CN103191420A - Preparation method of periodic wuchereria malayi compound multivalent DNA (deoxyribonucleic acid) vaccine - Google Patents

Abstract

The invention discloses a preparation method of a periodic wuchereria malayi compound multivalent DNA (deoxyribonucleic acid) vaccine. The preparation method comprises the following steps of: designing a primer, amplifying BmGAPDH (reduced glyceraldehyde-phosphate dehydrogenase) and BmCPI genes by PCR (polymerase chain reaction), purifying segments of BmGAPDH and BmCPI genes, connecting and converting a T carrier, screening and identifying an I positive recombinant clone, building and identifying an I recombinant expression vector, extracting and purifying a mass of recombinant plasmid and the like. The preparation method is convenient in preparation and good in effect. The periodic wuchereria malayi which is popular in China is taken as a research object, and the periodic wuchereria malayi cysteine proteinase inhibitor/3-phosphoglyceraldehyde dehydrogenase eukaryotic expression recombinant plasmid is built. The test result proves that the compound multivalent DNA vaccine can induce the mouse to generate the specific immune response reaction, so that a satisfied effect can be obtained, and the periodic wuchereria malayi compound multivalent DNA vaccine can be successfully prepared.

Description

The preparation method of the compound Multivalent DNA Vaccine of preiodic type Wuchereria malayi

Technical field

The present invention relates to a kind of preparation method of preiodic type Wuchereria malayi vaccine.

Background technology

Filaricide is a kind of insect-borne diseases of serious harm human health, classified as one of whole world keypoint control six big tropical diseases except malaria, schistosomicide, leishmaniasis, african trypanosomiasis, leprosy jointly by United Nations Development Programme/World Bank/World Health Organization (WHO), there are more than 70 countries and regions to have the lymph filaricide popular at present in the world, the threat that 1,100,000,000 populations are infected.Check chronic filariasis mainly causes elephatiasis, hydrocele of tunica vaginalis and chyluria etc., can make patient's disability in various degree, is classified as second disabling condition in the whole world by World Health Organization (WHO), is only second to mental disease.Whole world lymph filaricide patient reaches more than 9,000 ten thousand, and China is the country that mainly gets involved.Though the control of filaricide has obtained huge achievement in the world, but because nocturnal periodicity and the microfilaremia person of nature, biology and society such as climate warming and lymph filaricide do not have all multifactor extensive existence such as clinical symptoms more, the trend that the epidemic situation resume combustion occurs and spread in some countries and regions in recent years, the popular people's the health of not only giving of filaricide is brought serious harm, also becomes one of major reason of driving into poverty by medical crises.The lymph filaricide China popular have more than 2,000 year historical, through arduous for many years preventing and controlling, domestic filaricide obtains certain control, but still there are a considerable amount of filaricide patients in the whole nation so far, comprises that Jiangsu Province only has clinical manifestation person just to reach people more than 1,390,000.Because lymph filaricide communication media extensively exists, a large amount of external recurrent populations' pours in addition, especially coastal economy developed regions, more microfilariae of ohchocerciasis positive person among the external person of working of various places report in the recent period, but the risk factor weighted silk parasitosis that external source is imported is popular.The current research report shows that the microfilaremia of the remaining source of infection of the infected is sustainable more than 20 years, and intermediate density and higher density microfilaremia person have the effect of propagating filaricide.The main method for the treatment of filaricide is to adopt chemicals at present, though medicine can reduce infection rate and sickness rate, but because frequent application, still can not get rid of filaricide has potential drug-fast danger, adds that repetition chemotherapy and long-term monitoring measure incur great expense and large quantities of professional and technical personnel.Therefore, for seeking the auxiliary or alternative method of control filaricide, the research of filaricide vaccine becomes the problem that people pay close attention to day by day.

Compare with traditional vaccine, preparation nucleic acid vaccine (dna vaccination) has special advantages and potential: (1) can the comprehensive immunne response of excitating organism, and the protective immune response of its conservative antigen has the intersection resistant function to the pathogen of different subtype; (2) nucleic acid vaccine has identical physicochemical property, for combined immunization provides possibility.Combined immunization, can be with the synantigen not of encoding gene constructed in same plasmid, or with the multiple recombiant plasmid use in conjunction of different antigen genes, the multi-functional vaccine of preparation " combination vaccine " or " polyvalent vaccine " reaches once immunity and can obtain and the identical immune effect of different immunity repeatedly; (3) antigen of nucleic acid vaccine expression is near native conformation, and its submission process and natural infection are quite similar, and antigenicity is strong, so good immune effect, and immunne response is lasting; (4) danger that does not exist the attenuated live vaccine virulence to go up, and can cause that CTL replys; (5) the existing preventive effect of nucleic acid vaccine also has therapeutical effect; (6) easy to prepare, with low cost, be convenient to storage and transportation, dry DNA granule is at room temperature relatively stable, do not need refrigerating equipment, nucleic acid vaccine is dissolved in water before giving and just can restores to the original state simply, and therefore also comparatively convenient to backwoodsman use, this all is favourable with the public health aspect economically.

Filaricide is the many cells parasite of human, life cycle and antigen molecule complexity, and with the process of host's long-term evolution in produced the panimmunity escape mechanism, so univalent vaccine is difficult to successfully induce enough effectively immune protective efficiency in vivo.Filter out and effectively have protective antigen and carry out organic assembling; different antigen molecules are designed to unite maybe will the encode amino acid whose genetic fragment of functional epi-position of different antigen molecules to be coupled together; take the means of compound polyvalent vaccine; make vaccine after inoculation, can stimulate body to produce at the different life cycle specific immune response that period, polypide was all worked; this vaccine will have versatility and reliable protectiveness, be important content and the developing direction of vaccine development.Glyceraldehyde 3-phosphate dehydro-genase (GAPDH) is a kind of physiological metabolism enzyme that extensively is present in prokaryote and the eukaryote, and important effect is arranged in the carbohydrate metabolism of organism.Parasite cystatin (CPI) has the ability that some cytokine generated and induced anti-inflammatory response of regulating as immunomodulator.They have different physiological roles in the filaricide growth course, also be important dominance antigen, make the important target molecule that becomes vaccine and drug research.

Summary of the invention

The object of the present invention is to provide the preparation method of the compound Multivalent DNA Vaccine of preiodic type Wuchereria malayi a kind of easy to prepare, effective.

Technical solution of the present invention is:

The preparation method of the compound Multivalent DNA Vaccine of a kind of preiodic type Wuchereria malayi is characterized in that: comprise the following steps:

(1) design of primers

Known array according to preiodic type Wuchereria malayi glyceraldehyde 3-phosphate dehydro-genase gene among the GeneBank, use Primer Premier5.0 software design primer, introduce BamH I and Xho I restriction enzyme site respectively at 5 of upstream and downstream primer ' end, initial, termination codon and protectiveness base;

BmGAPDH-P1: forward primer 5'-CC GGATCCACC ATG ATT AAC ATT GAC TAT-3', the underscore sequence is BamH I restriction enzyme site

BmGAPDH-P2: downstream primer 5'-CC CTCGAGTTA GGT TGC TGT AGC CAT AT-3', the underscore sequence is Xho I restriction enzyme site

Known array according to preiodic type Wuchereria malayi cystatin gene among the GeneBank, use Primer Premier5.0 software design primer, introduce Nhe I and BamH I restriction enzyme site, initial termination codon and protectiveness base respectively at the 5' of upstream and downstream primer end;

BmCPI-P1: forward primer 5'-CC GCTAGCAAC GAT GTC AAT AAA AGA AG-3',

The underscore sequence is Nhe I restriction enzyme site

BmCPI-P2: downstream primer 5'-CC GGA TCCCTA TGC ACC AAT TAA AAC-3',

The underscore sequence is BamH I restriction enzyme site;

(2) pcr amplification BmGAPDH, BmCPI gene

Calculate primer Tm value, P1Tm=62.02, P2Tm=64.94, according to the form below is set up the PCR reaction system of 25 μ l in the 0.5m1Eppendorf pipe:

Reaction system is in modulation on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45 seconds, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis;

