CN105153290A - Schistosoma japonicum chymotrypsin-like protease (SjCTRL) as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a Schistosoma japonicum chymotrypsin-like protease (SjCTRL), a protein with an amino acid sequence shown in the SEQ ID NO.2, or a protein which has the same function and is formed through replacement, deletion or insertion of one or more amino acids for the protein. Besides, the invention further discloses a preparation method of the SjCTRL. The preparation method comprises steps such as transmembrane structure removed gene sequence amplification of the SjCTRL, recombinant plasmid construction and identification, induction expression and purification of the SjCTRL and the like, and the immunoprotection of the SjCTRL in anti-schistosoma-infection of mice is evaluated. It is preliminarily verified that the SjCTRL can reduce the number of adults of C57BL/6 mice infected by Schistosoma japonicum and the number of eggs in livers to a certain extent and can serve as a target of a potential anti-schistosoma vaccine.

Description

Schistosoma japonicum SjCTRL recombinant antigen protein and its production and use

Technical field

The present invention relates to molecule, RESEARCH ON CELL-BIOLOGY field, be specifically related to Schistosoma japonicum chymotrypsin-like recombinant antigen protein (chymotrypsin-likeprotease, SjCTRL) and preparation method thereof.In addition, the invention still further relates to the purposes of this Schistosoma japonicum SjCTRL recombinant antigen protein.

Background technology

Schistosomicide (schistosomiasis) is a kind of parasitic zoonoses (parasiticzoonoses), it is the second largest parasitosis being only second to malaria in the world, popular in 76 countries and regions, the whole world, mainly be distributed in Asia, Africa and Latin America, compromised number 600,000,000, number of the infected is more than 200,000,000.China was once by one of more serious country of Schistosoma japonicum harm, was once popular in 12 provinces on the south the Changjiang river, and patient is more than 1,200 ten thousand.Only there is Schistosoma japonicum popular in China, be mainly distributed in the Jiangsu based on Marshland endemic regions, Anhui, Jiangxi, Hubei, Hunan 5 province and based on the Sichuan of hilly endemic area Endemic Area, Yunnan 2 province at present.According to epidemic situation data presentation in 2013, schistosomiasis in China epidemic situation declined further, but realization " national prevention and control schistosomicide medium-term and long-term plans outline (2004-2015) " target still has certain pressure; Even the epidemic situation of attainment areas is unstable, also needs to continue to strengthen preventing and controlling, and carry out more effective monitoring.The current treatment for Schistosoma japonicum also stays in the application of chemicals, along with deepening continuously and the introducing of molecular biology and gene engineering of antischistosomal drug study on mechanism, to schistosomicide physiological metabolism, gene and and the physiopathologic relation of host will be further explained, some bilharzial lethal genes and weak metabolism link and target molecule will be found, thus for the drug effect that plays medicine better with reduce providing theoretical and ensureing of toxic side effects.

Schistosoma japonicum SjCTRL mono-unknown function albumen is the new protein sequence of Schistosoma japonicum.The present invention goes out Schistosoma japonicum SjCTRL gene by pcr amplification; and in intestinal bacteria this gene recombinant expressed; obtain the recombinant protein that size is about 24kDa; confirm that gained recombinant protein can reduce one-tenth borer population and the liver worm's ovum number of the C57BL/6 mouse infecting Schistosoma japonicum to a certain extent through animal protection test; be potential vaccine candidate target, thus complete the present invention.

Before making the present invention, also do not occur relating to Schistosoma japonicum SjCTRL recombinant antigen protein of the present invention and the open report for the preparation of anti schistosoma vaccine thereof.

Summary of the invention

One of the technical problem to be solved in the present invention is to provide a kind of Schistosoma japonicum SjCTRL recombinant antigen protein.

Two of the technical problem to be solved in the present invention is to provide a pair Auele Specific Primer for the amplification of Schistosoma japonicum SjCTRL gene PCR.

