, the synthesis of cDNA
1. total serum IgE 5ul is taken, it is rear to be added at 10mM dNTPmix 1ul, 0.5ug/ul oligo (dT) 12-18ul, DEPC Manage water 3ul, 65 DEG C of heating 5min, rear ice bath 5min.
2. 10 × Buffer 2ul is separately taken, 25mM MgCl2 4ul, 0.1M DTT 2ul, RNase inhibitor 1ul mixing, 42 DEG C of incubation 2min.
3. step 2 mixed liquor addition step 1 is mixed, adds 1ul reverse transcriptase Superscript II and mix, 42 DEG C It is incubated for 50min, 70 DEG C of 15min terminate reaction, and -70 DEG C save backup.
Four, PCR amplification Bm-myosin gene
P1=56.84, P2=66.90, reaction system cDNA 1.5ul, 10 × PCR Buffer 2.5ul, 2.5mM DNTPmix 4.0ul, P1 P2 each 0.7ul, 5U/ul TaqDNA polymeric protease 0.2ul, ddH2O 15.4ul.It mixes in DNA PCR is carried out on amplification instrument PTC-100.Loop parameter are as follows: 94 DEG C of 3min, 94 DEG C of 50s, 51 DEG C of 45s, 72 DEG C of 90s, totally 33 are followed Ring, last time recycle 72 DEG C of extension times as 7min, and 4 DEG C of coolings terminate reaction.Negative control is set up simultaneously.PCR is produced Object is identified through 1.0% agarose gel electrophoresis.
Five, the connection and conversion of Bm-myosin genetic fragment purifying and carrier T
1. purifying Bm-myosin genetic fragment: taking 50ulPCR product through 1.0% agarose gel electrophoresis, in the UV lamp The gel containing target gene is cut, is illustrated by plastic recovery kit, purification and recovery product.
2. the preparation of competent E.coli: bacillus coli DH 5 alpha 37 DEG C of overnight incubations on fresh LB agar plate.It chooses Single bacterium is taken to fall in 3ml LB culture medium, 37 DEG C of shaken overnights.300ul bacterium solution is taken to be added in 30ml LB culture medium, 37 DEG C 220rpm vibrate 4h, ice bath 1h, after be dispensed into 4 DEG C of 1.5ml Eppendorf pipe from 8000rpm 5min.Supernatant is abandoned, ice is added It bathes 0.1mol/L MgCl2 100ul to suspend, 4 DEG C of centrifugations, 8000rpm 5min abandons supernatant, and ice bath 0.1mol/L is added CaCl2- 10% glycerite 300ul suspends, and -70 DEG C save backup.
The connection of 3.Bm-myosin gene and pGEM-T carrier: taking the PCR product 3ul of purifying, and 2 × quick connect buffers Liquid 5ul, pGEM-T carrier (50ng) 1ul, T4DNA ligase 3weiss/ul 1ul, total 10ul mixing, 4 DEG C of connection 20h.
4. conversion: it takes ligation reaction 10ul that freshly prepared bacillus coli DH 5 alpha competent cell 300ul is added and mixes, Ice bath 30min, 42 DEG C of heat shock 90s are quickly moved to ice bath 3min, and 200ul LB culture medium, 37 DEG C of incubation 45min are added.It will Culture 100ul is coated in containing X-gal, IPTG, on 1.5% agar plate of Amp, is inverted plate, 37 DEG C are incubated overnight.
Six, the screening and identification of Positive recombinant clones
1. the screening of Positive recombinant clones: the good white bacterial plaque of picking growth conditions, and extract plasmid.
2.PCR identification: it using recombinant plasmid Bm-myosin/pGEM-T as template, is carried out with the primer P1 P2 of Bm-myosin PCR reaction, reaction system 25ul, Bm-myosin/pGEM-T recombinant plasmid 1.5ul, 10 × PCR Buffer 2.5ul, 2.5mM DNTPmix4.0ul, P1 P2 each 0.7ul, 5U/ul TaqDNA polymeric protease 0.2ul, ddH2O 15.4ul.Loop parameter Are as follows: 94 DEG C of 3min, 94 DEG C of 50s, 51 DEG C of 45s, 72 DEG C of 90s, totally 33 circulation, last time recycle 72 DEG C of extension times be 7min, 4 DEG C of cooling terminate are reacted.PCR product is identified through 1.0% agarose gel electrophoresis.
3. digestion is identified: the PCR that learns from else's experience identifies correct recombinant plasmid, with BamH I and I double digestion of Xho, with 1.0% fine jade Sepharose electrophoresis detection endonuclease bamhi.
Seven, Bm-MYOSIN secondary structure prediction
Respectively using on EXPASY server (http://www.expasy.ch/tools) SOPMA, GOR, nnPredict(University of California at SanFrancisco(UCSF))、HNN(Hierarchical NeuralNetwork method, Guermeur, 1997) method and EMBOSS program analysis prediction Bm-myosin second level Structure.
Eight, Bm-MYOSIN hydrophily, surface accessibility, antigenicity, polarity and flexibility parameter prediction
Using Hopp&Woods hydrophilic parameter, the surface Emini accessibility parameter, Jameson-Wolf antigenicity parameter, Zimmerman polarity parameters and flexibility parameter prediction (http://www.expasy.org/cgi-bin/protscale.pl)
Nine, Bm-MYOSIN t cell epitope is predicted
1. the prediction of source of people HLA class Ⅰmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server selects AA length for 9-mers, genotype HLA-A0201, HLA-A1101, predicts HLA class Ⅰmolecule Binding peptide retains the result of output.