Calculate primer Tm value, P1Tm=62.01, P2Tm=61.99, according to the form below is set up the PCR reaction system of 25 μ l in the 0.5m1Ep pipe:

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45s, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 35 circulations, last 72 ℃ are extended 10min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis;

(3) BmGAPDH, BmCPI genetic fragment purification and T carrier is connected and conversion

The colibacillary preparation of competence:

(1) get E.coli DH5 α and draw kind on 1.5% agar LB flat board with inoculating loop, 37 ℃ of constant temperature are inverted and are cultured to single bacterium colony appearance;

(2) next day, choose the monoclonal bacterium colony of an about 2mm of diameter from the LB flat board, be seeded in the 3ml LB culture medium, spend the night in 37 ℃ of 250rpm shaken cultivation;

(3) get bacterium liquid 0.3ml and add in the 30m1LB fluid medium, 37 ℃ of 250rpm vibrated 4-6 hour, made it to be in exponential phase, ice bath 1h;

(4) it is centrifugal the culture fluid branch to be installed to 4 ℃ of 1.5ml Ep pipes, and 4000rpm * 5min abandons supernatant, adds ice bath 0.1mol/L MgCl ₂100 μ l suspend, ice bath 30min;

(5) again in 4 ℃ centrifugal, 4000rpm * 5min abandons supernatant, adds ice bath 0.1mol/L CaCl ₂-10% glycerite 300 μ l suspend, and-70 ℃ of preservations are standby;

Purification BmGAPDH, BmCPI genetic fragment:

(1) get 50 μ l PCR products through 1.0% agarose gel electrophoresis, cutting contains the gel of genes of interest under uviol lamp, in the Ep pipe that immigration 1.5ml weighs in advance, claims glue heavy;

(2) add Binding Buffer in 400 μ l/100mg agarose gel ratios, 50 ℃ of-60 ℃ of water-bath 10min thoroughly melt glue.When adding hot melt adhesive, every 2min mixing once;

(3) glue that melts is transferred in the centrifugal post, room temperature is placed 2min, the centrifugal l min of 8000rpm room temperature;

(4) take off centrifugal post, abandon the waste liquid in the collecting pipe, centrifugal post is put into same collecting pipe, add 500 μ l Wash Solution, the centrifugal lmin of 8000rpm room temperature;

(5) repeating step (4) once;

(6) take off centrifugal post, abandon the waste liquid in the collecting pipe, the centrifugal lmin of blank pipe l2000rpm;

(7) centrifugal post is placed new sterilization 1.5ml Ep pipe, central authorities add 30 μ l Elution Buffer at the pillar film, and room temperature is placed 2min;

(8) the centrifugal 1min of 12000rpm room temperature, the liquid in the centrifuge tube is the dna fragmentation of recovery.

BmGAPDH, BmCPI genetic fragment are connected with the pGEM-T carrier:

In a sterilization 0.5ml Ep pipe, set up following 10 μ l linked systems:

In a sterilization 0.5ml Ep pipe, set up following 10 μ l linked systems:

Mixing, 4 ℃ connect 20h, arrange simultaneously not contain the coupled reaction system of PCR product as negative control; The conversion of pGEM-T/BmGAPDH, pGEM-T/BmCPI coupled reaction thing:

(1) get the DH5 α competent cell that a pipe is stored in-70 ℃ of refrigerators, ice bath melts;

(2) get the bacillus coli DH 5 alpha competence that coupled reaction thing 10 μ l add prepared fresh, set up negative control and positive control simultaneously, mixing gently, ice bath 20min;

(3) in 42 ℃ of heat shock 90s, change over to rapidly in the ice bath, continue ice bath 3min;

(4) add 200 μ l LB fluid mediums, hatch 45min in 37 ℃ of slow shaking;

(5) get culture, be applied to the 1.5% agar LB flat board for preparing in advance, add 100mg/ml Amp25 μ l, 24mg/ml IPTG40 μ l and 20mg/ml X-gal50 μ l in the 25ml agar LB liquid, positive in room temperature placement 30min, when treating that the glue surface does not have liquid flow, be inverted flat board, behind 37 ℃ of incubator incubated overnight 12～16h, place 4 ℃ of 2h;

(4) screening and the evaluation of the positive recombinant clone of pGEM-T/BmGAPDH, pGEM-T/BmCPI

The screening of positive recombinant clone:

(1) white single speckle of picking diameter 2mm from the agar plate is inoculated in the 3ml LB fluid medium, 37 ℃ of constant temperature 250rpm concussion overnight incubation 12h, extracting plasmid;

(2) get the bacterium liquid 1.5ml that spends the night and add in the Ep pipe, the centrifugal 2min of l2000rpm thoroughly abandons supernatant;

(3) add the solution I that 100 μ l comprise 50mmol/L glucose, 5mmol/L Tris Tris.HCl, 1.0mmol/L ethylenediaminetetraacetic acid, with the thorough suspension bacteria liquid of agitator;

(4) add the solution II that 200 μ l contain 0.4mol/L NaOH, 2%SDS, gentle mixing makes the antibacterial cracking immediately, and room temperature is placed 2min;

(5) add the solution III that 200 μ l contain 5mol/L potassium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL, centrifuge tube 5-10 time of turning upside down immediately makes abundant neutralization, the room temperature placement, and behind the 5min, the centrifugal 5min of 12000rpm;

(6) in the step (5), please be transferred in the interior centrifugal post of collecting pipe the centrifugal 1min of 10000rpm room temperature;

(7) abandon waste liquid, in centrifugal post, add the Wash Solution of 500 μ l, the centrifugal 1min of 12000rpm room temperature;

(8) repeating step (7) again;

(9) take off centrifugal post, abandon the waste liquid in the collecting pipe, the centrifugal l min of blank pipe l2000rpm;

(10) abandon waste liquid, centrifugal post is placed new sterilization 1.5ml Ep pipe, add 40 μ l Elution Buffer, behind 50 ℃ of placement 2min, the centrifugal 1min of 12000rpm room temperature.Solution in the centrifuge tube is the plasmid DNA of institute's extracting;

(11) agarose gel electrophoresis observed result;

The evaluation of the positive recombinant clone of pGEM-T/BmGAPDH, pGEM-T/BmCPI:

(1) PCR identifies:

PGEM-T/BmGAPDH PCR reaction system

PGEM-T/BmCPI PCR reaction system

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45s, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling cessation reactions; After amplification finished, product was identified through 1.0% agarose gel electrophoresis;

(2) enzyme action is identified:

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with BamH I and Xho I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and BamH I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

Seal opening with sealing film, 37 ℃ of water-bath enzyme action 4h open and seal, and continue the adding system;

The double digestion reaction system

37 ℃ of water-bath enzyme action 4h, 65 ℃ of deactivation 10min; Identify through 1.0% agarose gel electrophoresis;

(3) the nucleotide sequence order-checking is identified:

To contain the colibacillary bacterium liquid order-checking of pGEM-T/BmGAPDH, pGEM-T/BmGAPDH recombiant plasmid;

(5) pcDNA3.1 (+)/BmGAPDH/BmCPI construction of recombinant expression plasmid and evaluation

PcDNA3.1 (+)/BmGAPDH/BmCPI construction of recombinant plasmid:

(1) preparation of expression vector and purpose fragment

Recombiant plasmid pGEM-T/BmGAPDH is carried out double digestion with BamH I and Xho I, recombiant plasmid pGEM-T/BmCPI carries out double digestion with Nhe I and BamH I, expression vector pcDNA3.1 (+) plasmid carries out double digestion with Xho I and Nhe I, enzyme action system and condition are the same, through the purification recovery after the LMP sepharose electrophoresis respectively of BmGAPDH fragment, BmCPI fragment and pcDNA3.1 (+) fragment behind the double digestion;

(2) expression vector and purpose fragment is connected

Prepare an agarose gel, with sample on the sample difference equivalent that has just reclaimed, the concentration of DNA that UV spectrophotometer measuring reclaims in order to do reference for the connection ratio that goes on foot down, reclaims fragments with above-mentioned three and connects with the 4:4:1 ratio; The mol ratio of genes of interest and carrier is 4:4:1, sets up 25 μ l linked systems:

The coupled reaction system

Connect 20h under 16 ℃ of condition water-baths of PCR instrument;