Three of the technical problem to be solved in the present invention is to provide the preparation method of this Schistosoma japonicum SjCTRL recombinant antigen protein.

Four of the technical problem to be solved in the present invention is to provide this Schistosoma japonicum SjCTRL recombinant antigen protein and is preparing the application in anti schistosoma vaccine.

The present invention screen from Schistosoma japonicum full-length genome database one new be 79% with Schistosoma mansoni serine protease SmSP5 (the GeneBank accession number AHI46409.1) nucleotide sequence similarity reported, protein sequence similarity has the Schistosoma japonicum serine protease gene of 72%, through annotation called after Schistosoma japonicum chymotrypsin-like protein gene (S.japonicumchymotrypsin-likeprotease is called for short SjCTRL gene).SmSP5 albumen is in a new branch in Trematoda serine stretch protein S1 family system evolutionary tree, this branch between the trypsinase, Quimotrase of bilharzial cercaria elastoser and other families of S1, and not relevant bibliographical information its can be used as the potential vaccine target spot of anti-Schistosoma mansoni.

The present invention utilizes Protocols in Molecular Biology to carry out pcr amplification to Schistosoma japonicum SjCTRL gene, purifying amplified production, products therefrom, plasmid vector pET-28a (+) are carried out enzyme respectively cut, reclaim, connect recombinant plasmid pET-28a (+)-SjCTRL being built into SjCTRL gene prokaryotic, pass through transformation and selection, extracting recombinant plasmid, cuts after qualification confirmation through PCR, order-checking and enzyme, is converted in host cell colibacillus and expresses; Get the clone that expression amount is higher, fermentation, for thalline, identifies the solvability of recombinant protein; The recombinant protein of expression is dissolved in denaturing agent, carries out purifying with Ni-NTA agarose affinity column, finally obtain the recombinant protein SjCTRL of purifying, complete the body outer clone Expression and purification of SjCTRL gene.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

The present invention first aspect is to provide a kind of Schistosoma japonicum SjCTRL recombinant antigen protein, its albumen with aminoacid sequence shown in SEQIDNO.2 or the albumen with identical function by this albumen, one or more amino acid whose displacement, disappearance or insertion occurring and formed; This Schistosoma japonicum SjCTRL recombinant antigen protein is adopted and is prepared with the following method:

1) amplification (remove prediction transmembrane structure part) of Schistosoma japonicum SjCTRL gene order: the nucleotide sequence of design shown in SEQIDNO:3 or the nucleotide sequence shown in its complementary strand and SEQIDNO:4 or its complementary strand are primer, with containing Schistosoma japonicum cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;

2) structure of Schistosoma japonicum SjCTRL recombinant plasmid and qualification;

3) by this recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein of expressing;

4) recombinant protein of purifying expression is carried out with Ni-NTA agarose affinity column.

The present invention second aspect there is provided a pair Auele Specific Primer for the amplification of Schistosoma japonicum SjCTRL gene PCR (removing the transmembrane structure part of prediction); its sequence is (single underscore represents protection base, and double underline is respectively BamH I restriction enzyme site and Xho I restriction enzyme site):

Upstream: 5 '- cG aATTTAGAATATCGTATACAAAATGGTT-3 ' (as shown in SEQIDNO:3) or its complementary strand;

Downstream: 5 '- cC tCAACCACTGCCTGCTATTG-3 ' (as shown in SEQIDNO:4) or its complementary strand.

Third aspect of the present invention provides a kind of preparation method of Schistosoma japonicum SjCTRL recombinant antigen protein, comprises the steps:

1) amplification of Schistosoma japonicum SjCTRL gene order (removes the transmembrane structure part of prediction, from Schistosoma japonicum cDNA, obtain the DNA fragmentation of SjCTRL with round pcr): the nucleotide sequence of design shown in SEQIDNO:3 or the nucleotide sequence shown in its complementary strand and SEQIDNO:4 or its complementary strand are primer, with containing Schistosoma japonicum cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;

2) structure of Schistosoma japonicum SjCTRL recombinant plasmid and qualification: with pET-28a (+) vector construction Schistosoma japonicum SjCTRL recombinant expression plasmid pET-28a (+)-SjCTRL;

3) by pET-28a (+)-SjCTRL recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein SjCTRL expressed;

4) with Ni-NTA agarose affinity column purify express Schistosoma japonicum SjCTRL recombinant protein.

