CN100335636C - Thioredoxin gene of Schistosoma japonicum (Chinese Mainland Strain) and its cloning expression method and application - Google Patents
Thioredoxin gene of Schistosoma japonicum (Chinese Mainland Strain) and its cloning expression method and application Download PDFInfo
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Abstract
The present invention relates to a sequence of a thioredoxin (SjcTrx) gene of schistosoma japonicum (Chinese Mainland Strain), a cloning expression method thereof and an application thereof. The SjcTrx gene disclosed by the present invention has a nucleotide sequence shown in the sequence 1 and a coded corresponding amino acid sequence. The present invention also discloses an expression method of the SjcTrx gene in colibacillus. Purified recombination protein and DNA vaccines are prepared and used for immunizing C57BL/6 mice, and results show that the SjcTrx gene of the present invention can be used for preparing recombinant protein vaccines and DNA vaccines for preventing and treating schistosomiasis japonicum.
Description
Technical field
The invention belongs to biological technical field, be specifically related to sequence and the cloning expression method and the application of Schistosoma japonicum (Chinese mainland strain) Trx (SjcTrx) gene.
Background technology
Schistosomiasis japanica once be widely current in China Yangtze valley and on the south 12 provinces, at the beginning of nineteen fifty generation, the whole nation has 1,160 ten thousand schistosomicide patients approximately, intermediate host's snail distribution area 14,800,000,000 m
2Through the over half a century unremitting effort, the prevention and cure of snail fever work of China has obtained remarkable achievement, and to nineteen ninety-five, 5 provinces (district, city) such as existing Guangdong, Shanghai, Fujian, Guangxi, Zhejiang have reached schistosomiasis propagation blocking-up standard.But in recent years, China's schistosomiasis endemic scope enlarges to some extent, and prevention and cure of schistosomiasis work situation is relatively severeer, and outstanding behaviours exists: the schistosomiasis infection number increases, and partial area acute infection occurs and break out epidemic situation; Some areas epidemic situation is revivable, and the epidemic-stricken area scope obviously enlarges and to Urban Sprawl; The diffusion of oncomelania big area, human poultry infection's danger increases.Reported in recent years that patient's number was in rising trend, wherein, the acute infection number obviously increases, and increasing by 59%, 2003 year than calendar year 2001 in 2002 increased by 22% than 2002 again, and an acute schistosomicide is broken out epidemic situation surplus having taken place 30.2003, the whole nation also had 110 popular schistosomicide in county, and patient 84.3 ten thousand, oncomelania area 3,500,000,000 m
2More than; Still had patient 84.25 ten thousand in 2004, oncomelania area 38.46 hundred million m
2All occupy first of the popular country of schistosomiasis japanica more than the popular scope of schistosomicide and have spiral surface long-pending wide, patient's number.The extensive chemotherapy of spiral shell and the praziquantel of going out facts have proved the prevention and cure of snail fever problem that at all do not solve.The problem of superinfection seriously exists, the life-time service of only effective chemotherapeutics praziquantel may produce drug resistance strain and the existence of a large amount of Mammals reservoir hosts, is the subject matter that China's prevention and cure of snail fever work faces.Thereby seek long-acting prophylactico-therapeutic measures and become major objective, in view of the long-lasting of vaccine prevention effect and economic characteristics, be used as one of schistosomicide integrated control important measures and receive much attention: chemotherapy means (fugitive) combine with long lasting measure (vaccine).Seeking the new candidate antigens molecule of vaccine is one of emphasis of schistosomiasis japanica vaccine research.
Trx (Trx) is a kind of small molecular weight protein that redoxomorphism is arranged, and has anti-peroxidation in vivo, removes radical, effects such as protective tissue organ, and implant with apoptosis, immunomodulatory, embryo and grow relevant.Up to the present, Shang Weijian relates to the report of schistosoma japonicum Chinese strain Trx (SjcTrx) gene in this specification sheets and applied research thereof.
Summary of the invention
Technical problem to be solved by this invention is open schistosoma japonicum Chinese strain Trx (SjcTrx) gene order, and the clone of SjcTrx gene and the expression method in intestinal bacteria, and the application of open SjcTrx gene in preparation control Schistosoma japonicum disease vaccine.
The present invention is according to the Schistosoma japonicum Philippines strain thioredoxin gene sequences Design primer of announcing, total RNA is a template with Schistosoma japonicum continent strain adult, the complete encoder block of reverse transcription PCR (RT-PCR) amplification SjcTrx gene, be cloned into respectively among prokaryotic expression carrier pET28a and the carrier for expression of eukaryon pcDNA3, made up recombinant plasmid pET28a-SjcTrx and pcDNA3-SjcTrx respectively, and the cloned genes nucleotide sequence has been measured.
SjcTrx gene disclosed by the invention has the coding amino acid sequence corresponding shown in nucleotide sequence shown in the sequence 1 and the sequence 2.106 amino acid of SjcTrx gene open reading frame coding, according to sequencing result deduced amino acid and Schistosoma japonicum Philippines strain Trx 97% homology (seeing accompanying drawing 1) is arranged, 43% homology (seeing accompanying drawing 2) is arranged with the Schistosoma mansoni Trx.