(3) connect product transformed competence colibacillus DH5 α bacterium

It is the same to connect method for transformation, gets to connect liquid transformed competence colibacillus bacillus coli DH 5 alpha, coats on LB-Amp (100mg/ml) flat board 37 ℃ of incubated overnight;

PcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid is identified:

(1) Preliminary Identification

Several single bacterium colonies of picking shake the bacterium cultivation on the conversion flat board, extract plasmid DNA respectively and carry out agarose gel electrophoresis, and obvious band Preliminary screening greater than the empty carrier plasmid is the reorganization clone strain;

(2) PCR identifies

With pcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid of the positive clone of above-mentioned Preliminary Identification as template, carry out the PCR reaction with the PCR reaction system of BmGAPDH, BmCPI respectively, the PCR of the positive recombinant clone of PCR reaction system and condition and pGEM-T/BmGAPDH, pGEM-T/BmCPI identifies identical, and all what amplify genes of interest is positive recombinant clone.Be that template is done negative control with the empty carrier plasmid simultaneously;

(3) enzyme action is identified

The recombinant plasmid dna of getting Preliminary Identification carries out double digestion, does tee pipe, uses BamH I and Xho I respectively, and Nhe I and BamH I and Xho I and Nhe I are carried out the double digestion reaction, do contrast with corresponding plasmid simultaneously.Detect the endonuclease bamhi size with 1.0% LMP agarose gel electrophoresis;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Xho I and BamH I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

Seal opening with sealing film, 37 ℃ of water-bath enzyme action 4h open and seal, and continue the adding system;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and BamH I, sets up 41 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and Xho I, sets up 40 μ l enzyme action systems, 37 ℃ of water-bath enzyme action 6h, 65 ℃ of deactivation 10min; Detect endonuclease bamhi with 1.0% agarose gel electrophoresis;

(6) a large amount of extracting and purifyings of pcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid

(1) gets 50ul after the strain room temperature that will contain recombiant plasmid is melted and join the shaking table that places 37 ℃ of constant temperature, 250rpm in the 100ml LB solution that contains 50ul100mg/ml Amp and cultivate 12-16h, place 4 degree refrigerators coolings afterwards;

(2) after bacterium liquid is cultivated 12-16h, use ultraviolet spectrophotometer to measure its OD600 value, if OD600 numerical value between 0.6-0.8, shows that antibacterial is in the exponential phase of vigorous growth, then can be used for carrying out the extracting of plasmid;

(3) get and spend the night bacterium to the 50ml centrifuge tube, centrifugal 1 minute of 5000rpm, collecting precipitation is abandoned supernatant;

(4) every pipe adds the suspension of a large amount of extraction agent boxes of 5ml plasmid, the re-suspended cell precipitation;

(5) every pipe adds the lysate of a large amount of extraction agent boxes of 5ml plasmid afterwards, puts upside down centrifuge tube 4-6 time, and room temperature placement 3-5 minute makes the complete cracking of antibacterial, and solution is transparent;

(6) every pipe add a large amount of extraction agent boxes of 5ml plasmid in conjunction with liquid, put upside down 4-6 mixing of centrifuge tube immediately, visible white floccule generation, the centrifugal 10-20 of 5000rpm room temperature minute then;

(7) supernatant is poured into or is drawn in the plasmid purification column, centrifugal 2 minutes of room temperature is abandoned the collection liquid in pipe;

(8) in plasmid purification column, add the cleaning mixture of a large amount of extraction agent boxes of 7ml plasmid, centrifugal 2 minutes of room temperature, flush away impurity is abandoned the collection liquid in pipe, and centrifugal 2 minutes of 5000rpm room temperature is again removed residual liquid and trace ethanol is volatilized fully afterwards;

(9) plasmid purification column is placed on the 50ml centrifuge tube, add the eluent of a large amount of extraction agent boxes of 2ml plasmid to managing on the inner cylinder, placed 2 minutes, the 5000rpm room temperature is centrifugal 2 minutes then, add 10ml DNA purification in the gained liquid in conjunction with liquid, be added to behind the mixing in the former plasmid purification column;

(10) room temperature is abandoned the collection liquid in pipe after centrifugal 2 minutes again;

(11) cleaning mixture of a large amount of extraction agent boxes of adding 15ml plasmid in plasmid purification column, centrifugal 2 minutes of 5000rpm room temperature behind nearly step flush away impurity, is abandoned the collection liquid in pipe;

(12) the 5000rpm room temperature is centrifugal 2 minutes, removes residual liquid and trace ethanol is volatilized fully;

(13) plasmid purification column is placed on the 50ml centrifuge tube, add the 2ml eluent to managing on the inner cylinder, placed 2 minutes;

(14) the 5000rpm room temperature is centrifugal 2 minutes, and gained liquid is ultrapure plasmid;

(15) with normal saline the plasmid of purification being diluted to final concentration is 1mg/mL, and-20 ℃ of preservations are standby.

Described quick connection buffer is made up of 132mM Tris-HCl, 20mM MgCl2,2mM DTT, 2mM ATP, 15% Polyethylene Glycol.

Immunological adjuvant is that non-methylated cytosine and guanylic acid are the oligodeoxynucleotide (CpG ODN) of core, and sequence is as follows:

CpG ODN1826 5 '-TCCATGACGTTCCTGACGTT-3 ', and full chain D2EHDTPA backbone modification strengthen its nuclease resistance; Be mixed with 1mg/mL with physiological saline solution.

The present invention is easy to prepare, and is effective; Be object of study with the popular preiodic type Wuchereria malayi of China, made up preiodic type Wuchereria malayi cystatin/glyceraldehyde 3-phosphate dehydro-genase eukaryotic expression recombiant plasmid (pcDNA3.1 (+)-BmCPI/BmGAPDH), and should compound Multivalent DNA Vaccine immunity inoculation BALB/c mouse, detect the immune effect of its vaccine with methods such as RT-PCR, MTT, ELISA.But result of the test has proved this compound Multivalent DNA Vaccine inducing mouse and has produced the antigen-specific immune responses reaction that obtain promising result, the compound Multivalent DNA Vaccine of manufacturing cycle type Wuchereria malayi is succeedd.

The invention will be further described below in conjunction with drawings and Examples.

Fig. 1 is the sketch map of using standard substance diluent proportional diluted standard substance in the immunization trial of the present invention.

The specific embodiment

1 design of primers

Known array according to preiodic type Wuchereria malayi glyceraldehyde 3-phosphate dehydro-genase gene (BmGAPDH) among the GeneBank; use Primer Premier5.0 software design primer; introduce BamH I and Xho I restriction enzyme site respectively at 5 of upstream and downstream primer ' end; initial, termination codon and protectiveness base, it is synthetic that primer is given birth to worker bio-engineering corporation by Shanghai.

BmGAPDH-P1: forward primer 5'-CC GGATCCACC ATG ATT AAC ATT GAC TAT-3' underscore sequence is BamH I restriction enzyme site (Tm=62.02)

BmGAPDH-P2: downstream primer 5'-CC CTCGAGTTA GGT TGC TGT AGC CAT AT-3' underscore sequence is Xho I restriction enzyme site (Tm=64.94)

Known array according to preiodic type Wuchereria malayi cystatin (BmCPI) gene among the GeneBank; use Primer Premier5.0 software design primer; introduce Nhe I and BamH I restriction enzyme site respectively at the 5' of upstream and downstream primer end; initial termination codon and protectiveness base, primer are given birth to worker bio-engineering corporation by Shanghai and are synthesized.

BmCPI-P1: forward primer 5'-CC GCTAGCAAC GAT GTC AAT AAA AGA AG-3'

The underscore sequence is Nhe I restriction enzyme site (Tm=62.01)

BmCPI-P2: downstream primer 5'-CC GGA TCCCTA TGC ACC AAT TAA AAC-3'

The underscore sequence is BamH I restriction enzyme site (Tm=61.99)

2PCR amplification BmGAPDH, BmCPI gene

Calculating primer Tm value (P1Tm=62.02, P2Tm=64.94), according to the form below is set up the PCR reaction system of 25 μ l in 0.5m1Eppendorf pipe (Ep pipe):

Table 1BmGAPDH PCR reaction system

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45 seconds (s), 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis.