As the preferred technical scheme of the present invention, step 1) amplification of Schistosoma japonicum SjCTRL gene order, be specially:

With the cDNA clone containing Schistosoma japonicum SjCTRL gene order for template, SEQIDNO.3 (CG aATTTAGAATATCGTATACAAAATGGTT) or its complementary strand be 5 ' primer, SEQIDNO.4 ( cC tCAACCACTGCCTGCTATTG) or its complementary strand be 3 ' primer, carry out pcr amplification, gained PCR primer through agarose gel electrophoresis, use glue recovery test kit ( gelExtractionKit) purifying is reclaimed.The reaction conditions of described pcr amplification is 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1min, 35 circulations, and 72 DEG C extend 10min; Finally be stored in 4 DEG C.

As the preferred technical scheme of the present invention, step 2) build recombinant plasmid pET-28a (+)-SjCTRL that can express Schistosoma japonicum SjCTRL encoding gene in host cell, be specially:

By step 1) the PCR primer fragment of gained and plasmid vector pET-28a (+) carry out double digestion with restriction enzyme BamH I and XhoI respectively, and reclaim purifying; Be connected with the concentration of carrier endonuclease bamhi according to the goal gene fragment after purifying, be built into recombinant plasmid pET-28a (+)-SjCTRL of SjCTRL encoding gene prokaryotic expression; This recombinant plasmid is passed through CaCl ₂method is transformed into bacillus coli DH 5 alpha competent cell, and coat after cultivation containing on kantlex LB agar plate, the bacterium colony on the above-mentioned flat board of random picking, collects thalline after cultivation, contained recombinant plasmid in extracting thalline, carries out PCR qualification, order-checking qualification.

As the preferred technical scheme of the present invention, step 3) expression of recombinant plasmid pET-28a (+)-SjCTRL in colibacillus (host cell), being specially: transformed step 2 by calcium) extracting gained pET-28a (+)-SjCTRL recombinant plasmid transformed enters in e. coli bl21 (DE3), coats on the LB agar plate containing kantlex after cultivation.Bacterium colony on the above-mentioned flat board of random picking, cultivates bacterium liquid to logarithmic phase, adds inductor (IPTG) and continues to cultivate.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies abduction delivering result, directly with whole cell electrophoresis showed.

As the preferred technical scheme of the present invention, step 4) purifying Schistosoma japonicum SjCTRL recombinant protein, be specially: get the frozen bacterial classification of expressing Schistosoma japonicum SjCTRL recombinant protein amount higher and line on the LB agar plate containing kantlex, after overnight incubation, colony inoculation on the above-mentioned flat board of random picking is to the same liquid nutrient medium containing kantlex, cultivate bacterium liquid to logarithmic phase, add inductor (IPTG) and continue to cultivate, finally collect thalline; In above-mentioned thalline, add Protein Extraction agent cracking bacterium, centrifugal rear reservation supernatant liquor and precipitation, by SDS-PAGE electroresis appraisal result.According to the above results, by denaturing agent cracking gained precipitation, precipitation is dissolved in denaturing agent, and carry out recovery with Ni-NTA agarose affinity column and purify, SDS-PAGE checks purification result.

The present invention the 4th aspect provides a kind of Schistosoma japonicum SjCTRL recombinant antigen protein and is preparing the application in anti schistosoma vaccine, the albumen that described Schistosoma japonicum SjCTRL recombinant antigen protein has aminoacid sequence shown in SEQIDNO.2 or the albumen with identical function by this albumen, one or more amino acid whose displacement, disappearance or insertion occurring and formed.