2. the prediction of source of mouse MHC class Ⅰmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server selects AA length for 9-mers, and genotype is that (including H2-Kd, H2-Ld and H2-Dd are H2-d type to H2-d The MHC class Ⅰmolecule of mouse expression), it predicts MHC class Ⅰmolecule binding peptide, retains the result of output.
3. the prediction of source of people HLA class Ⅱmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server, selects genotype for HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1*0401, HLA-DRB1* 0701, HLA-DRB1*0801, HLA-DRB1*0901, HLA-DRB1*1101, HLA-DRB1*1301, HLA-DRB1*1501, in advance HLA class Ⅱmolecule binding peptide is surveyed, the result of output is retained.
4. the prediction of source of mouse MHC class Ⅱmolecule combination epitope: by the AA sequence of myosin with the input of FASTA format Rankpep server selects genotype for I-Ad, I-Ed, predicts source of mouse MHC class Ⅱmolecule binding peptide, retains output As a result.
Ten, the selection of epitope gene segment
According to B cell Antigen Epitope Prediction and t cell epitope prediction result, selection is rich in the genetic fragment of epitope The segment using plasmid pcDNA3.1 (+)-BmM55 as template, and is cloned into prokaryotic expression by (562bp-1269bp) design primer In carrier pET-28a (+), pET-BmM29 is constructed.Using 5 software Design primers of Primer.
Upstream (P1): 5 '-GCGGATCCGCTAATGAATTGAATATGC-3 '
(wherein GGATCC is I restriction enzyme site of BamH)；
Downstream (P2): 5 '-CGGAATTCCTATGGTGAACGGAGTACGGT
(wherein GAATTC is I restriction enzyme site of EcoR).
Primer is synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd, and ddH is used before use2O dissolution, concentration are 10 μm of ol/L.
11, epitope gene segment PCR amplification
1. P1, P2 are respectively upstream and downstream primer using pcDNA3.1 (+)-BmM55 as template, reaction system is shown in Table 1.
1 pcr amplification reaction system of table
Amplified reaction parameter are as follows:
5 μ l samples are taken to detect PCR amplification target gene with 1% agarose gel electrophoresis.
12, target gene and carrier connection and identification
1.PCR product and pET-28a (+) vector plasmid double digestion system are shown in Table 2.
2 double enzyme digestion reaction system of table
Target gene is connect with carrier, and specific reaction system is shown in Table 3.
3 target gene of table and carrier coupled reaction system
It mixes, reacts at room temperature 30min or more, product is for converting.
The preparation of 2.E.coli DH5 α competent cell
(1) it is gone bail for oese and is stored in -80 DEG C of DH5 α bacterium solutions, draw bacterium on not antibiotic LB plate, 37 DEG C are inverted About 14-16h is cultivated to single colonie appearance.
(2) the LB liquid that single bacterium drops down onto 5ml non-resistant, 37 DEG C of shaken cultivation about 10-12h are chosen.
(3) for the bacterium solution for taking 1ml to be incubated overnight to containing in 100ml nonreactive LB liquid, 37 DEG C of shaken cultivation about 2-2.5h arrive OD value Up to 0.5, ice bath 5-10min.
(4) 2 50ml are dispensed into, sterile centrifuge tube is pre-chilled, 4 DEG C, 1600rpm is centrifuged 7min.
(5) cell is resuspended with 10ml ice-cold 0.1M CaCl2 solution, 4 DEG C, 1100rpm is centrifuged 5min.
(6) cell is resuspended with 10ml ice-cold 0.1M CaCl2 solution, after placing 30min on ice, 4 DEG C, 1100rpm centrifugation 5min。
(7) cell is resuspended with 2ml ice-cold 0.1M CaCl2 solution, 15% glycerol freezes in -80 DEG C after packing.
3. conversion and identification
(1) 100 μ l are taken to be stored in -80 DEG C of calcification bacterium, ice bath melts；10 μ l connection products are added, mix gently, ice bath 30min。
(2) heat shock, 42 DEG C of water-bath 30-90s, is not moved.
(3) it is quickly transferred in ice bath, cooling 1-2min.
(4) plus 2.25ml LB liquid is to test tube, then plus 0.25ml mixture, slant setting, 37 DEG C, 100rpm shaken cultivation 45min。
(5) 5000rpm is centrifuged 1min.
(6) part supernatant is abandoned, is poured on after mixing on LB plate (containing Amp), is applied uniformly, be stored at room temperature with spreader, until Liquid is absorbed, and 37 DEG C of incubators are inverted culture 12-16h.
(7) monoclonal is chosen to 5ml LB liquid (containing Amp) with white pipette tips, 37 DEG C, 220rpm shaken cultivation 12-16h, until OD value Between 0.6-0.8.
(8) positive control is done by above-mentioned steps simultaneously, negative control transformation reacts.
(9) it extracts plasmid and carries out plasmid electroresis appraisal and PCR identification.
(10) it chooses PCR and identifies positive bacterium solution, it is limited to serve Hai Yingjun biotechnology after expanding in LB liquid medium Company is sequenced.