The expression method of SjcTrx gene disclosed by the invention in intestinal bacteria is that the SjcTrx gene clone is arrived among the prokaryotic expression plasmid pET28a (+), and construction recombination plasmid pET28a-SjcTrx after conversion BL21 expresses bacterium, uses the IPTG abduction delivering.The SDS-PAGE electrophoretic analysis shows that recombinant plasmid obtains to efficiently express the about 14kDa of expressing protein molecular weight, called after reSjcTrx.
Western Blotting test demonstration recombinant protein can be discerned by schistosoma japonicum infection rabbit anteserum and reSjcTrx recombinant antigen immune serum, and reactionless with normal rabbit serum and normal mouse serum, illustrates that reSjcTrx has good antigenicity.With Ni post affinity chromatography purification of recombinant proteins reSjcTrx, with the recombinant protein of purifying as antigen, immune C
57BL/6 mouse, immunizing dose are 15 μ g/ mouse, and immunity is 3 times altogether, at interval 2 weeks.3 weeks were used the schistosoma japonicum cercariae challenge infection after the last immunity, cutd open mouse extremely behind the challenge infection 6 weeks, were counted as worm's ovum number in borer population and the liver.The result shows, compares with infecting control group, and worm reduction rate and hepatic tissue egg reduction rate that reSjcTrx adds behind the ISA720 adjuvant immunity group mouse challenge infection are respectively 22.8% and 29.5%.
The present invention discovers that the SjcTrx gene can be used for preparation control Schistosoma japonicum disease vaccine.The SjcTrx gene clone in eukaryon expression plasmid pcDNA3, is built into dna vaccination pcDNA3-SjcTrx.Nucleic acid vaccine injection C
57The BL/6 mouse, SjcTrx can express in the myocyte of mouse muscle injection site.At C
57In the protection test of BL/6 mouse immune, every mouse is injected 100 μ g nucleic acid vaccines through quadriceps muscle of thigh, injects altogether 3 times, at interval 2 weeks.3 weeks were used the schistosoma japonicum cercariae challenge infection after the last immunity, cutd open mouse extremely behind the challenge infection 6 weeks, were counted as worm's ovum number in borer population and the liver.The result shows, compares with infecting control group, and nucleic acid vaccine immunity group worm reduction rate and hepatic tissue egg reduction rate are respectively 45.7% and 41.4%, and nucleic acid vaccine and protein vaccine combined immunization group worm reduction rate and hepatic tissue egg reduction rate are respectively 37.0% and 31.6%.
The invention will be further described below in conjunction with embodiment.
Description of drawings
Fig. 1: schistosoma japonicum Chinese strain Trx and Schistosoma japonicum Philippines strain Trx homology compare of analysis
Fig. 2: Schistosoma japonicum continent strain Trx and Schistosoma mansoni Trx homology compare of analysis
Fig. 3: RT-PCR amplifying target genes and pET28a-SjcTrx recombinant plasmid double digestion and PCR identify
1:1kb DNA marker; The 2:pET28a empty plasmid; The 3:pET28a-SjcTrx recombinant plasmid; 4:pET28a empty plasmid double digestion (BamHI and SalI); 5:pET28a-SjcTrx recombinant plasmid double digestion (BamHI and SalI); 6:pET28a empty plasmid DNA is a template PCR product; The 7:pET28a-SjcTrx recombinant plasmid dna is a template PCR product; 8:SjcTrx RT-PCR product; 9:100bp DNA Marker
Fig. 4: SDS-PAGE analyzes the abduction delivering and the purifying of recombinant protein
M: protein standard molecular weight; Before 1:pET28a/BL21 induces; 2:pET28a/BL21 induces back 4h; Before 3:pET28a-SjcTrx/BL21 induces; 4:pET28a-SjcTrx/BL21 induces back 4h; 5: the recombinant protein reSjcTrx of purifying
Fig. 5: Western Blotting is to the analysis of recombinant protein reSjcTrx
M: protein standard molecular weight; 1: react with the schistosoma japonicum infection rabbit anteserum; 2: react with the Schistosoma japonicum normal rabbit serum; 3: react with reSjcTrx recombinant antigen immune mouse serum; 4: with the normal mouse sero-reaction
Fig. 6: ELISA detects reorganization Trx (reSjcTrx) vaccine immune mouse serological specificity IgG antibody horizontal
Fig. 7: RT-PCR amplifying target genes and pcDNA3-SjcTrx recombinant plasmid double digestion and PCR identify
1:1kb DNA marker; The 2:pcDNA3 empty plasmid; The 3:pcDNA3-SjcTrx recombinant plasmid; 4:pcDNA3 empty plasmid double digestion (BamHI and SalI); 5:pcDNA3-SjcTrx recombinant plasmid double digestion (BamHI and SalI); 6:pcDNA3 empty plasmid DNA is a template PCR product; The 7:pcDNA3-SjcTrx recombinant plasmid dna is a template PCR product; 8:SjcTrx RT-PCR product; 9:100bp DNA Marker
Fig. 8: the immunostaining test detects nucleic acid vaccine and expresses in the mouse muscle tissue
A：pcDNA3-SjcTrx B：pcDNA3
Fig. 9: ELISA detects nucleic acid vaccine (pcDNA3-SjcTrx) immune serum specific IgG antibodies
Embodiment
1. material
1.1 Schistosoma japonicum continent strain cercaria and adult: the positive oncomelania that contains ripe cercaria is provided by key lab of this institute, ordinary method escape cercaria.Schistosoma japonicum continent strain adult (male and female mixing) is taken from the rabbit that schistosoma japonicum cercariae infected for 6 weeks.