The C57BL/6 mouse in 12 4 ~ 6 week ages is divided into 2 groups by the present invention at random, emulsion dorsal sc multi-point injection is made after the SjCTRL recombinant protein (25 μ g) of experimental group purifying and Freund's complete adjuvant balanced mix, the SjCTRL recombinant protein (25 μ g) of 2 weeks rear purifying is injected with Freund's incomplete adjuvant (50 μ l/ only) subcutaneous 3 of mixing and emulsifying back part, only uses purifying protein booster immunization 1 time after 4 weeks.Vehicle control group is with adjuvant immunity (50 μ l/ only).Two groups of mouse belly challenge infection (40 ± 2) schistosoma japonicum cercariae respectively after 6 weeks.Within after challenge infection the 35th day, cut open and kill mouse, adult is collected in thoracic aorta perfusion, calculates liver worm reduction rate.Get every rat liver to weigh, add 5ml physiological saline tissue homogenate, with 5ml10%KOH in 37 DEG C of digestion 6h, get the whole worm's ovum number of microscopy after 20 μ l smears.Employing double-blind method counts, and everyone microscopy 3 smears, calculate mean value and the egg reduction rate of every gram of excrement worm's ovum number of every mouse.Worm reduction rate (per-cent)=[1 – (and protein immunization group on average every mouse obtain adult number/vehicle control group on average every mouse obtain into borer population)] × 100%, liver egg reduction rate (per-cent)=[1 – (protein immunization group is the on average worm's ovum number contained by every gram of liver of worm's ovum number/vehicle control group contained by every gram of liver on average)] × 100%.Through the checking of the animal protection test of above-mentioned Schistosoma japonicum SjCTRL recombinant protein, use cercaria challenge infection after Schistosoma japonicum SjCTRL recombinant antigen protein immunity C57BL/6 mouse of the present invention, 25.4% worm reduction rate and 80% liver egg reduction rate can be obtained.Protection of animal test-results shows this albumen and can be used as potential anti-schistosome vaccine target spot, particularly Be very effective in egg reduction rate, has the significance that control schistosomiasis is propagated.

Accompanying drawing explanation

Fig. 1 is the amplification schematic diagram of Schistosoma japonicum SjCTRL gene order in embodiment 1.In Fig. 1, M:DNA molecular weight standard; 1:SjCTRL.

Fig. 2 is Schistosoma japonicum SjCTRL gene recombination plasmid bacterium colony PCR qualification result schematic diagram in embodiment 3.In Fig. 2, M:DNA molecular weight standard; 1-4:SjCTRL; Through the BL21 E. coli clones pcr amplification transformed, 1-4 represents 4 bacterium colonies of random selecting, all successfully connects conversion.

Fig. 3 is the SDS-PAGE analytical results schematic diagram of Schistosoma japonicum SjCTRL in embodiment 4.In Fig. 3, M: Protein Marker; 1: do not induce the full bacterium of contrast; 2: non-induced precipitation 3 ~ 5: the full bacterium of 4h (1mM/LIPTG), upper cleer and peaceful precipitation after induction.

Fig. 4 is the SDS-PAGE analytical results schematic diagram of Schistosoma japonicum SjCTRL recombinant protein purification effect in embodiment 5.In Fig. 4, M: Protein Marker; The full bacterium lysate of pET-28a/SjCTRL after 1:IPTG induction; 2: affinity purification passes liquid; 3: SjCTRL recombinant protein after purifying.

Embodiment

Following examples are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in embodiment, usually conveniently condition, or according to the condition that manufacturer advises.