13, BmM29 epitope gene construction of eukaryotic expression vector
It purifies purpose epitope gene and is subcloned to gram of the BamH I and EcoR I of eukaryotic expression vector pcDNA3.1 (+) Grand site.The molar ratio of target gene and carrier is 5:1, and reaction system 25ul, 16 DEG C of water-bath connections are overnight.Take connection liquid conversion Competent E.coli DH5 α is coated on LB-Amp (100mg/ml) plate, and 37 DEG C are incubated overnight.From LB-Amp (100mg/ Ml) random picking single bacterium colony on plate, is inoculated in LB-Amp culture medium, 37 DEG C of shaken overnight cultures respectively, extracts recombination Plasmid, through I digestion of BamH I and EcoR, rear 1.0% agarose gel electrophoresis identification.
14, BmM29 epitope gene vaccine immune response is tested
1.pcDNA3.1 (+) and pcDNA3.1 (+)-BmM29 plasmid largely extract
(1) 50ml centrifuge tube is added in the bacterium solution for taking 100ml to be incubated overnight, and 12000rpm is centrifuged 3min and collects bacterium, as far as possible Abandon supernatant to the greatest extent.
(2) 12000rpm is centrifuged 3min, and remaining a small amount of liquid is sucked out with pipettor.
(3) solution 1 of a large amount of extraction agent boxes of 10ml plasmid is added, it is outstanding by cell using turbula shaker and pipettor It is floating, no small bacteria block.
(4) solution 2 of a large amount of extraction agent boxes of 10ml plasmid is added, leniently spins upside down 6-8 times, is stored at room temperature immediately 5min。
(5) solution 4 of a large amount of extraction agent boxes of 10ml plasmid is added, leniently spins upside down 6-8 times, is stored at room temperature immediately 10min, 12000rpm are centrifuged 20min, and solution is all poured into Filter column, slowly push push handle, filtrate collect 50ml from In heart pipe.
(6) add 8ml isopropanol into filtrate, adsorption column is transferred to after mixing, 12000rpm is centrifuged 2min, abandons in collecting pipe Waste liquid.
(7) add 3ml to remove endotoxin buffer into adsorption column, be stored at room temperature 2min, 12000rpm is centrifuged 2min, abandons and collects Waste liquid in pipe.
(8) rinsing liquid W2 is added into adsorption column, 12000rpm is centrifuged 2min, abandons the waste liquid in collecting pipe.It is repeated once.
(9) adsorption column is placed back in into collecting pipe, 12000rpm is centrifuged 5min, removes liquid remaining in adsorption column.
(10) adsorption column is placed in clean 50ml collecting pipe, adds 1ml eluent TE, is stored at room temperature 5min, 12000rpm from Heart 2min.
(11) plasmid of purifying is diluted to final concentration of 1mg/ml with physiological saline, -20 DEG C save backup.
2. the preparation of immunological adjuvant CpG ODN
Using the cytimidine of non-methylation and guanylic acid as the oligodeoxynucleotide (CpG of core in novel adjuvant ODN) most representative.The sequence of CpG ODN synthesis is as follows: CpG ODN 1,826 5 '-TCCATGACGTTCCTGACGTT-3 ', And full chain phosphorothioate backbone modification, to enhance its nuclease resistant, by Shanghai, Ying Jun biotech firm is synthesized.It is water-soluble with physiology salt Solution is configured to 1mg/ml.
3. experimental animal is grouped
48 BALB/c experiment mices are weighed in sequence, according to 4 groups of random digits table point, every group 12, A:PBS Control group；B: adjuvant CpG control group；C: empty carrier pcDNA3.1 (+)/CpG group；D: recombinant plasmid pcDNA3.1 (+)-BmM29/ CpG group, immunization protocol 0,2,4 week each immune primary.
4. inoculation position pre-processes
DNA vaccination injection site is mouse back leg tibialis anterior, and recombinant protein vaccine is subcutaneous multi-point injection.Lml is left-handed Bupivacaine hydrochloride injection is diluted in 14ml physiological saline, obtains 0.5mg/ml dilution.Every time before inoculation for 24 hours, in connecing It injects left-handed 50 μ l of bupivacaine HCl and is pre-processed in kind position.
5. immunity inoculation
100 μ l of A:PBS；B: adjuvant CpG 30 μ g；100 μ g/30 μ g of C:pcDNA3.1 (+)/CpG；D:pcDNA3.1 (+)‐BmM29/CpG 100μg/30μg.It is immunized 3 times altogether, every minor tick 2 weeks.
14, immune response detects
1. serum collection: 4 after initial immunity, collecting mice serum with eyeball excise method within 6,8 weeks, every group takes 4 Mouse.It is stored at room temperature 1h, sets 4 DEG C overnight, serum to be precipitated, 4 DEG C, 4000rpm is centrifuged 10min.Serum is dispensed, is stored in -80 ℃。
2. IgG is detected in immune serum
(1) antigen coat: with antigen coat buffer by antigen diluent at 5 μ g/mL, with 100 hole μ l/ coated elisa plates, 4 DEG C overnight；
(2) liquid is abandoned, is washed 3 times with PBST.It is closed using 100 μ l/ hole confining liquids, is stored at room temperature 1h；
(3) liquid is abandoned, after serum antibody diluent is according to 1:100 dilution proportion, 100 holes μ l/, room temperature 2h；
(4) it is washed 3 times with PBST；
(5) after the IgG antibody for diluting HRP coupling with sample diluting liquid 1:2000,100 holes μ l/, room temperature 1h；
(6) it is washed 3 times using PBST；
(7) 100 hole μ l/ substrate solutions are added, after acting on 15min, add 50 μ l 2mol/L H2SO4Terminate reaction；
(8) light absorption value is measured at 450nm, saves data.