Calculating primer Tm value (P1Tm=62.01, P2Tm=61.99), according to the form below is set up the PCR reaction system of 25 μ l in the 0.5m1Ep pipe:

Table 2BmCPI PCR reaction system

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45s, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 35 circulations, last 72 ℃ are extended 10min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis.

Being connected and conversion of 3BmGAPDH, BmCPI genetic fragment purification and T carrier

3.1 the colibacillary preparation of competence

(1) get E.coli DH5 α and draw kind on 1.5% agar (LB) flat board with inoculating loop, 37 ℃ of constant temperature are inverted and are cultured to single bacterium colony appearance (about 16h).

(2) next day, choose the monoclonal bacterium colony of an about 2mm of diameter from the LB flat board, be seeded in the 3ml LB culture medium, spend the night in 37 ℃ of 250rpm shaken cultivation.

(3) get bacterium liquid 0.3ml and add in the 30m1LB fluid medium, 37 ℃ of 250rpm vibrated 4-6 hour, made it to be in exponential phase, ice bath 1h.

(4) it is centrifugal the culture fluid branch to be installed to 4 ℃ of 1.5ml Ep pipes, and 4000rpm * 5min abandons supernatant, adds ice bath 0.1mol/L MgCl ₂100 μ l suspend, ice bath 30min.

(5) again in 4 ℃ centrifugal, 4000rpm * 5min abandons supernatant, adds ice bath 0.1mol/L CaCl ₂-10% glycerite 300 μ l suspend, and-70 ℃ of preservations are standby.

3.2 purification BmGAPDH, BmCPI genetic fragment

(1) get 50 μ l PCR products through 1.0% agarose gel electrophoresis, cutting contains the gel of genes of interest under uviol lamp, in the Ep pipe that immigration 1.5ml weighs in advance, claims glue heavy.

(2) add Binding Buffer in 400 μ l/100mg agarose gel ratios, 50 ℃ of-60 ℃ of water-bath 10min thoroughly melt glue.When adding hot melt adhesive, every 2min mixing once.

(3) glue that melts is transferred in the centrifugal post, room temperature is placed 2min, the centrifugal l min of 8000rpm room temperature.

(4) take off centrifugal post, abandon the waste liquid in the collecting pipe, centrifugal post is put into same collecting pipe, add 500 μ l Wash Solution, the centrifugal l min of 8000rpm room temperature.

(5) repeating step (4) once.

(6) take off centrifugal post, abandon the waste liquid in the collecting pipe, the centrifugal l min of blank pipe l2000rpm.

(7) centrifugal post is placed new sterilization 1.5ml Ep pipe, central authorities add 30 μ l Elution Buffer at the pillar film, and room temperature is placed 2min.

(8) the centrifugal 1min of 12000rpm room temperature, the liquid in the centrifuge tube is the dna fragmentation of recovery.

3.3BmGAPDH, the BmCPI genetic fragment is connected with the pGEM-T carrier

In a sterilization 0.5ml Ep pipe, set up following 10 μ l linked systems:

Table 3T-A connects

* 132mM Tris-HCl, 20mM MgCl2,2mM DTT, 2mM ATP, 15% Polyethylene Glycol (PEG6000)

In a sterilization 0.5ml Ep pipe, set up following 10 μ l linked systems:

Table 4T-A connects

* 132mM Tris-HCl, 20mM MgCl2,2mM DTT, 2mM ATP, 15% Polyethylene Glycol (PEG6000)

Mixing, 4 ℃ connect 20h, arrange simultaneously not contain the coupled reaction system of PCR product as negative control.

3.4pGEM-T/BmGAPDH, the conversion of pGEM-T/BmCPI coupled reaction thing

(1) get the DH5 α competent cell that a pipe is stored in-70 ℃ of refrigerators, ice bath melts.

(2) get the bacillus coli DH 5 alpha competence that coupled reaction thing 10 μ l add prepared fresh, set up negative control (negative control during connection) and positive control (complete plasmid DNA) simultaneously, mixing gently, ice bath 20min.

(3) in 42 ℃ of heat shock 90s, change over to rapidly in the ice bath, continue ice bath 3min.

(4) add 200 μ l LB fluid mediums, hatch 45min in 37 ℃ of slow shaking.

(5) it is an amount of to get culture, be applied to the 1.5% agar LB flat board for preparing in advance, (adding 100mg/ml Amp25 μ l, 24mg/ml IPTG40 μ l and 20mg/ml X-gal50 μ l in the 25ml agar LB liquid), positive in room temperature placement 30min, when treating that the glue surface does not have liquid flow, be inverted flat board, behind 37 ℃ of incubator incubated overnight 12～16h, place 4 ℃ of 2h.

Screening and the evaluation of the positive recombinant clone of 4pGEM-T/BmGAPDH, pGEM-T/BmCPI

4.1 the screening of positive recombinant clone

(1) white single speckle of the about 2mm of picking diameter from the agar plate is inoculated in the 3ml LB fluid medium, 37 ℃ of constant temperature 250rpm concussion overnight incubation 12h, extracting plasmid.

(2) get the bacterium liquid 1.5ml that spends the night and add in the Ep pipe, the centrifugal 2min of l2000rpm thoroughly abandons supernatant.

(3) add 100 μ l solution I (50mmol/L glucose, 5mmol/L Tris (Tris) Tris.HCl (PH8.0), 1.0mmol/L ethylenediaminetetraacetic acid (PH8.0)), with the thorough suspension bacteria liquid of agitator.

(4) add 200 μ l solution II (0.4mol/L NaOH, 2%SDS[sodium lauryl sulphate], mix with preceding equal-volume), gentle mixing makes the antibacterial cracking immediately, room temperature placement 2min.

(5) add 200 μ l solution III (5mol/L potassium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL), centrifuge tube 5-10 time of turning upside down immediately makes abundant neutralization, the room temperature placement.Behind the 5min, the centrifugal 5min of 12000rpm.

(6) in the step (5), please be transferred in the interior centrifugal post of collecting pipe the centrifugal 1min of 10000rpm room temperature.

(7) abandon waste liquid, in centrifugal post, add the Wash Solution of 500 μ l, the centrifugal 1min of 12000rpm room temperature.

(8) repeating step (7) again.

(9) take off centrifugal post, abandon the waste liquid in the collecting pipe, the centrifugal l min of blank pipe l2000rpm.

(10) abandon waste liquid, centrifugal post is placed new sterilization 1.5ml Ep pipe, add 40 μ l Elution Buffer, behind 50 ℃ of placement 2min, the centrifugal 1min of 12000rpm room temperature.Solution in the centrifuge tube is the plasmid DNA of institute's extracting.

(11) agarose gel electrophoresis observed result.

4.2pGEM-T/BmGAPDH, the evaluation of the positive recombinant clone of pGEM-T/BmCPI

(1) PCR identifies:

Table 5pGEM-T/BmGAPDH PCR reaction system

Table 6pGEM-T/BmCPI PCR reaction system

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45s, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis.

(2) enzyme action is identified:

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with BamH I and Xho I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis

Table 7 double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and BamH I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis.

Table 8 double digestion reaction system

Seal opening with sealing film, 37 ℃ of water-bath enzyme action 4h open and seal, and continue the adding system

Table 9 double digestion reaction system

37 ℃ of water-bath enzyme action 4h, 65 ℃ of deactivation 10min.Identify through 1.0% agarose gel electrophoresis.

(3) the nucleotide sequence order-checking is identified:

The colibacillary bacterium liquid that will contain pGEM-T/BmGAPDH, pGEM-T/BmGAPDH recombiant plasmid is served the order-checking of sea living worker bio-engineering corporation.

(5pcDNA3.1+)/BmGAPDH/BmCPI construction of recombinant expression plasmid and evaluation

(5.1pcDNA3.1+)/BmGAPDH/BmCPI construction of recombinant plasmid

(1) preparation of expression vector and purpose fragment:

Recombiant plasmid pGEM-T/BmGAPDH is carried out double digestion with BamH I and Xho I, recombiant plasmid pGEM-T/BmCPI carries out double digestion with Nhe I and BamH I, expression vector pcDNA3.1 (+) plasmid carries out double digestion with Xho I and Nhe I, enzyme action system and condition are the same, through the purification recovery after the LMP sepharose electrophoresis respectively of BmGAPDH fragment, BmCPI fragment and pcDNA3.1 (+) fragment behind the double digestion.