Main agents is as shown in table 1:

Table 1

Key instrument equipment is as shown in table 2:

Table 2

PCR instrument	U.S. Bio-Rad
		Protein electrophoresis instrument	U.S. Bio-Rad
Table-type high-speed refrigerated centrifuge	Germany Eppendorf

High speed freezing centrifuge (CR22F)	FDAC
		Milli-Q pure water system	U.S. Millipore

Test materials:

Schistosoma japonicum cDNA, host strain E. coli DH5 α, BL21 (DE3), plasmid pET-28a (+), provide by Prevention & Control Station of Parasitic Disease, China Diseases Prevention & C.

The clone of embodiment 1 Schistosoma japonicum SjCTRL gene

The amplification of 1.1SjCTRL gene fragment and purifying:

1.1.1 according to the sequence (as shown in SEQIDNO.1 sequence) of SjCTRL gene (GenbankFN317561.1), PrimerPremier5.0 software design a pair Auele Specific Primer is utilized, as follows:

PF:5 '- cG aATTTAGAATATCGTATACAAAATGGTT-3 ' (as shown in SEQIDNO.3);

PR:5 '- cC tCAACCACTGCCTGCTATTG-3 ' (as shown in SEQIDNO.4);

Single underscore be the protectiveness base of upstream and downstream primer, double underline is the BamHI restriction enzyme site of upstream primer and the XhoI restriction enzyme site of downstream primer.Auele Specific Primer is synthesized by prompt base (Shanghai) trade Co., Ltd in the English Weihe River.

1.1.2 with Schistosoma japonicum cDNA for template, carry out PCR reaction, amplification SjCTRL gene, reaction system is as follows:

Reaction conditions:

With 1.2% sepharose (GoldView ^tMnucleic acid dye) electrophoresis detection PCR primer, observe whether there is object band, result as shown in Figure 1, M:DNA molecular weight standard, 1:SjCTRL.The result display of PCR reaction, there is a band clearly at about 663bp place, conforms to, show successfully from cDNA, to amplify SjCTRL gene with the size of expection fragment.

1.1.3PCR the purifying of product reclaims (AxyGEN company glue reclaims test kit)

1), after electrophoresis terminates, with clean, sharp blade, DNA object fragment is cut down from sepharose.

2) added by the glue of cutting and weigh in EP pipe, then both weighed, twice weight subtracts each other to obtain net weight.

3) add 300 μ lBufferDE-A according to every 100mg (about 100 μ l) gel, heat with 75 DEG C after mixing, be interrupted mixing (every 2-3min), until blob of viscose melts (6-7min) completely;

4) add the BufferDE-B of 0.5 BufferDE-A volume, mix.When the DNA fragmentation be separated is less than 400bp, then add the Virahol of 1 gel volume;

5) draw the mixed solution of previous step, transfer to DNA preparation pipe (being placed in 2ml centrifuge tube), 12,000 × g centrifugal 1min, abandon filtrate;

6) putting back 2ml centrifuge tube by preparing pipe, adding 500 μ lBufferW1,12,000 × g centrifugal 30s, abandon filtrate;

7) putting back 2ml centrifuge tube by preparing pipe, adding 700 μ lBufferW2,12,000 × g centrifugal 30s, abandon filtrate; Same method 700 μ lBufferW2 wash once, 12,000 × g centrifugal 1min;

8) put back in 2ml centrifuge tube by preparing pipe, 12,000 × g centrifugal 1min;

9) be placed in clean 1.5ml centrifuge tube by preparing pipe, add 25-30 μ lElutionbuffer (preheating 65 DEG C) or deionized water preparing film central authorities, room temperature leaves standstill 1min, 12,000 × g centrifugal 1min.

10) detect organic efficiency with agarose gel electrophoresis, and estimate the concentration of DNA fragmentation.

The structure of embodiment 2SjCTRL gene recombination plasmid pET-28a (+)-SjCTRL

2.1PCR product double digestion and recovery

The PCR object fragment of recovery spent the night in 37 DEG C of water-bath double digestions, endonuclease reaction system is as follows:

Digestion products carries out 1.2% sepharose (GoldView ^tMnucleic acid dye) electrophoresis, cut object band, again reclaim DNA molecular, way of recycling, with 1.1.3 in embodiment 1, reclaims product and is stored in-20 DEG C.