3. mouse boosting cell acquires
(1) 8 weeks execution mouse after initial immunity；
(2) sterile 10ml screw socket pipe is taken, 3ml lymphocyte separation medium, 37 DEG C of preheating 2h are added；
(3) mouse of execution 2-3min in 75% ethanol solution is set to carry out disinfection；
(4) upward by the left veutro of mouse, the left skin of abdomen of mouse is gently lifted with tweezers, cuts about 1cm with operating scissors The long edge of a knife is torn skin of abdomen with hand, exposure peritonaeum, it is seen that red strip spleen, by its careful separation.
(5) 100 mesh copper sieve is put into the plate for containing 3ml Hank ' s liquid, spleen is placed on copper sieve, is first torn spleen It is broken, then be lightly ground with glass bar, until only remaining connective tissue on copper sieve, then copper sieve is rinsed with 3mlHank ' s liquid；
(6) the screw socket pipe of the lymphocyte separation medium containing 3ml is tilted 45 °, with dropper by the cell suspension in plate along pipe Wall slowly shifts (lymphocyte separation medium: cell suspension=1:2), not break through the interface of lymphocyte separation medium；
(7) room temperature 2000rpm is centrifuged 20min, and liquid is divided into 4 layers in pipe；
(8) the 2nd layer of extracting waste, is transferred in the 50ml centrifuge tube of the s liquid of the Hank ' containing 10ml, room temperature 1000rpm centrifugation 10min；
(9) supernatant liquid is abandoned, 10ml Hank ' s liquid is added, gently piping and druming mixes, and room temperature 1000rpm is centrifuged 10min；
(10) supernatant liquid is abandoned, 10ml Hank ' s liquid is added, gently piping and druming mixes, and is counted with tally；
(11) room temperature 1000rpm is centrifuged 10min, abandons supernatant liquid, appropriate complete medium is added and makes the end of cell dense Degree is 2 × 106cells/ml.
4. cell factor induces
2 × 106cells/ml cell suspension, 475 hole μ l/ is added in 24 orifice plates, and every hole adds 100 μ l/ml ConA, 25 μ l extremely 37 DEG C of incubation 48h in final concentration of 5 μ g/ml, 5%CO2 incubator.5000rpm is centrifuged 10min and collects culture solution, takes supernatant quasi- Standby detection.
5.INF- γ and IL-4 assay
(1) shift to an earlier date the ELISA kit that 20min taking-up is stored in 4 DEG C, balance to room temperature；
(2) concentrated cleaning solution ddH2O is diluted 20 times；
(3) take out ELISA Plate from having balanced into the hermetic bag of room temperature, be added standard sample (160pg/ml, 80pg/ml, 40pg/ml, 20pg/ml, 10pg/ml, 0pg/ml) each 50ul；
(4) ELISA Plate is taken out into the hermetic bag of room temperature from having balanced, in addition to blank well, be separately added into sample diluting liquid 40ul and sample to be tested 10ul；
(5) in addition to blank well, the detection antibody of horseradish peroxidase-labeled is added in every hole in standard sample wells and sample well 100 μ l seal reacting hole, 37 DEG C of incubation 30min with sealing plate film；
(6) liquid is discarded, is patted dry on blotting paper, cleaning solution is filled it up in every hole, is stood 1min, is got rid of cleaning solution, absorbing water It pats dry on paper, so repeats board-washing 5 times；
(7) each 50 μ l of color developing agent A, B is first added in every hole, and 37 DEG C are protected from light incubation 15min；
(8) every hole is added in terminate liquid 50 μ l, 15min, and the OD value in each hole is measured at 450nm wavelength；
(9) calculate OD value: the OD value of each standard items and sample subtracts the OD value of blank well；
(10) it draws standard curve: abscissa being made with standard concentration, corresponding OD value makees ordinate, draws out standard items Linear regression curves；
(11) concentration of the sample can be found on standard curve by the OD value of sample.
6. data are analyzed
Sample data is analyzed using SPSS19.0 statistics software, measurement data is indicated with mean ± standard deviation, mouse blood The OD of clear antibody450The comparison among groups of cell factor IFN-γ and IL-4 level compare use in value, mouse boosting cell culture supernatant One-way analysis of variance, Multiple range test uses SNK (Student-Newman-Keuls) method between group.P < 0.05 is that difference has statistics Learn meaning.
The measurement of 4 immune serum antibody OD450 value of table
Note: * expression is respectively compared with three control groups, and difference is statistically significant, P < 0.05.
The different immune grouping mouse cytokine INF- γ and IL-4 of table 5 are horizontal (pg/ml)
Note: * expression is respectively compared with three control groups, and difference is statistically significant, P < 0.05.