(2) being connected of expression vector and purpose fragment:

Prepare an agarose gel, with sample on the sample difference equivalent that has just reclaimed, relative concentration after rough estimate three's recovery once, the concentration of while DNA that UV spectrophotometer measuring reclaims, so that for the connection ratio that goes on foot is down done reference, reclaim fragments with above-mentioned three and connect with the 4:4:1 ratio.

The mol ratio of genes of interest and carrier is 4:4:1, sets up 25 μ l linked systems:

Table 10 coupled reaction system

Connect 20h under 16 ℃ of condition water-baths of PCR instrument.

(3) connect product transformed competence colibacillus DH5 α bacterium

It is the same to connect method for transformation, gets to connect liquid transformed competence colibacillus bacillus coli DH 5 alpha, coats on LB-Amp (100mg/ml) flat board 37 ℃ of incubated overnight.

5.2pcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid is identified

(1) Preliminary Identification

Several single bacterium colonies of picking shake the bacterium cultivation on the conversion flat board, extract plasmid DNA respectively and carry out agarose gel electrophoresis, and obvious band Preliminary screening greater than the empty carrier plasmid is the reorganization clone strain.

(2) PCR identifies

With pcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid of the positive clone of above-mentioned Preliminary Identification as template, carry out the PCR reaction with the PCR reaction system of BmGAPDH, BmCPI respectively, the PCR of the positive recombinant clone of PCR reaction system and condition and pGEM-T/BmGAPDH, pGEM-T/BmCPI identifies identical, and all what amplify genes of interest is positive recombinant clone.Be that template is done negative control with the empty carrier plasmid simultaneously.

(3) enzyme action is identified

The recombinant plasmid dna of getting Preliminary Identification carries out double digestion, does tee pipe, uses BamH I and Xho I respectively, and Nhe I and BamH I and Xho I and Nhe I are carried out the double digestion reaction, do contrast with corresponding plasmid simultaneously.Detect the endonuclease bamhi size with 1.0% LMP agarose gel electrophoresis.

Table 11 double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Xho I and BamH I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis.

Table 12 double digestion reaction system

Seal opening with sealing film, 37 ℃ of water-bath enzyme action 4h open and seal, and continue the adding system

Table 13 double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and BamH I, sets up 41 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis.

Table 14 double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and X ho I, sets up 40 μ l enzyme action systems, 37 ℃ of water-bath enzyme action 6h, 65 ℃ of deactivation 10min.Detect endonuclease bamhi with 1.0% agarose gel electrophoresis.

A large amount of extracting and purifyings of (6pcDNA3.1+)-BmCPI/BmGAPDH recombiant plasmid

(1) getting 50ul after the strain room temperature that will contain recombiant plasmid is melted joins and contains 50ul Amp(ampicillin, place the shaking table overnight incubation (12-16h) of 37 ℃ of constant temperature, 250rpm in 100ml LB solution 100mg/ml), place 4 degree refrigerator coolings afterwards;

(2) after bacterium liquid is cultivated 12-16h, use ultraviolet spectrophotometer to measure its OD600 value, if OD600 numerical value between 0.6-0.8, shows that antibacterial is in the exponential phase of vigorous growth, then can be used for carrying out the extracting of plasmid;

(3) get and spend the night bacterium to the 50ml centrifuge tube, centrifugal 1 minute of 5000rpm, collecting precipitation is abandoned supernatant.(but repetitive operation);

(4) every pipe adds 5ml suspension (a large amount of extraction agent boxes of plasmid), the re-suspended cell precipitation;

(5) every pipe adds 5ml lysate (a large amount of extraction agent boxes of plasmid) afterwards, puts upside down centrifuge tube 4-6 time, and room temperature was placed 3-5 minute, made the complete cracking of antibacterial, and solution is transparent;

(6) every pipe adds 5ml in conjunction with liquid (a large amount of extraction agent boxes of plasmid), puts upside down 4-6 mixing of centrifuge tube immediately, and visible white floccule produces the centrifugal 10-20 of 5000rpm room temperature minute then;

(7) supernatant is poured into or is drawn in the plasmid purification column, centrifugal 2 minutes of High-speed room-temperature is abandoned the collection liquid in pipe;

(8) in plasmid purification column, add 7ml cleaning mixture (a large amount of extraction agent boxes of plasmid), centrifugal 2 minutes of High-speed room-temperature, flush away impurity is abandoned the collection liquid in pipe, centrifugal 2 minutes of 5000rpm room temperature is again removed residual liquid and trace ethanol is volatilized fully afterwards.

(9) plasmid purification column is placed on the 50ml centrifuge tube, add 2ml eluent (a large amount of extraction agent boxes of plasmid) to managing on the inner cylinder, placed 2 minutes, the 5000rpm room temperature is centrifugal 2 minutes then, adds 10ml solution VI(DNA purification in the gained liquid in conjunction with liquid) be added in the former plasmid purification column behind the mixing;

(10) room temperature is abandoned the collection liquid in pipe after centrifugal 2 minutes again;

(11) in plasmid purification column, add 15ml cleaning mixture (a large amount of extraction agent boxes of plasmid), centrifugal 2 minutes of 5000rpm room temperature,

Behind nearly step flush away impurity, abandon the collection liquid in pipe;

(12) the 5000rpm room temperature is centrifugal 2 minutes, removes residual liquid and trace ethanol is volatilized fully.

(13) plasmid purification column is placed on the 50ml centrifuge tube, add the 2ml eluent to managing on the inner cylinder, placed 2 minutes;

(14) the 5000rpm room temperature is centrifugal 2 minutes, and gained liquid is ultrapure plasmid.

(15) with normal saline the plasmid of purification being diluted to final concentration is 1mg/mL, and-20 ℃ of preservations are standby.

The preparation of 7 immunological adjuvant CpG ODN

Be that the oligodeoxynucleotide (CpG ODN) of core is the most representative with non-methylated cytosine and guanylic acid in the novel adjuvant, CpG ODN is synthetic by Shanghai Ying Jun biotech firm, and synthetic sequence is as follows:

CpG ODN18265 '-TCCATGACGTTCCTGACGTT-3 ', and full chain D2EHDTPA backbone modification strengthen its nuclease resistance.Be mixed with 1mg/mL with physiological saline solution.

The immunity inoculation of 8DNA vaccine

8.1 laboratory animal grouping

Divide 4 groups at random with BALB/c mouse, 10 every group, the A group: the PBS matched group, the B group:

PcDNA3.1 (+)/CpG group, C group: pcDNA3.1-BmCPI/CpG group, D group: pcDNA3.1 – BmCPI/BmGAPDH/CpG group.

8.2 inoculation position pretreatment

Nucleic acid vaccination is taked left back lower limb tibialis anterior injection system.Bupivacaine hydrochloride inj is diluted to 0.5mg/ml, 24h before each inoculation, 50 μ l carry out pretreatment in inoculation position injection bupivacaine hydrochloride.

8.3 immunity inoculation

The nucleic acid vaccine that dilution is good and adjuvant are through left back lower limb tibialis anterior immune mouse, injected dose: A group: PBS (100 μ l), B group: plasmid pcDNA3.1 (+)/CpG (100 μ g), C group: plasmid pcDNA3.1-BmCPI/CpG (100 μ g), D group: plasmid pcDNA3.1-BmCPI/BmGAPDH/CpG(100 μ g/30 μ g), immunity is 3 times altogether, and each immunity is 2 weeks at interval.

9 vaccination sample collection and detections

9.1 sample collection

1. the extraction of immune mouse injection site muscle

4 weeks after the last immunity, every group of left back lower limb tibialis anterior muscle of getting 1 mice ,-70 ℃ of preservations.