1.2 bacterial strain and plasmid: e. coli bl21 strain and carrier pET28a are available from Novagen company.
1.3 laboratory animal: 6 the week age C
57The BL/6 female mice is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center.
1.4 main agents: total RNA extraction agent box, plasmid DNA extraction agent box, PCR product purification test kit, enzyme is cut the product purification test kit, the Taq archaeal dna polymerase, dNTPs, ThermoScript II (AMV), RNasin, the T4DNA ligase enzyme, restriction enzyme (BamHI and SalI), sheep anti-mouse igg-HRP etc. are U.S. Promega company product, Tris alkali, agarose, O-Phenylene Diamine (OPD), benzidine amine (DAB) and 4-chloro-1-naphthols etc. are U.S. Sigma product, the protein purification test kit is purchased the company in Qiagen, the MontanideISA720 adjuvant is provided by French Seppic company, pvdf membrane is a U.S. BIO-RAD company, Tryptone and Yeast Extract are OXOID company product, and other reagent is homemade analytical pure.
1.5 goal gene primer design: analyze Schistosoma japonicum Philippines strain Trx encoding gene, design a pair of primer:
Upstream primer 1:5 '-GC
GGATCCATGAGTAACGTACTGCATATAG-3 '
Downstream primer 1:
5′-GC
GTCGACTTAGCCATATTTCATATATAAGAGGCAC-3′
Upstream primer 5 ' end is introduced restriction enzyme BamHI restriction enzyme site (line part) and initiator codon (black matrix), downstream primer 5 ' introducing restriction enzyme SalI restriction enzyme site (line part) and terminator codon (black matrix).Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
2. method
2.1SjcTrx the amplification of gene:
Extract the total RNA of Schistosoma japonicum adult by total RNA extraction agent cassette method, reverse transcription generates cDNA first chain, and operation is undertaken by the test kit specification sheets.With the reverse transcription product is template, pcr amplification SjcTrx gene.Reaction system is:
10×reaction Buffer 5.0μl
Upstream primer 11.0 μ l
Downstream primer 11.0 μ l
dNTPs 2.0μl
Reverse transcription product 5.0 μ l
Taq archaeal dna polymerase 0.5 μ l
Deionized water 35.5 μ l
Add above reactant successively, centrifugal in short-term behind the mixing, carry out pcr amplification.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of 45sec, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 72 ℃ prolong 7min.
2.2 the structure of recombinant expression vector pET28a-SjcTrx:
Above-mentioned PCR product carries out 1% low melting-point agarose gel electrophoresis, with PCR product purification test kit to the purpose fragment purifying of tapping rubber.Purified pcr product and plasmid pET28a be with restriction enzyme BamHI and SalI double digestion, and the dna fragmentation behind the double digestion is with 1% low melting-point agarose gel electrophoresis, cuts the product purification test kit purifying of tapping rubber with enzyme.Double digestion plasmid pET28a (+) DNA is connected through the T4DNA ligase enzyme with PCR product endonuclease bamhi, and 4 ℃ of connections are spent the night.Connect product and transform BL21 (DE3) competence bacterium (cold Calcium Chloride Method preparation), bacterium liquid is coated on the LB solid medium that contains kantlex (Kan) (30 μ g/ml), in 37 ℃ of overnight incubation.
2.3 the evaluation of recombinant plasmid pET28a-SjcTrx:
Adopt restriction enzyme digestion enzyme BamHI and SalI double digestion, PCR and agarose gel electrophoresis that recombinant plasmid dna is identified.The recombinant plasmid that screens acquisition is further carried out nucleotide sequencing identify, determine whether the external source target gene fragment is correctly inserted in pET28a (+) carrier.
2.4 the abduction delivering of recombinant protein and purifying:
Recombinant plasmid pET28a-SjcTrx transforms BL21 bacterium liquid and draws LB solid culture substrate (containing Kan 30 μ g/ml) overnight incubation, the single colony inoculation of picking is in 3ml LB liquid nutrient medium (containing Kan 30 μ g/ml) overnight incubation, diluted the incubated overnight bacterium in LB liquid nutrient medium (containing Kan 30 μ g/ml) with 1: 100, cultivate 4h in 37 ℃ 250 rev/mins, add isopropylthiogalactoside (IPTG) to final concentration 1mmol/L, abduction delivering 4h.Centrifugal collection thalline, behind the ultrasonic thalline that breaks, the centrifuging and taking supernatant liquor is by the affinity chromatography column purification target protein of Ni-NTA, and the albumen of purifying is with the uv-absorbing standard measure.