By testing successfully amplify Periodic Brugia myosin genetic fragment.Through 1.0% Ago-Gel electricity Swimming detection, Bm-myosin amplified fragments size are consistent substantially with expected 1292bp.It is provided on gene sequencing and GeneBank Sequence blast comparison result, homology 98%, it was demonstrated that cloned sequence is correct.It is 431 amino acid that it, which expresses albumen,.It is comprehensive Close the B cells epitopes such as secondary structure prediction and hydrophily, surface accessibility, polarity and the flexibility parameter of Bm-MYOSIN albumen Prediction result and t cell epitope forecasting research as a result, selection be rich in epitope genetic fragment (562bp-1269bp), Eukaryotic expression vector pcDNA3.1 (+) plasmid is connect with BmM29 target gene fragment, building pcDNA3.1 (+)-BmM29 is true Nuclear expression carrier prepares Periodic Brugia M29 epitope gene DNA vaccination.
By the epitope gene DNA vaccination immunity inoculation BALB/c mouse back leg tibialis anterior, vaccine is carried out to immune mouse and is exempted from Epidemic disease effect detection measures IgG and splenocyte cell factor INF- γ and IL-4 content in immune serum.As the result is shown: exempting from Epidemic disease mouse generates high-level specific IgG antibodies, hence it is evident that is higher than control group (the equal < 0.05 of P).With the increase of immune time, exempt from The OD450 value of epidemic disease mouse IgG antibody is in rising trend.Immune cell factor testing result is shown: the training of immune group mouse boosting cell The level of cell factor IFN-γ and IL-4 in supernatant are supported obviously higher than control group, be statistically significant (P < 0.05).The above the results show Periodic Brugia M29 epitope gene DNA vaccination immunity inoculation can induce mouse and generate The humoral and cellular immune response responsing reaction of specificity obtains promising result.Periodic Brugia M29 epitope gene DNA vaccination Prepare success.
SEQUENCE LISTING
<110>Nantong University
<120>purposes of Periodic Brugia M29 epitope gene DNA vaccination
<130> 2014.10.21
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ccggatccat gaaatgcaaa aaat 24
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccctcgagat ggtgaacgga gtacg 25
<210> 3
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gcggatccgc taatgaattg aatatgc 27
<210> 4
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
cggaattcct atggtgaacg gagtacggt 29

Claims (1)

1. a kind of preparation method of Periodic Brugia M29 epitope gene DNA vaccination, it is characterized in that: described includes following step It is rapid:
(1) design of primers
According in GenBank Periodic Brugia myosin gene it is highly conserved
Sequence area using Primer Premier5.0 software Design primers, and introduces I/BamH of Xho, I restriction enzyme site respectively, Originate termination codon and protectiveness base；
P1: 5 '-CC of upstreamGGA TCC ATG AAA TGC AAA AAA T-3’
Underlined sequences are Tm=56.84 DEG C of I restriction enzyme site of BamH
P2: 5 '-CC of downstreamCTC GAG ATG GTG AAC GGA GTA CG-3’
Underlined sequences are Tm=66.90 DEG C of I restriction enzyme site of Xho
(2) collection of Periodic Brugia polypide and microfilaria and the extraction of total serum IgE and measurement
(1) adult for being derived from meriones unguiculatus peritoneal fluid and microfilaria are moved into 1.5ml homogenizer, 1mlTrizol reagent is added Grinding, is stored at room temperature 5min；
(2) 0.2ml chloroform is added, stands 2min after shaking 15s, 4 DEG C, 12000rpm centrifugation 15min takes supernatant；
(3) 0.5ml isopropanol is added to mix, is stored at room temperature 10min, 4 DEG C, 12000rpm is centrifuged 10min and abandons supernatant；
(4) 1ml75% ethyl alcohol is added to mix, 4 DEG C, 9000rpm is centrifuged 5min and abandons supernatant；
(5) DEPC processing water 30ul, 60 DEG C of 10min are dissolved in after dry 5min；
(6) it takes 2ul total serum IgE to dilute 200 times, OD260 and OD280 value is surveyed on ultraviolet specrophotometer, determine its concentration and pure Degree, and carry out formaldehyde electrophoresis detection；
(3) synthesis of cDNA
(1) total serum IgE 5ul is taken, rear that 10mM dNTPmix 1ul, 0.5ug/ul oligo (dT) 12-18ul is added, DEPC handles water 3ul, 65 DEG C of heating 5min, rear ice bath 5min；
(2) 10 × Buffer 2ul is separately taken, 25mM MgCl2 4ul, 0.1M DTT 2ul, RNase inhibitor 1ul are mixed, and 42 DEG C It is incubated for 2min；
(3) step (2) mixed liquor is added in step (1) substance and is mixed, it is mixed to add 1ul reverse transcriptase Superscript II It is even；42 DEG C of incubations 50min, 70 DEG C of 15min terminate reaction, and -70 DEG C save backup；
(4) PCR amplification Bm-myosin gene
Tm=56.84 DEG C of P1, Tm=66.90 DEG C of P2, reaction system cDNA 1.5ul, 10 × PCR Buffer 2.5ul, 2.5mM dNTPmix 4.0ul, P1 each 0.7ul of P2,5U/ul Taq DNA polymerase 0.2ul, ddH2O 15.4ul；It mixes PCR is carried out on DNA cloning instrument PTC-100；Loop parameter are as follows: 94 DEG C of 3min, 94 DEG C of 50s, 51 DEG C of 45s, 72 DEG C of 90s, altogether 33 circulations, last time recycle 72 DEG C of extension times as 7min, and 4 DEG C of coolings terminate reaction；Negative control is set up simultaneously； PCR product is identified through 1.0% agarose gel electrophoresis；