The preparation (AxyPrep Plasmid Miniprep Kit) of 2.2pET-28a (+) empty plasmid

Single bacterium colony of pET-28a (+) plasmid is contained in 5mlLB substratum (kantlex containing 50 μ g/ml), 37 DEG C of overnight incubation at the upper picking of LB flat board (kantlex containing 50 μ g/ml).Next day, get 1ml nutrient solution and proceed in 1.5ml centrifuge tube, be stored in 4 DEG C as bacterial classification.By remaining nutrient solution in 5000rpm, centrifugal 10 minutes, abandon supernatant.The resuspended bacterial precipitation of BufferS1 (suspend and need evenly, should not leave little bacterium block, otherwise the cracking of thalline can be affected) of RNaseA1 has been added with 250 μ l.Add 250 μ lBufferS2, gentle but spin upside down mixing fully 6 times, until form bright solution.Add 400 μ lBufferS3, gentleness also spins upside down mixing 10 times fully, and room temperature leaves standstill 2 minutes, centrifugal 10 minutes of 14000rpm (if still have suspended substance, can put upside down the rear recentrifuge of mixing gently 3 minutes).Being transferred to by supernatant liquor after centrifugal prepares in pipe, is placed in 2ml centrifuge tube, centrifugal 1 minute of 1400rpm.Filtrate is transferred to again preparation pipe to repeat in the same way to combine once.Abandon filtrate, put get back to preparing pipe in former centrifuge tube, add 500 μ lBufferW1 and wash, centrifugal 1 minute of 14000rpm.Abandon filtrate, put get back to preparing pipe in former centrifuge tube, add 700 μ lBufferW2 (having added the dehydrated alcohol of proper volume), centrifugal 1 minute of 14000rpm, abandons filtrate; Wash once with 700 μ lBufferW2 more in the same way, abandon filtrate.Put preparing pipe and get back in 2ml centrifuge tube, centrifugal 1 minute of 14000rpm.Moving into new 1.5ml centrifuge tube by preparing pipe, adding 20 μ l elutriants (elutriant is heated to 65 DEG C in advance, can improve elution efficiency) in the film central authorities of preparing pipe, room temperature leaves standstill 1 minute; 14000rpm carries out wash-out in centrifugal 1 minute.Rejoined by elutriant and prepare periosteum central authorities, room temperature leaves standstill 1 minute, centrifugal 1 minute of 14000rpm eluted dna again.Elutriant is placed in-20 DEG C of preservations, detects the plasmid DNA extracted with agarose gel electrophoresis.(with reference to AxyPrep Plasmid Miniprep Kit operational manual)

The double digestion of 2.3pET-28a empty plasmid

Add digestion with restriction enzyme pET-28a (+) empty plasmid, 37 DEG C of water-baths spend the night, and reaction system is as follows:

Digestion products carries out 1% sepharose (GoldView ^tMnucleic acid dye) electrophoresis, cut object fragment and carry out recovery purifying, way of recycling is with 1.1.3 in embodiment 1, and sample retention is in-20 DEG C.

The structure of 2.4 recombinant plasmids

Be connected with expression vector fragment by exogenous genetic fragment after recovery according to the ratio of 3:1 (mol ratio), linked system is as follows:

Reaction system connects in 16 DEG C of water-baths spends the night, and builds pET-28a (+)-SjCTRL recombinant plasmid.

The qualification of embodiment 3 Schistosoma japonicum SjCTRL expression vector

3.1 connect product conversion E.coliDH5 α competent cell

Connection product in 2.4 of embodiment 2 is mixed gently with E.coliDH5 α competent cell, ice bath 30 minutes, in 42 DEG C of water-bath heat shocks 1.5 minutes, ice bath 5 minutes again.In culture tube, the SOC substratum of 900 μ l is added, 37 DEG C, 200rpm, shaking culture 1 hour under aseptic condition.By cultured bacterium liquid in 3500rpm, centrifugal 3 minutes, discard most of supernatant, stay the resuspended thalline of about 100 μ l substratum.Re-suspension liquid is spread evenly across on LB flat board (kantlex containing 50 μ g/ml), is inverted overnight incubation for 37 DEG C, observes colony growth situation.