2.5 western blot test (Western blotting):
Purifying Schistosoma japonicum continent strain Trx recombinant protein (reSjcTrx) is behind the SDS-PAGE electrophoresis, and electrotransfer changes the film condition and shifts 1h for the 30V constant voltage to pvdf membrane.The pvdf membrane of adhesion protein is immersed in the 0.01mol/L phosphoric acid buffer (PBS) that contains 1% skim-milk 4 ℃ of sealings and spends the night.Immerse 37 ℃ of slight jolting reaction 1h in the anti-reaction solution (dilution in 1: 100 of schistosoma japonicum infection rabbit anteserum or the dilution in 1: 500 of reSjcTrx immune serum, 5% calf serum, diluent are PBS); With the slight jolting washing of PBS-T (0.1%Tween-20) room temperature 3 times, each 5min; Immerse two anti-reaction solutions (HRP mark goat anti-rabbit igg or sheep anti-mouse igg dilution in 1: 1000,5% calf serum, diluent are PBS), 37 ℃ of slight jolting reaction 1h, PBS-T washing 3 times, each 5min; With PBS washing 1 time, whether benzidine amine (DAB) and the colour developing of 4-chloro-1-naphthols are observed the recombinant protein band and are discerned by above serum antibody.
2.6reSjcTrx the observation of immanoprotection action:
With 30 6 the week age female C
57The BL/6 mouse is divided into 3 groups at random, 10 every group: reSjcTrx vaccine immunity group, ISA720 adjuvant control group and infection control group.Every mouse of vaccine immunity group is through item back multiple spot subcutaneous injection emulsive 15 μ g reSjcTrx recombinant antigen and ISA720 adjuvants, and immunity is 3 times altogether, at interval 2 weeks; The adjuvant control group mice is then injected emulsive physiological saline and is added the ISA720 adjuvant, infects control group mice and does not inject any antigen or adjuvant.Last is after 3 weeks of immunity, and the every mouse of experimental group and control group infects schistosoma japonicum cercariae (30 ± 1) bar through skin of abdomen.The cercaria challenge infection was dissected the perfusion of mouse portal vein and is collected imago of blood fluke after 6 weeks, counted the imago of blood fluke number of collecting in every mouse, calculated the worm reduction rate of vaccine immune mouse:
According to a conventional method with the digestion of 8% potassium hydroxide, count schistosome ovum number in every murine liver tissue after every murine liver tissue shreds respectively, calculate the hepatic tissue egg reduction rate:
2.7ELISA detect the immune serum specific IgG antibodies:
Vaccine immunity group and 2 control group mice respectively at immunity before, before the challenge infection and cut open kill before blood sampling, separation of serum ,-20 ℃ of preservations are standby.Measure the special IgG antibody horizontal of mice serum Schistosoma japonicum Trx with the ELISA method.ReSjcTrx recombinant antigen 1 μ g/ hole bag quilt; Add mice serum diluent (mice serum to be measured 1: 80 dilution, diluent is 0.01mol/LPBS) 100 μ l/ holes and hatch 1h in 37 ℃, then with PBS-T washing 3 times, each 5min; Add sheep anti-mouse igg diluent (dilution in 1: 1000, diluent is the same), hatch 1h, PBS-T washing 3 times, each 5min in 37 ℃; The 20min that develops the color in the OPD colour developing liquid adds 2mol/LH
2SO
4Termination reaction records and respectively organizes A
492Value.
2.8 statistical method:
With the SAS software package data are carried out the t check analysis.
3 results
3.1RT-PCR amplifying target genes and pET28a-Trx recombinant plasmid are identified:
SjcTrx encoding gene pcr amplification product detects about 334bp (seeing accompanying drawing 3) through 1% agarose gel electrophoresis, big or small consistent with expectation.The pET28a-Trx recombinant plasmid dna is that template PCR reaction product is through agarose gel electrophoresis through BamHI and SalI double digestion and with the recombinant plasmid dna, all see the similar band of RT-PCR product size, and pET28a empty plasmid DNA there is no this band through double digestion (BamHI and SalI) and PCR.
3.2SjcTrx gene nucleotide series is measured and is analyzed:
Sequencing result shows that recombinant plasmid pET28a-SjcTrx successfully constructs, confirm SjcTrx coding 106 amino acid (sequence 2), the relative molecular weight 14 000 (14kDa of expressing protein, contain 6 Histidines of carrier), find that behind the homology compare of analysis homology with Schistosoma japonicum Philippines strain Trx aminoacid sequence is 97% (seeing accompanying drawing 1), with the homology of Schistosoma mansoni Trx aminoacid sequence be 43% (seeing accompanying drawing 2).
3.3 the purifying of recombinant chou abduction delivering and recombinant antigen (reSjcTrx):
Behind pET28a-SjcTrx/BL21 engineering bacteria usefulness IPTG abduction delivering, locate a special band of expression at Mr14 000 (14kDa), and pET28a/BL21 empty plasmid contrast bacterium induces the back not have this band.Expressing protein also has a band (accompanying drawing 4) at Mr14 000 place behind the Ni-NTA purifying.
3.4Western Blotting：
Purifying reSjcTrx and schistosoma japonicum infection rabbit anteserum and recombinant antigen immune serum have significant reaction (accompanying drawing 5).This explanation reorganization Trx has antigenicity and immunoreactivity preferably.