(5) connection and conversion of Bm-myosin genetic fragment purifying and carrier T
(1) it purifies Bm-myosin genetic fragment: taking 50ulPCR product through 1.0% agarose gel electrophoresis, cut in the UV lamp The gel containing target gene is cut, is illustrated by plastic recovery kit, purification and recovery product；
(2) preparation of competent E.coli: bacillus coli DH 5 alpha 37 DEG C of overnight incubations on fresh LB agar plate；Picking list Bacterium colony is in 3ml LB culture medium, 37 DEG C of shaken overnights；300ul bacterium solution is taken to be added in 30ml LB culture medium, 37 DEG C of 220rpm 4h, ice bath 1h are vibrated, 1.5ml Eppendorf pipe, 4 DEG C of centrifugations, 8 000rpm 5min are then dispensed into；Supernatant is abandoned, ice is added It bathes 0.1mol/L MgCl2 100ul to suspend, 4 DEG C of centrifugations, 8000rpm5min abandons supernatant, and ice bath 0.1mol/L CaCl is added2- 10% glycerite 300ul suspends, and -70 DEG C save backup；
(3) the PCR product 3ul of purifying, 2 × rapid ligation buffer the connection of Bm-myosin gene and pGEM-T carrier: are taken 5ul, pGEM-T carrier 1ul, T4DNA ligase 3weiss/ul1ul, total 10ul mixing, 4 DEG C of connection 20h；
(4) is converted: it takes ligation reaction 10ul that freshly prepared bacillus coli DH 5 alpha competent cell 300ul is added and mixes, ice 30min, 42 DEG C of heat shock 90s are bathed, ice bath 3min is quickly moved to, 200ul LB culture medium, 37 DEG C of incubation 45min are added；It will training It supports object 100ul to be coated in containing X-gal, IPTG, on 1.5% agar plate of Amp, is inverted plate, 37 DEG C are incubated overnight；
(6) screening and identification of Positive recombinant clones
(1) screening of Positive recombinant clones: the good white bacterial plaque of picking growth conditions, and extract plasmid；
(2) PCR is identified: using recombinant plasmid Bm-myosin/pGEM-T as template, carrying out PCR with primer P1, P2 of Bm-myosin Reaction, reaction system 25ul, Bm-myosin/pGEM-T recombinant plasmid 1.5ul, 10 × PCR Buffer 2.5ul, 2.5mM Each 0.7ul of dNTPmix4.0ul, P1, P2,5U/ul Taq DNA polymerase 0.2ul, ddH2O 15.4ul；Loop parameter are as follows: 94 DEG C 3min, 94 DEG C of 50s, 51 DEG C of 45s, 72 DEG C of 90s, totally 33 circulations, last time recycle 72 DEG C of extension times and are 7min, 4 DEG C of cooling terminate are reacted；PCR product is identified through 1.0% agarose gel electrophoresis；
(3) digestion is identified: the PCR that learns from else's experience identifies correct recombinant plasmid, with BamH I and I double digestion of Xho, with 1.0% agarose Detected through gel electrophoresis endonuclease bamhi；
(7) Bm-MYOSIN secondary structure prediction
Respectively using SOPMA, GOR, nnPredic, HNN method and the analysis prediction of EMBOSS program on EXPASY server The secondary structure of Bm-myosin；
(8) Bm-MYOSIN hydrophily, surface accessibility, antigenicity, polarity and flexibility parameter prediction
Using Hopp&Woods hydrophilic parameter, the surface Emini accessibility parameter, Jameson-Wolf antigenicity parameter, Zimmerman polarity parameters and flexibility parameter prediction；
(9) Bm-MYOSIN t cell epitope is predicted
(1) the AA sequence of myosin the prediction of source of people HLA class Ⅰmolecule combination epitope: is inputted into Rankpep with FASTA format Server selects AA length for 9-mers, genotype HLA-A0201, HLA-A1101, predicts HLA class Ⅰmolecule binding peptide, protects Stay the result of output；
(2) the AA sequence of myosin the prediction of source of mouse MHC class Ⅰmolecule combination epitope: is inputted into Rankpep with FASTA format Server selects AA length for 9-mers, genotype H2-Kd, H2-Ld and H2-Dd, for the MHC I of H2-d type mouse expression Class molecule；It predicts MHC class Ⅰmolecule binding peptide, retains the result of output；
(3) the AA sequence of myosin the prediction of source of people HLA class Ⅱmolecule combination epitope: is inputted into Rankpep with FASTA format Server, select genotype for HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*0801, HLA-DRB1*0901, HLA-DRB1*1101, HLA-DRB1*1301, HLA-DRB1*1501 predict HLA Class Ⅱmolecule binding peptide retains the result of output；
(4) the AA sequence of myosin the prediction of source of mouse MHC class Ⅱmolecule combination epitope: is inputted into Rankpep with FASTA format Server selects genotype for I-Ad, I-Ed, predicts source of mouse MHC class Ⅱmolecule binding peptide, retains the result of output；
(10) selection of epitope gene segment
According to B cell Antigen Epitope Prediction and t cell epitope prediction result, selection is rich in the genetic fragment 562bp- of epitope The segment using plasmid pcDNA3.1 (+)-BmM55 as template, and is cloned into prokaryotic expression carrier pET- by 1269bp design primer In 28a (+), pET-BmM29 is constructed；Using Primer5 software Design primers；