2. collect mice serum respectively at 2,4,6 weeks after the last immunity with the eyeball excise method, get 4 mices for every group:

(1) left hand is caught mice, and head inclination is downward, and holds cervical region with forefinger and thumb and make the eyeball evagination.Owing to the pressure to cervical region makes cerebrovascular congestion, can make the temporary transient forfeiture of sensation.

(2) right hand is held aseptic elbow tweezer, extracts the eyeball of evagination fast, rapidly drop of blood is gone in the aseptic 1.5ml Ep pipe, and when reaching aequum (about 500 μ l), left hand loosens, with the hemostasis of aseptic dry cotton ball compressing eye socket.

(3) room temperature is placed 1h, and the centrifugal 3000rpm * 15min of room temperature moves to upper serum in the new 1.5ml Ep pipe, posts label, is stored in-20 ℃.

3. immune mouse spleen cell collection (operating on the super-clean bench):

(1) after the last immunity 4,6 all every group get 4 mices respectively, cervical vertebra dislocation causes death.

(2) get aseptic 10ml screw socket pipe, add the 4ml lymphocyte separation medium, place 37 ℃ of preheating 2h.

(3) neck is evaded the deadly mice of mortar and put 2～3min in the liquor-saturated solution of 75% second, sterilization skin.

(4) routine disinfection white mice abdominal part, left veutro is up; With surgical forceps mice left side skin of abdomen is mentioned gently, cut about 1cm rimmer knife mouth with operating scissors; With hands skin of abdomen is torn, exposed peritoneum, red color visible strip spleen; Mention mouse peritoneum with the aseptic operation tweezer, cut off to expose fully the abdominal cavity with the aseptic operation cutter; Careful separation goes out spleen.

(5) spleen is placed the little plate of containing an amount of aseptic Hank ' s liquid, clean spleen.

(6) aseptic 100 order copper sieve is put into the plate that another contains an amount of aseptic Hank ' s liquid, spleen is placed on it, grind the connective tissue of remaining spleen only on copper sieves gently with aseptic Glass rod.

(7) the 10ml screw socket pipe with preheating tilts 45 °, and aseptic dropper pastes tube wall suspension in the plate slowly is transferred in the pipe, and attention can not be broken through the interface of lymph separating medium.

(8) the centrifugal 2000rpm * 20min of room temperature, suspension is divided into 4 layers.

(9) get the 2nd layer of splenocyte suspension, move in the 50ml centrifuge tube that contains 10mlHank ' s liquid the centrifugal 1000rpm * 10min of room temperature.

(10) discard supernatant liquid, add 10mlHank ' s liquid, piping and druming mixing, the centrifugal 1000rpm * 10min of room temperature.

(11) discard supernatant liquid, add 10mlHank ' s liquid, the piping and druming mixing is got 10 μ l and is added in the counting chamber counting.

(12) the centrifugal 1000rpm * 10min of room temperature discards supernatant liquid, adds the 5ml complete medium, the piping and druming mixing.

9.2 detect

1.RT-PCR the expression of method validation plasmid.

2. lymphocyte transformation test (mtt assay):

(1) use trypan blue dyeing counting living cells is answered the lymphocyte suspension more than 95%, and adjusting cell concentration is 2 * 10 ⁶Individual/m1.

(2) add cell suspension 100 μ l in the every hole of 96 well culture plates, 3 multiple holes are established in every hole, add ConA in 1 hole.

(3) 5 μ l put and contain 5% CO ₂Incubator is cultivated 72h for 37 ℃.

(4) cultivate the preceding 4h of end, add MTT10 μ l, mixing 1min on agitator puts and continues to cultivate 4h in the incubator.

(5) take out culture plate, every hole adds 100 μ l DMSO, behind mixing 1min on the agitator, leaves standstill 20min, and purple crystal is dissolved fully.

(6) survey the OD value in the 570nm place with enzyme-linked immunosorbent assay instrument.

(stimulation index SI=adds concanavalin A (ConA) stimulates A respectively to organize lymphocyte stimulation indices ₅₇₀Meansigma methods/do not add ConA stimulate A ₅₇₀Meansigma methods).

3. the mensuration of serum I NF-γ:

(1) in advance 20min takes out INF-γ from 4 ℃ and measures test kits, with balance to room temperature.

(2) concentrated cleaning solution is diluted (1:20) with distilled water.

(3) with standard substance diluent proportional diluted standard substance (showing as Fig. 1).

(4) take out lath from balance to the sealing bag of room temperature, except blank well, respectively serum specimen or variable concentrations standard substance are added each hole (100 μ l/ hole), seal reacting hole with the shrouding gummed paper, 37 ℃ of incubators are hatched 90min.

(5) wash plate, get rid of liquid in the most hole, every hole adds cleaning mixture 350 μ l, gets rid of most liquid after leaving standstill 30s, pats dry in absorbent paper, washes plate 5 times.

(6) except blank well, each hole adds biotinylated antibody working solution (100 μ l/ hole), seals reacting hole with the shrouding gummed paper, and 37 ℃ of incubators are hatched 60min.

(7) wash plate 5 times.

(8) except blank well, each hole adds enzyme conjugates working solution (100 μ l/ hole), seals reacting hole with the shrouding gummed paper, and 37 ℃ of incubators are hatched 30min.

(9) wash plate 5 times.

(10) every hole adds developer (100 μ l/ hole), and 37 ℃ of incubators of lucifuge are hatched 10-15min.

(11) every hole adds stop buffer (100 μ l/ hole), measures OD with microplate reader in the 5min behind the mixing ₄₅₀Value.

4. the mensuration of serum il-4 (step is with the mensuration of serum I NF-γ).

5. data analysis

Use SPSS11.5 statistics software sample data is carried out statistical analysis, adopt the t check.

Table 15 mouse lymphocyte breeder reaction result of the test (mtt assay, A ₅₇₀Value)

Annotate: #and* difference has statistical significance, P＜0.05.

The different immunity of table 16 time mouse cell factor IFN-γ level (pg/ml)

Annotate: #and* difference has statistical significance, P＜0.05.

The different immunity of table 17 time mouse cell factor IL-4 level (pg/ml)

Annotate: #and* difference has statistical significance, P＜0.05.

Amplify preiodic type Wuchereria malayi BmGAPDH, BmCPI genetic fragment by success of the test.Detect through 1.0% agarose gel electrophoresis, BmGAPDH, BmCPI amplified fragments size conform to substantially with 877bp, the 621bp of expection.BmGAPDH gene sequencing result is total to 877bp from beginning codon ATG to termination codon, and its expressing protein is 292 aminoacid.BmCPI gene sequencing result is total to 621bp from start codon ATG to termination codon, its expressing protein is 200 aminoacid.The sequence blast that provides on two gene sequencing and GeneBank result relatively, homology is 99%, proves that cloned sequence is correct.Carrier for expression of eukaryon pcDNA3.1 (+) plasmid is connected with BmGAPDH, BmCPI target gene fragment, prepares the compound Multivalent DNA Vaccine of preiodic type Wuchereria malayi (pcDNA3.1 (+)-BmCPI/BmGAPDH).

Should compound Multivalent DNA Vaccine immunity inoculation BALB/c mouse back leg tibialis anterior, immune group injected in mice position muscle is carried out the RT-PCR checking, obtain and target gene fragment band of the same size, prove that the complex gene dna vaccination that makes up can be at the mice expression in vivo.Detect the immune effect of its vaccine with MTT, ELISA method, the lymphocyte transformation test result shows: the immune group mouse spleen lymphocyte is external after ConA stimulates, its lymphocyte stimulation indices raises gradually with the prolongation of immunity time, apparently higher than matched group (P＜0.05); The immune cell factor testing result shows: immune group mice serum IFN-γ level 2 weeks after immunity just begin apparently higher than matched group, and statistical significance (P＜0.05) is arranged; The IL-4 level of immune group mice serum 4 weeks after immunity begin apparently higher than matched group, statistical significance (P＜0.05) is arranged, but the compound Multivalent DNA Vaccine inducing mouse of above experimental result proof preiodic type Wuchereria malayi produces the antigen-specific immune responses reaction, obtains promising result.