3.5reSjcTrx immune mouse protection effect observation:
Cutd open in 6 weeks behind the immune mouse challenge infection and kill, collect adult and counting through the portal vein perfusion, vaccine immunity group (reSjcTrx adds the ISA adjuvant) is with ISA720 adjuvant control group and infect control group relatively, seizes into the equal significance of borer population difference (P＜0.05).Compare with infecting control group, the worm reduction rate of vaccine immunity group mouse is 22.8% (table 1).
Cut open mouse extremely behind the challenge infection 6 weeks, collect and separate japonice ovum and counting in the murine liver tissue, vaccine immunity group (reSjcTrx adds the ISA adjuvant) is with ISA720 adjuvant control group and infect control group relatively, and worm's ovum is counted the equal significance of difference (P＜0.05).Compare with infecting control group, the egg reduction rate of vaccine immunity group mouse is 29.5% (table 1).
Table 1 reSjcTrx (reSjcTrx) vaccine immune mouse subtract worm
Rate and egg reduction rate
| Group Group | On average seize into borer population (the Average no. ofWorms of x ± s) (x ± s) | Worm reduction rate (%) Worm reduction rate (%) | Average hepatic tissue worm's ovum number (the Average no.of eggs in liver tissue of x ± s) (x ± s) | Egg reduction rate (%) Egg reduction rate (%) |
| ReSjcTrx+ISA720 adjuvant immunity group | 14.2±4.1 * | 22.8 | 8521±3278 * | 29.5 |
| ISA720 adjuvant control group | 19.9±7.3 | - | 11502±2642 | 4.9 |
| Infect control group | 18.4±3.8 | - | 12095±4172 | - |
Annotate: compare with ISA720 adjuvant control group and infection control group,
*P＜0.053.6ELISA detects recombinant protein reSjcTrx immune serum specific IgG antibodies:
Serological specificity IgGA value is than adjuvant control group with infect control group mice (P＜0.01) (accompanying drawing 6) that significantly raise behind the recombinant protein vaccine immune group mouse immune.
The structure of embodiment 2, Schistosoma japonicum Trx nucleic acid vaccine and the protection test of nucleic acid vaccine mouse immune
1 material
1.1 Schistosoma japonicum continent strain cercaria and adult: the positive oncomelania that contains ripe cercaria is provided by key lab of this institute, ordinary method escape cercaria.Schistosoma japonicum continent strain adult (male and female mixing) is taken from the rabbit that schistosoma japonicum cercariae infected for 6 weeks.
1.2 bacterial strain and plasmid: the e. coli jm109 strain available from Invotrogen company and carrier pcDNA3 available from Novagen company.
1.3 laboratory animal: 6 the week age C
57The BL/6 female mice is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center.
1.4 main agents: it is U.S. Promega company product that total RNA extraction agent box, plasmid DNA extraction agent box, PCR product purification test kit, enzyme are cut product purification test kit, Taq archaeal dna polymerase, dNTPs, ThermoScript II (AMV), RNasin, T4DNA ligase enzyme, restriction enzyme (BamHI and EcoRI), sheep anti-mouse igg-HRP etc., Tris alkali, agarose, penbritin and O-Phenylene Diamine (OPD) etc. are U.S. Sigma product, Tryptone and Yeast Extract are OXOID company product, and other reagent is homemade analytical pure.
1.5 goal gene primer design: analyze Schistosoma japonicum Philippines strain thioredoxin gene, analyze reading frame, design a pair of primer:
Upstream primer 2:5 '-gc
GgatccATG AGTAACGTACTGC-3 '
Downstream primer 2:5 '-gc
GaattcTCATTTGTGTTTCCGG-3 '
Upstream primer is introduced the BamHI restriction enzyme site, and downstream primer is introduced the EcoRI restriction enzyme site.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
2. method
2.1SjcTrx the amplification of gene:
Extract the total RNA of Schistosoma japonicum adult by total RNA extraction agent cassette method, the ordinary method reverse transcription generates cDNA first chain.With the reverse transcription product is template, pcr amplification SjcTrx gene.Reaction system is:
10×reaction Buffer 5.0μl
dNTPs 2.0μl
Reverse transcription product 5.0 μ l
Taq archaeal dna polymerase 0.5 μ l
Deionized water 35.5 μ l
Add above reactant successively, centrifugal in short-term behind the mixing, carry out pcr amplification.The PCR reaction conditions is: 94 ℃ of pre-sex change 5min; 94 ℃ of 45sec, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; Last 72 ℃ prolong 7min.
2.2 the structure of recombinant expression vector pcDNA3-SjcTrx:
Above-mentioned PCR product carries out 1% low melting-point agarose gel electrophoresis, with PCR product purification test kit to the purpose fragment purifying of tapping rubber.Purified pcr product and plasmid pcDNA3 be with restriction enzyme BamHI and EcoRI double digestion, and the dna fragmentation behind the double digestion is with 1% low melting-point agarose gel electrophoresis, cuts the product purification test kit purifying of tapping rubber with enzyme.Purifying pcDNA3 double digestion dna fragmentation is connected through the T4DNA ligase enzyme with the purified pcr product endonuclease bamhi, and 4 ℃ of connections are spent the night.Connect product and transform JM109 competence bacterium (cold Calcium Chloride Method preparation), bacterium liquid is coated on the LB solid medium that contains penbritin (Amp) (100 μ g/ml), in 37 ℃ of overnight incubation.Next day, picking was separated good single bacterium colony respectively at cultivating amplification in the LB nutrient solution that contains Amp (100 μ g/ml), and the extracting plasmid DNA is used for further evaluation.