Upstream P1:5 '-GCGGATCCGCTAATGAATTGAATATGC-3 '
Wherein GGATCC is I restriction enzyme site of BamH；
Downstream P2:5 '-CGGAATTCCTATGGTGAACGGAGTACGGT-3 '
Wherein GAATTC is I restriction enzyme site of EcoR；
Primer is dissolved using preceding with ddH2O, and concentration is 10 μm of ol/L；
(11) epitope gene segment PCR amplification
(1) using pcDNA3.1 (+)-BmM55 as template, P1, P2 are respectively upstream and downstream primer, reaction system:
Pcr amplification reaction system
Amplified reaction parameter are as follows:
5 μ l samples are taken to detect PCR amplification target gene with 1% agarose gel electrophoresis；
(12) target gene is connect and identification with carrier
A, PCR product and pET-28a (+) vector plasmid double digestion system:
Double enzyme digestion reaction system
Target gene is connect with carrier, specific reaction system:
Target gene and carrier coupled reaction system
It mixes, reacts at room temperature 30min or more, product is for converting；
B, the preparation of E.coli DH5 α competent cell
(1) it is gone bail for oese and is stored in -80 DEG C of DH5 α bacterium solutions, draw bacterium on not antibiotic LB plate, 37 DEG C of inversions are cultivated 14-16h to single colonie occur；
(2) the LB liquid that single bacterium drops down onto 5ml non-resistant, 37 DEG C of shaken cultivation 10-12h are chosen；
(3) bacterium solution for taking 1ml to be incubated overnight is to containing in 100ml nonreactive LB liquid, 37 DEG C of shaken cultivation 2-2.5h, to OD value up to 0.5, Ice bath 5-10min；
(4) 2 50ml are dispensed into, sterile centrifuge tube is pre-chilled, 4 DEG C, 1600rpm is centrifuged 7min；
(5) cell is resuspended with 10ml ice-cold 0.1M CaCl2 solution, 4 DEG C, 1100rpm is centrifuged 5min；
(6) cell is resuspended with 10ml ice-cold 0.1M CaCl2 solution, after placing 30min on ice, 4 DEG C, 1100rpm centrifugation 5min；
(7) the 0.1M CaCl ice-cold with 2ml2Cell is resuspended in solution, and 15% glycerol freezes in -80 DEG C after packing；
C, conversion and identification
(1) 100 μ l are taken to be stored in -80 DEG C of calcification bacterium, ice bath melts；10 μ l connection products are added, mix gently, ice bath 30min；
(2) heat shock, 42 DEG C of water-bath 30-90s, is not moved；
(3) it is quickly transferred in ice bath, cooling 1-2min；
(4) plus 2.25ml LB liquid is to test tube, then plus 0.25ml mixture, slant setting, 37 DEG C, 100rpm shaken cultivation 45min；
(5) 5000rpm is centrifuged 1min；
(6) part supernatant is abandoned, is poured on after mixing on the LB plate containing Amp, is applied uniformly, is stored at room temperature with spreader, until liquid It is absorbed, 37 DEG C of incubators are inverted culture 12-16h；
(7) LB liquid of the monoclonal to 5ml containing Amp is chosen with white pipette tips, 37 DEG C, 220rpm shaken cultivation 12-16h, until OD value exists Between 0.6-0.8；
(8) positive control is done by above-mentioned steps simultaneously, negative control transformation reacts；
(9) it extracts plasmid and carries out plasmid electroresis appraisal and PCR identification；
(10) it chooses PCR and identifies positive bacterium solution, be sequenced after being expanded in LB liquid medium；
(13) BmM29 epitope gene construction of eukaryotic expression vector
It purifies purpose epitope gene and is subcloned to the clone position of the BamH I and EcoR I of eukaryotic expression vector pcDNA3.1 (+) Point；The molar ratio of target gene and carrier is 5:1, and reaction system 25ul, 16 DEG C of water-bath connections are overnight；Take connection liquid conversion impression State bacillus coli DH 5 alpha is coated on LB-Amp plate, wherein containing Amp100mg in every mlLB, 37 DEG C are incubated overnight；From LB- Random picking single bacterium colony, is inoculated in respectively in LB-Amp culture medium on Amp plate, 37 DEG C of shaken overnight cultures, extracts recombination Plasmid, through I digestion of BamH I and EcoR, rear 1.0% agarose gel electrophoresis identification；
The test of (14) BmM29 epitope gene vaccine immune response
A.pcDNA3.1 (+) and pcDNA3.1 (+)-BmM29 plasmid largely extract
(1) 50ml centrifuge tube is added in the bacterium solution for taking 100ml to be incubated overnight, and 12000rpm is centrifuged 3min and collects bacterium, abandons as far as possible to the greatest extent Supernatant；
(2) 12000rpm is centrifuged 3min, and remaining a small amount of liquid is sucked out with pipettor；
(3) solution 1 of a large amount of extraction agent boxes of 10ml plasmid is added, is suspended using turbula shaker and pipettor by cell, nothing Small bacteria block；
(4) solution 2 of a large amount of extraction agent boxes of 10ml plasmid is added, leniently spins upside down 6-8 times, is stored at room temperature immediately 5min；
(5) solution 4 of a large amount of extraction agent boxes of 10ml plasmid is added, leniently spins upside down 6-8 times, is stored at room temperature immediately 10min, 12000rpm are centrifuged 20min, and solution is all poured into Filter column, slowly push push handle, filtrate collect 50ml from In heart pipe；