Claims (3)

1. the preparation method of the compound Multivalent DNA Vaccine of preiodic type Wuchereria malayi is characterized in that: comprise the following steps:

(1) design of primers

Known array according to preiodic type Wuchereria malayi glyceraldehyde 3-phosphate dehydro-genase gene among the GeneBank, use Primer Premier5.0 software design primer, introduce BamH I and Xho I restriction enzyme site respectively at 5 of upstream and downstream primer ' end, initial, termination codon and protectiveness base;

BmGAPDH-P1: forward primer 5'-CC GGATCCACC ATG ATT AAC ATT GAC TAT-3', the underscore sequence is BamH I restriction enzyme site

BmGAPDH-P2: downstream primer 5'-CC CTCGAGTTA GGT TGC TGT AGC CAT AT-3', the underscore sequence is Xho I restriction enzyme site

Known array according to preiodic type Wuchereria malayi cystatin gene among the GeneBank, use Primer Premier5.0 software design primer, introduce Nhe I and BamH I restriction enzyme site, initial termination codon and protectiveness base respectively at the 5' of upstream and downstream primer end;

BmCPI-P1: forward primer 5'-CC GCTAGCAAC GAT GTC AAT AAA AGA AG-3',

The underscore sequence is Nhe I restriction enzyme site

BmCPI-P2: downstream primer 5'-CC GGA TCCCTA TGC ACC AAT TAA AAC-3',

The underscore sequence is BamH I restriction enzyme site;

(2) pcr amplification BmGAPDH, BmCPI gene

Calculate primer Tm value, P1Tm=62.02, P2Tm=64.94, according to the form below is set up the PCR reaction system of 25 μ l in the 0.5m1Eppendorf pipe:

Reaction system is in modulation on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45 seconds, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis;

Calculate primer Tm value, P1Tm=62.01, P2Tm=61.99, according to the form below is set up the PCR reaction system of 25 μ l in the 0.5m1Ep pipe:

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45s, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 35 circulations, last 72 ℃ are extended 10min again, 4 ℃ of cooling cessation reactions.After amplification finished, product was identified through 1.0% agarose gel electrophoresis;

(3) BmGAPDH, BmCPI genetic fragment purification and T carrier is connected and conversion

The colibacillary preparation of competence:

(1) get E.coli DH5 α and draw kind on 1.5% agar LB flat board with inoculating loop, 37 ℃ of constant temperature are inverted and are cultured to single bacterium colony appearance;

(2) next day, choose the monoclonal bacterium colony of an about 2mm of diameter from the LB flat board, be seeded in the 3ml LB culture medium, spend the night in 37 ℃ of 250rpm shaken cultivation;

(3) get bacterium liquid 0.3ml and add in the 30m1LB fluid medium, 37 ℃ of 250rpm vibrated 4-6 hour, made it to be in exponential phase, ice bath 1h;

(4) it is centrifugal the culture fluid branch to be installed to 4 ℃ of 1.5ml Ep pipes, and 4000rpm * 5min abandons supernatant, adds ice bath 0.1mol/L MgCl ₂100 μ l suspend, ice bath 30min;

(5) again in 4 ℃ centrifugal, 4000rpm * 5min abandons supernatant, adds ice bath 0.1mol/L CaCl ₂-10% glycerite 300 μ l suspend, and-70 ℃ of preservations are standby;

Purification BmGAPDH, BmCPI genetic fragment:

(1) get 50 μ l PCR products through 1.0% agarose gel electrophoresis, cutting contains the gel of genes of interest under uviol lamp, in the Ep pipe that immigration 1.5ml weighs in advance, claims glue heavy;

(2) add Binding Buffer in 400 μ l/100mg agarose gel ratios, 50 ℃ of-60 ℃ of water-bath 10min thoroughly melt glue.When adding hot melt adhesive, every 2min mixing once;

(3) glue that melts is transferred in the centrifugal post, room temperature is placed 2min, the centrifugal l min of 8000rpm room temperature;

(4) take off centrifugal post, abandon the waste liquid in the collecting pipe, centrifugal post is put into same collecting pipe, add 500 μ l Wash Solution, the centrifugal lmin of 8000rpm room temperature;

(5) repeating step (4) once;

(6) take off centrifugal post, abandon the waste liquid in the collecting pipe, the centrifugal lmin of blank pipe l2000rpm;

(7) centrifugal post is placed new sterilization 1.5ml Ep pipe, central authorities add 30 μ l Elution Buffer at the pillar film, and room temperature is placed 2min;

(8) the centrifugal 1min of 12000rpm room temperature, the liquid in the centrifuge tube is the dna fragmentation of recovery.

BmGAPDH, BmCPI genetic fragment are connected with the pGEM-T carrier:

In a sterilization 0.5ml Ep pipe, set up following 10 μ l linked systems:

In a sterilization 0.5ml Ep pipe, set up following 10 μ l linked systems:

Mixing, 4 ℃ connect 20h, arrange simultaneously not contain the coupled reaction system of PCR product as negative control; The conversion of pGEM-T/BmGAPDH, pGEM-T/BmCPI coupled reaction thing:

(1) get the DH5 α competent cell that a pipe is stored in-70 ℃ of refrigerators, ice bath melts;

(2) get the bacillus coli DH 5 alpha competence that coupled reaction thing 10 μ l add prepared fresh, set up negative control and positive control simultaneously, mixing gently, ice bath 20min;

(3) in 42 ℃ of heat shock 90s, change over to rapidly in the ice bath, continue ice bath 3min;

(4) add 200 μ l LB fluid mediums, hatch 45min in 37 ℃ of slow shaking;

(5) get culture, be applied to the 1.5% agar LB flat board for preparing in advance, add 100mg/ml Amp25 μ l, 24mg/ml IPTG40 μ l and 20mg/ml X-gal50 μ l in the 25ml agar LB liquid, positive in room temperature placement 30min, when treating that the glue surface does not have liquid flow, be inverted flat board, behind 37 ℃ of incubator incubated overnight 12～16h, place 4 ℃ of 2h;

(4) screening and the evaluation of the positive recombinant clone of pGEM-T/BmGAPDH, pGEM-T/BmCPI

The screening of positive recombinant clone:

(1) white single speckle of picking diameter 2mm from the agar plate is inoculated in the 3ml LB fluid medium, 37 ℃ of constant temperature 250rpm concussion overnight incubation 12h, extracting plasmid;

(2) get the bacterium liquid 1.5ml that spends the night and add in the Ep pipe, the centrifugal 2min of l2000rpm thoroughly abandons supernatant;

(3) add 100 μ l and comprise 50mmol/L glucose, 5mmol/L Tris

The solution I of Tris.HCl, 1.0mmol/L ethylenediaminetetraacetic acid is with the thorough suspension bacteria liquid of agitator;

(4) add the solution II that 200 μ l contain 0.4mol/L NaOH, 2%SDS, gentle mixing makes the antibacterial cracking immediately, and room temperature is placed 2min;

(5) add the solution III that 200 μ l contain 5mol/L potassium acetate 60mL, glacial acetic acid 11.5mL, water 28.5mL, centrifuge tube 5-10 time of turning upside down immediately makes abundant neutralization, the room temperature placement, and behind the 5min, the centrifugal 5min of 12000rpm;

(6) in the step (5), please be transferred in the interior centrifugal post of collecting pipe the centrifugal 1min of 10000rpm room temperature;

(7) abandon waste liquid, in centrifugal post, add the Wash Solution of 500 μ l, the centrifugal 1min of 12000rpm room temperature;

(8) repeating step (7) again;

(9) take off centrifugal post, abandon the waste liquid in the collecting pipe, the centrifugal lmin of blank pipe l2000rpm;

(10) abandon waste liquid, centrifugal post is placed new sterilization 1.5ml Ep pipe, add 40 μ l Elution Buffer, behind 50 ℃ of placement 2min, the centrifugal 1min of 12000rpm room temperature.Solution in the centrifuge tube is the plasmid DNA of institute's extracting;

(11) agarose gel electrophoresis observed result;

The evaluation of the positive recombinant clone of pGEM-T/BmGAPDH, pGEM-T/BmCPI:

(1) PCR identifies:

PGEM-T/BmGAPDH PCR reaction system

PGEM-T/BmCPI PCR reaction system

Reaction system is being modulated on ice, mixing, and centrifugal rearmounted PCR instrument, behind 94 ℃ of pre-degeneration 3min, 94 ℃ of degeneration 45s, 57 ℃ of annealing 45s, 72 ℃ are extended 90s, 30 circulations, last 72 ℃ are extended 7min again, 4 ℃ of cooling cessation reactions; After amplification finished, product was identified through 1.0% agarose gel electrophoresis;

(2) enzyme action is identified:

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with BamH I and Xho I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and BamH I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

Seal opening with sealing film, 37 ℃ of water-bath enzyme action 4h open and seal, and continue the adding system;

The double digestion reaction system

37 ℃ of water-bath enzyme action 4h, 65 ℃ of deactivation 10min; Identify through 1.0% agarose gel electrophoresis;

(3) the nucleotide sequence order-checking is identified:

To contain the colibacillary bacterium liquid order-checking of pGEM-T/BmGAPDH, pGEM-T/BmGAPDH recombiant plasmid;

(5) pcDNA3.1 (+)/BmGAPDH/BmCPI construction of recombinant expression plasmid and evaluation

PcDNA3.1 (+)/BmGAPDH/BmCPI construction of recombinant plasmid:

(1) preparation of expression vector and purpose fragment

Recombiant plasmid pGEM-T/BmGAPDH is carried out double digestion with BamH I and Xho I, recombiant plasmid pGEM-T/BmCPI carries out double digestion with Nhe I and BamH I, expression vector pcDNA3.1 (+) plasmid carries out double digestion with Xho I and Nhe I, enzyme action system and condition are the same, through the purification recovery after the LMP sepharose electrophoresis respectively of BmGAPDH fragment, BmCPI fragment and pcDNA3.1 (+) fragment behind the double digestion;

(2) expression vector and purpose fragment is connected

Prepare an agarose gel, with sample on the sample difference equivalent that has just reclaimed, the concentration of DNA that UV spectrophotometer measuring reclaims in order to do reference for the connection ratio that goes on foot down, reclaims fragments with above-mentioned three and connects with the 4:4:1 ratio;

The mol ratio of genes of interest and carrier is 4:4:1, sets up 25 μ l linked systems:

The coupled reaction system

Connect 20h under 16 ℃ of condition water-baths of PCR instrument;

(3) connect product transformed competence colibacillus DH5 α bacterium

It is the same to connect method for transformation, gets to connect liquid transformed competence colibacillus bacillus coli DH 5 alpha, coats on LB-Amp (100mg/ml) flat board 37 ℃ of incubated overnight;

PcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid is identified:

(1) Preliminary Identification

Several single bacterium colonies of picking shake the bacterium cultivation on the conversion flat board, extract plasmid DNA respectively and carry out agarose gel electrophoresis, and obvious band Preliminary screening greater than the empty carrier plasmid is the reorganization clone strain;

(2) PCR identifies

With pcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid of the positive clone of above-mentioned Preliminary Identification as template, carry out the PCR reaction with the PCR reaction system of BmGAPDH, BmCPI respectively, the PCR of the positive recombinant clone of PCR reaction system and condition and pGEM-T/BmGAPDH, pGEM-T/BmCPI identifies identical, and all what amplify genes of interest is positive recombinant clone.Be that template is done negative control with the empty carrier plasmid simultaneously;

(3) enzyme action is identified

The recombinant plasmid dna of getting Preliminary Identification carries out double digestion, does tee pipe, uses BamH I and Xho I respectively, and Nhe I and BamH I and Xho I and Nhe I are carried out the double digestion reaction, do contrast with corresponding plasmid simultaneously.Detect the endonuclease bamhi size with 1.0% LMP agarose gel electrophoresis;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Xho I and BamH I, sets up 25 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

Seal opening with sealing film, 37 ℃ of water-bath enzyme action 4h open and seal, and continue the adding system;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and BamH I, sets up 41 μ l enzyme action systems, detects endonuclease bamhi with 1.0% agarose gel electrophoresis;

The double digestion reaction system

The PCR that learns from else's experience identifies correct recombiant plasmid, carries out double digestion with Nhe I and Xho I, sets up 40 μ l enzyme action systems, 37 ℃ of water-bath enzyme action 6h, 65 ℃ of deactivation 10min; Detect endonuclease bamhi with 1.0% agarose gel electrophoresis;

(6) a large amount of extracting and purifyings of pcDNA3.1 (+)-BmCPI/BmGAPDH recombiant plasmid

(1) gets 50ul after the strain room temperature that will contain recombiant plasmid is melted and join the shaking table that places 37 ℃ of constant temperature, 250rpm in the 100ml LB solution that contains 50ul100mg/ml Amp and cultivate 12-16h, place 4 degree refrigerators coolings afterwards;

(2) after bacterium liquid is cultivated 12-16h, use ultraviolet spectrophotometer to measure its OD600 value, if OD600 numerical value between 0.6-0.8, shows that antibacterial is in the exponential phase of vigorous growth, then can be used for carrying out the extracting of plasmid;

(3) get and spend the night bacterium to the 50ml centrifuge tube, centrifugal 1 minute of 5000rpm, collecting precipitation is abandoned supernatant;

(4) every pipe adds the suspension of a large amount of extraction agent boxes of 5ml plasmid, the re-suspended cell precipitation;

(5) every pipe adds the lysate of a large amount of extraction agent boxes of 5ml plasmid afterwards, puts upside down centrifuge tube 4-6 time, and room temperature placement 3-5 minute makes the complete cracking of antibacterial, and solution is transparent;

(6) every pipe add a large amount of extraction agent boxes of 5ml plasmid in conjunction with liquid, put upside down 4-6 mixing of centrifuge tube immediately, visible white floccule generation, the centrifugal 10-20 of 5000rpm room temperature minute then;

(7) supernatant is poured into or is drawn in the plasmid purification column, centrifugal 2 minutes of room temperature is abandoned the collection liquid in pipe;

(8) in plasmid purification column, add the cleaning mixture of a large amount of extraction agent boxes of 7ml plasmid, centrifugal 2 minutes of room temperature, flush away impurity is abandoned the collection liquid in pipe, and centrifugal 2 minutes of 5000rpm room temperature is again removed residual liquid and trace ethanol is volatilized fully afterwards;

(9) plasmid purification column is placed on the 50ml centrifuge tube, add the eluent of a large amount of extraction agent boxes of 2ml plasmid to managing on the inner cylinder, placed 2 minutes, the 5000rpm room temperature is centrifugal 2 minutes then, add 10ml DNA purification in the gained liquid in conjunction with liquid, be added to behind the mixing in the former plasmid purification column;

(10) room temperature is abandoned the collection liquid in pipe after centrifugal 2 minutes again;

(11) cleaning mixture of a large amount of extraction agent boxes of adding 15ml plasmid in plasmid purification column, centrifugal 2 minutes of 5000rpm room temperature behind nearly step flush away impurity, is abandoned the collection liquid in pipe;

(12) the 5000rpm room temperature is centrifugal 2 minutes, removes residual liquid and trace ethanol is volatilized fully;

(13) plasmid purification column is placed on the 50ml centrifuge tube, add the 2ml eluent to managing on the inner cylinder, placed 2 minutes;

(14) the 5000rpm room temperature is centrifugal 2 minutes, and gained liquid is ultrapure plasmid;

(15) with normal saline the plasmid of purification being diluted to final concentration is 1mg/mL, and-20 ℃ of preservations are standby.

2. the preparation method of the compound Multivalent DNA Vaccine of preiodic type Wuchereria malayi according to claim 1, it is characterized in that: described quick connection buffer is made up of 132mM Tris-HCl, 20mM MgCl2,2mM DTT, 2mM ATP, 15% Polyethylene Glycol.

3. the preparation method of the compound Multivalent DNA Vaccine of preiodic type Wuchereria malayi according to claim 1, it is characterized in that: immunological adjuvant is that non-methylated cytosine and guanylic acid are the oligodeoxynucleotide (CpG ODN) of core, and sequence is as follows:

CpG ODN1826 5 '-TCCATGACGTTCCTGACGTT-3 ', and full chain D2EHDTPA backbone modification strengthen its nuclease resistance; Be mixed with 1mg/mL with physiological saline solution.

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