2.3 the evaluation of eukaryotic expression recombinant plasmid pcDNA3-SjcTrx and a large amount of preparation:
Adopt restriction enzyme BamHI and EcoRI double digestion, pcr amplification and agarose gel electrophoresis that recombinant plasmid dna is identified.The positive colony recombinant plasmid that screening obtains is further identified with the nucleotide sequence order-checking.Identify that the correct single colony inoculation of pcDNA3-SjcTrx/JM109 engineering bacteria cultivates amplification in the LB nutrient solution that contains Amp (100 μ g/ml), add LB liquid nutrient medium (containing Amp100 μ g/ml) in 1: 100 ratio next day and cultivate 4h in 37 ℃ 250 rev/mins.Centrifugal collection thalline extracts recombinant plasmid pcDNA3-SjcTrx in a large number with alkaline lysis, is dissolved in stroke-physiological saline solution, identifies through BamHI and EcoRI double digestion and nucleotide sequencing.Ultraviolet spectrophotometer is measured the A of plasmid
260And A
280, calculate content and purity check.Be equipped with pcDNA3 empty plasmid DNA in contrast with legal system.
2.4 the expression of dna vaccination in the local organization of intramuscular injection observed in immunoenzyme stain test (IEST):
Get 66 the week age female C
57The BL/6 mouse, every mouse is through quadriceps muscle of thigh injection pcDNA3-SjcTrx dna vaccination 100 μ g, get the intramuscular injection site local organization in back 24 hours, 48 hours and 72 hours respectively at injection and prepare frozen section, with the expression of goal gene in the IEST detection injection site tissue, the person is contrast (2 C with injection pcDNA3 empty plasmid
57The BL/6 mouse).Take out frozen section, return back to room temperature, 0.01mol/L PBS rinsing is sealed 2h with the 0.01mol/L PBS that contains 3% calf serum in 37 ℃; Add anti-reSjcTrx immune serum (dilution in 1: 50,5% calf serum) and hatch 1h for 37 ℃, with PBS-T (1%Tween-20) washing 3 times, each 5min; Add the sheep anti-mouse igg (dilution in 1: 100) of HRP mark, hatch 1h for 37 ℃; PBS-T washing 3 times adds DAB colour developing liquid, color development at room temperature 20min, and PBS-T washs 3 termination reactions, and opticmicroscope is observed down.
2.5 the mouse immune provide protection is observed:
With 30 6 the week age female C
57The BL/6 mouse is divided into 3 groups at random: pcDNA3-SjcTrx nucleic acid vaccine immunity group, pcDNA3 empty plasmid control group and challenge infection control group, every group of 10 mouse.Through abdominal injection 0.75% Sodital sodium solution anesthetized mice, every mouse of nucleic acid vaccine immunity group is injected 100 μ gpcDNA3-SjcTrx plasmid DNA through quadriceps muscle of thigh by 10 μ l/ gram body weight, injects altogether 3 times, at interval 2 weeks; Every mouse of empty plasmid control group is injected 100 μ gpcDNA3 plasmid DNA in the corresponding time through quadriceps muscle of thigh; Infect control group and then do not inject any plasmid DNA.In 3 weeks after the last immunity, each is organized every mouse and infects schistosoma japonicum cercariae (30 ± 1) bar through skin of abdomen.6 weeks behind challenge infection, cut open mouse extremely, adult is collected in the portal vein perfusion, and murine liver tissue digests the back in microscopically counting japonice ovum number with 8% potassium hydroxide, and calculates worm reduction rate and egg reduction rate.
Each organize mouse respectively at immunity before, before the challenge infection and cut open kill before blood sampling, separation of serum ,-20 ℃ of preservations are used for the detection of specific antibody.
2.6ELISA detect immune serum specific antibody (IgG) level:
Measure above-mentioned mice serum specific IgG level with the ELISA method.Recombinant antigen reSjcTrx 1 μ g/ hole bag quilt adds 37 ℃ of sealings of the 0.01mol/L PBS 2h that contains 5% calf serum; Add mice serum to be measured (dilution in 1: 80) and hatch 1h for 37 ℃; With PBS-T (containing 0.1%Tween-20) washing 3 times, each 5min; Add the sheep anti-mouse igg (dilution in 1: 1000) of HRP mark, hatch 1h for 37 ℃; PBS-T washing 3 times adds the colour developing of OPD colour developing liquid, 2mol/LH
2SO
4Termination reaction is measured and is respectively organized A
492Value.
2.7 statistical method:
With the SAS software package data are carried out the t check analysis.
3. result
3.1RT-PCR amplification SjcTrx gene and pcDNA3-SjcTrx recombinant plasmid are identified:
SjcTrx encoding gene pcr amplification product detects about 334bp (accompanying drawing 7) through agarose gel electrophoresis, and is consistent with the expectation size.The SjcTrx gene is connected with the pcDNA3 prokaryotic expression carrier and the recombinant plasmid (pcDNA3-Trx) that makes up is that template PCR reaction product is through agarose gel electrophoresis through BamHI and EcoRI double digestion and with the recombinant plasmid dna, all see similar size strip, and pcDNA3 empty plasmid DNA there is no this band through double digestion and PCR.