(6) add 8ml isopropanol into filtrate, adsorption column is transferred to after mixing, 12000rpm is centrifuged 2min, abandons useless in collecting pipe Liquid；
(7) add 3ml to remove endotoxin buffer into adsorption column, be stored at room temperature 2min, 12000rpm is centrifuged 2min, abandons in collecting pipe Waste liquid；
(8) rinsing liquid W2 is added into adsorption column, 12000rpm is centrifuged 2min, abandons the waste liquid in collecting pipe；It is repeated once；
(9) adsorption column is placed back in into collecting pipe, 12000rpm is centrifuged 5min, removes liquid remaining in adsorption column；
(10) adsorption column is placed in clean 50ml collecting pipe, adds 1ml eluent TE, is stored at room temperature 5min, 12000rpm centrifugation 2min；
(11) plasmid of purifying is diluted to final concentration of 1mg/ml with physiological saline, -20 DEG C save backup；
B. the preparation of immunological adjuvant CpG ODN
In novel adjuvant using the cytimidine of non-methylation and guanylic acid as the oligodeoxynucleotide CpG ODN of core most It is representative；The sequence of CpG ODN synthesis is as follows: CpG ODN 1,826 5 '-TCCATGACGTTCCTGACGTT-3 ', and full chain Phosphorothioate backbone is modified to enhance its nuclease resistant, is configured to 1mg/ml with physiological saline solution；
C. experimental animal is grouped
48 BALB/c experiment mices are weighed in sequence, according to 4 groups of random digits table point, every group 12, A:PBS control Group；B: adjuvant CpG control group；C: empty carrier pcDNA3.1 (+)/CpG group；D: recombinant plasmid pcDNA3.1 (+)-BmM29/CpG Group, immunization protocol 0,2,4 week each immune primary；
D. immunity inoculation
100 μ l of A:PBS；B: adjuvant CpG 30 μ g；100 μ g/30 μ g of C:pcDNA3.1 (+)/CpG；D:pcDNA3.1 (+)- BmM29/CpG 100μg/30μg；It is immunized 3 times altogether, every minor tick 2 weeks；
The detection of (15) immune response
1. serum collection: 4 after initial immunity, collecting mice serum with eyeball excise method within 6,8 weeks, every group takes 4 mouse； It is stored at room temperature 1h, sets 4 DEG C overnight, serum to be precipitated, 4 DEG C, 4000rpm is centrifuged 10min；Serum is dispensed, is stored in -80 DEG C；
2. IgG is detected in immune serum
(1) antigen coat: with antigen coat buffer by antigen diluent at 5 μ g/mL, with 100 hole μ l/ coated elisa plates, 4 DEG C of mistakes Night；
(2) liquid is abandoned, is washed 3 times with PBST；It is closed using 100 μ l/ hole confining liquids, is stored at room temperature 1h；
(3) liquid is abandoned, after serum antibody diluent is according to 1:100 dilution proportion, 100 holes μ l/, room temperature 2h；
(4) it is washed 3 times with PBST；
(5) after the IgG antibody for diluting HRP coupling with sample diluting liquid 1:2000,100 holes μ l/, room temperature 1h；
(6) it is washed 3 times using PBST；
(7) 100 hole μ l/ substrate solutions are added, after acting on 15min, add 50 μ l 2mol/L H2SO4Terminate reaction；
(8) light absorption value is measured at 450nm, saves data；
3. cell factor induces
2 × 106cells/ml cell suspension, 475 hole μ l/ is added in 24 orifice plates, and every hole adds 100 μ l/ml ConA, 25 μ l dense to end Degree is 37 DEG C of incubation 48h in 5 μ g/ml, 5%CO2 incubators；5000rpm is centrifuged 10min and collects culture solution, and supernatant is taken to prepare inspection It surveys；
4.INF- γ and IL-4 assay
(1) shift to an earlier date the ELISA kit that 20min taking-up is stored in 4 DEG C, balance to room temperature；
(2) concentrated cleaning solution ddH2O is diluted 20 times；
(3) ELISA Plate is taken out from having balanced into the hermetic bag of room temperature, standard sample 160pg/ml, 80pg/ml, 40pg/ is added Ml, 20pg/ml, 10pg/ml, 0pg/ml, each 50ul；
(4) take out ELISA Plate into the hermetic bag of room temperature from having balanced, in addition to blank well, be separately added into sample diluting liquid 40ul and Sample to be tested 10ul；
(5) in addition to blank well, 100 μ of detection antibody of horseradish peroxidase-labeled is added in every hole in standard sample wells and sample well L seals reacting hole, 37 DEG C of incubation 30min with sealing plate film；
(6) liquid is discarded, is patted dry on blotting paper, cleaning solution is filled it up in every hole, is stood 1min, cleaning solution is got rid of, on blotting paper It pats dry, so repeats board-washing 5 times；
(7) each 50 μ l of color developing agent A, B is first added in every hole, and 37 DEG C are protected from light incubation 15min；
(8) every hole is added in terminate liquid 50 μ l, 15min, and the OD value in each hole is measured at 450nm wavelength；
(9) calculate OD value: the OD value of each standard items and sample subtracts the OD value of blank well；
(10) it draws standard curve: abscissa being made with standard concentration, corresponding OD value makees ordinate, it is linear to draw out standard items Regression curve；
(11) concentration of the sample can be found on standard curve by the OD value of sample.
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