3.2SjcTrx gene nucleotide series is measured:
Through the nucleotide sequencing checking, SjcTrx encoding gene and above-mentioned protein vaccine gene (sequence 1 and 2) in full accord show that the cDNA of SjcTrx is cloned into carrier for expression of eukaryon pcDNA3 with correct direction, the success of reorganization pcDNA3-SjcTrx plasmid construction.
3.3 nucleic acid vaccine is expressed in the mouse muscle tissue:
After the pcDNA3-SjcTrx injection mouse muscle histogenic immunity, get injection site mouse muscle tissue preparation frozen section in different time, IEST detects local expression albumen situation.As a result, injection 24h just has tangible SjcTrx protein expression, the pale brown look dyeing of visible specificity sheet or point-like in the mouse myocyte, and do not see specific stain particle (accompanying drawing 8) in the empty plasmid immune mouse Skeletal Muscle Cell.Show that pcDNA3-SjcTrx can express in the injection site myocyte.
3.4 the nucleic acid vaccine immunity protective effect is observed:
6 weeks behind the challenge infection, vaccine immunity group, empty plasmid control group and challenge infection control group are on average seized into borer population and are respectively 10.0,15.2 and 18.0, the vaccine immunity group is compared with the challenge infection control group with the empty plasmid control group, and difference all has significance (P＜0.05).Compare with infecting control group, the worm reduction rate of vaccine immunity group and empty plasmid control group is respectively 45.7% and 17.4% (table 2).
The average worm's ovum number of vaccine immunity group, empty plasmid control group and challenge infection control group is respectively 7086,8098 and 12095, and the vaccine immunity group is compared difference with the infection control group highly significant (P＜0.01).Compare with infecting control group, the egg reduction rate of vaccine immunity group and empty plasmid control group is respectively 41.4% and 33.0% (table 2).
Table 2pcDNA3-SjcTrx nucleic acid vaccine immunity mouse worm reduction rate and egg reduction rate
| Group Group | On average seize into borer population, (the Average no. of worms of x ± s), (x ± s) | Worm reduction rate (%) Worm reduction rate (%) | Average hepatic tissue worm's ovum number (the Average no.of eggs in liver tissue of x ± s) (x ± s) | Egg reduction rate (%) Egg reduction rate (%) |
| PcDNA3-SjcTrx vaccine immunity group pcDNA3-SjcTrx vaccine | 10.0±5.1(1) | 45.7 | 7086±4200(1) | 41.4 |
| PcDNA3 empty plasmid control group pcDNA3control | 15.2±4.3(2) | 17.4 | 8098±3384(2) | 33.0 |
| Challenge infection control group Challenging infection control | 18.0±3.8 | - | 12095±4172 | - |
Compare (1) P＜0.01, (2) P＜0.05 with the infection control group
3.5 immune serum specific IgG level:
Serological specificity IgGA value significantly raises (P＜0.05) than empty plasmid and challenge infection control group behind the nucleic acid vaccine immunity group mouse immune, and empty plasmid control group and challenge infection control group no significant difference (P＞0.05) (accompanying drawing 9).
Sequence table
＜110〉Prevention ﹠ Control Station of Parasitic Disease, China Diseases Prevention ﹠ C
＜120〉schistosoma japonicum Chinese strain thioredoxin gene and cloning expression method thereof and application
＜130〉specification sheets, claims
<160>2
<170>PatentIn version 3.2
<210>1
<211>321
<212>DNA
＜213〉schistosoma japonicum Chinese strain Trx (SjcTrx) gene order
<220>
<221>CDS
<222>(1)..(318)
<400>1
atg agt aac gta ctg cat ata gaa acc gat gac gat ttt gat tct ttc 48
Met Ser Asn Val Leu His Ile Glu Thr Asp Asp Asp Phe Asp Ser Phe
1 5 10 15
tta aag gaa aat aag gat aaa tta att gtt gtt gat ttt ttc gca act 96
Leu Lys Glu Asn Lys Asp Lys Leu Ile Val Val Asp Phe Phe Ala Thr
20 25 30
tgg tgt ggg ccg tgt aaa aaa ata gct cct gct ttc gaa gcg ttg agc 144
Trp Cys Gly Pro Cys Lys Lys Ile Ala Pro Ala Phe Glu Ala Leu Ser
35 40 45
gct gat cgt tcg gca tta tat gtg aag gtt gac gtg gat aaa ctt gaa 192
Ala Asp Arg Ser Ala Leu Tyr Val Lys Val Asp Val Asp Lys Leu Glu
50 55 60
gaa act gcc aaa aga tac gat gta aca gct atg cca acg ttt att gtg 240
Glu Thr Ala Lys Arg Tyr Asp Val Thr Ala Met Pro Thr Phe Ile Val
65 70 75 80
ata aaa aat ggc gaa aaa gtc gat aca gtg gtt gga gct tct ata gaa 288
Ile Lys Asn Gly Glu Lys Val Asp Thr Val Val Gly Ala Ser Ile Glu
85 90 95
aat gtt gaa gct gcg atc agg aaa cac aaa taa 321
Asn Val Glu Ala Ala Ile Arg Lys His Lys
100 105
<210>2
<211>106
<212>PRT
＜213〉schistosoma japonicum Chinese strain Trx (SjcTrx) gene order
<400>2
Met Ser Asn Val Leu His Ile Glu Thr Asp Asp Asp Phe Asp Ser Phe
1 5 10 15
Leu Lys Glu Asn Lys Asp Lys Leu Ile Val Val Asp Phe Phe Ala Thr
20 25 30
Trp Cys Gly Pro Cys Lys Lys Ile Ala Pro Ala Phe Glu Ala Leu Ser
35 40 45
Ala Asp Arg Ser Ala Leu Tyr Val Lys Val Asp Val Asp Lys Leu Glu
50 55 60
Glu Thr Ala Lys Arg Tyr Asp Val Thr Ala Met Pro Thr Phe Ile Val
65 70 75 80
Ile Lys Asn Gly Glu Lys Val Asp Thr Val Val Gly Ala Ser Ile Glu
85 90 95
Asn Val Glu Ala Ala Ile Arg Lys His Lys
100 105
Claims (5)
1. nucleotide sequence of schistosoma japonicum Chinese strain Trx of encoding is characterized in that the corresponding aminoacid sequence of this nucleotide sequence and coding thereof is as follows:
atg agt aac gta ctg cat ata gaa acc gat gac gat ttt gat tct ttc 48
Met Ser Asn Val Leu His Ile Glu Thr Asp Asp Asp Phe Asp Ser Phe
1 5 10 15
tta aag gaa aat aag gat aaa tta att gtt gtt gat ttt ttc gca act 96
Leu Lys Glu Asn Lys Asp Lys Leu Ile Val Val Asp Phe Phe Ala Thr
20 25 30
tgg tgt ggg ccg tgt aaa aaa ata gct cct gct ttc gaa gcg ttg agc 144
Trp Cys Gly Pro Cys Lys Lys Ile Ala Pro Ala Phe Glu Ala Leu Ser
35 40 45
gct gat cgt tcg gca tta tat gtg aag gtt gac gtg gat aaa ctt gaa 192
Ala Asp Arg Ser Ala Leu Tyr Val Lys Val Asp Val Asp Lys Leu Glu
50 55 60
gaa act gcc aaa aga tac gat gta aca gct atg cca acg ttt att gtg 240
Glu Thr Ala Lys Arg Tyr Asp Val Thr Ala Met Pro Thr Phe Ile Val
65 70 75 80
ata aaa aat ggc gaa aaa gtc gat aca gtg gtt gga gct tct ata gaa 288
Ile Lys Asn Gly Glu Lys Val Asp Thr Val Val Gly Ala Ser Ile Glu
85 90 95
aat gtt gaa gct gcg atc agg aaa cac aaa taa 321
Asn Val Glu Ala Ala Ile Arg Lys His Lys
100 105。
2. the cloning process of the nucleotide sequence of the described coding schistosoma japonicum Chinese strain of claim 1 Trx is characterized in that designed primer sequence is in order to obtain the nucleotide sequence of complete ORF:
Upstream primer 1:5 '-GCGGATCCATGAGTAACGTACTGCATATAG-3 '
Downstream primer 1:5 '-GCGTCGACTTAGCCATATTTCATATATAAGAGGCAC-3 '
Or,
Upstream primer 2:5 '-GCGGATCCATGAGTAACGTACTGC-3 '
Downstream primer 2:5 '-GCGAATTCTCATTTGTGTTTCCGG-3 '.
3. the nucleotides sequence of the described coding schistosoma japonicum Chinese strain of claim 1 Trx is listed in the expression method in the intestinal bacteria, it is characterized in that the employed host bacterium of this method is e. coli bl21 (DE3), prokaryotic expression plasmid is pET28a+, and the recombinant plasmid of formation is pET28a-SjcTrx.
4. the nucleotides sequence of the described coding schistosoma japonicum Chinese strain of claim 1 Trx is listed in and prepares the application that prevents and treats in schistosomiasis japanica recombinant protein vaccine and the nucleic acid vaccine.
5. application according to claim 4 is characterized in that wherein said nucleic acid vaccine is made up of SjcTrx and eukaryon expression plasmid pcDNA3, and the SjcTrx gene clone in eukaryon expression plasmid pcDNA3, is built into dna vaccination pcDNA3-SjcTrx.
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| CN1235164A (en) * | 1998-05-08 | 1999-11-17 | 北京医科大学 | Peptide for vaccine of schistosomiasis |
| US6294341B1 (en) * | 1998-03-21 | 2001-09-25 | Korea Institute Of Science And Technology | Method for detecting a substance having an activity to inhibit HIV infection using immunoassay and variant protein used for said method |
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| US6294341B1 (en) * | 1998-03-21 | 2001-09-25 | Korea Institute Of Science And Technology | Method for detecting a substance having an activity to inhibit HIV infection using immunoassay and variant protein used for said method |
| CN1235164A (en) * | 1998-05-08 | 1999-11-17 | 北京医科大学 | Peptide for vaccine of schistosomiasis |
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| Title |
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| 日本血吸虫thioredoxin基因的克隆和表达 邵莜等，热带医学杂志，第2卷第4期 2004 * |
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