The PCR qualification of 3.2 recombinant plasmids

Single bacterium colony on random picking flat board carries out bacterium colony PCR qualification, PCR primer detects (result as shown in Figure 2 through agarose gel electrophoresis, in Fig. 2,1-4 represents 4 bacterium colonies of random selecting, all successfully connect conversion), there is object band in the molecular size range place being presented at expectation through PCR qualification, shows the insertion having exogenous genetic fragment.

The order-checking qualification of 3.3 recombinant plasmids

Whether single bacterium colony Song Yingweijie base trade Co., Ltd that selection can amplify object band checks order, to check the sequence of Insert Fragment correct.Sequencing analysis result shows that the exogenous genetic fragment sequence inserted is correct, construction of recombinant plasmid success.

The expression in E.coli of the recombinant plasmid of embodiment 4 Schistosoma japonicum SjCTRL and qualification

The abduction delivering of 4.1pET-28a (+)-SjCTRL recombinant plasmid

1) get pET-28a (+)-SjCTRL recombinant plasmid transformed E.coliBL21 (DE3) competent cell that 1 μ l checks order correct, method for transformation is with 3.1 in embodiment 3.

2) next day, respectively at the LB substratum (kantlex containing containing 50 μ g/mls) of each random picking 6 single colony inoculations on each flat board in 5ml, 37 DEG C, 200rpm, shaking culture is to OD ₆₀₀=0.6.

3) take out 1ml bacterium liquid as bacterial classification, then take out 2ml bacterium liquid and do not add IPTG in contrast, it is that the IPTG of 1mM induces that remaining 2ml bacterium liquid adds final concentration, and 37 DEG C, 250rpm, continues cultivation 4 hours.

4) by cultured bacterium liquid in 4 DEG C, centrifugal 10 minutes of 5000rpm collects thalline, supernatant discarded.

4.2SDS-PAGE identifies induced product

The resuspended bacterial sediment of 200 μ l1 × PBS is added respectively in induction pipe and control tube.From induction pipe and control tube, take out the re-suspension liquid of 5 μ l respectively, add 2 × SDS-PAGE sample-loading buffer 5 μ l, after mixing, boil sex change in 5 minutes in 100 DEG C.Add the sample of 8 μ l in each loading hole respectively, carry out SDS-PAGE analysis, separation gel is 12%, and concentrated glue is 5%.Gel formula is as shown in table 3 below:

Table 3

The voltage arranging concentrated glue is 80V, and the voltage of separation gel is that 100V carries out electrophoresis.Powered-down when bromophenol blue indicator effusion sheet glass lower rim.Take off gel staining fluid to dye 2 hours, then with destainer decolouring, until protein band is high-visible.

As shown in Figure 3, by pET-28a (+)-SjCTRL recombinant plasmid transformed E.coliBL21 (DE3) competent cell, thalline before comparatively inducing after IPTG abduction delivering has occurred that molecular weight is about the obvious band of expression of 24kDa, be the product of goal gene and Histag amalgamation and expression, size conforms to theoretical Mr.

4.3 recombinant protein solvability qualifications

To determine that E.coliBL21 (DE3) strain inoculation can expressing recombinant protein is in 100mlLB substratum (kantlex containing 50 μ g/ml), 37 DEG C, 200rpm is cultured to OD ₆₀₀=0.6.The bacterium liquid cultivated is taken out 1ml as bacterial classification, separately gets 2ml in contrast.In remaining bacterium liquid, add IPTG to final concentration is 1mM, 37 DEG C, and 200rpm continues cultivation 4 hours.

Bacterium liquid after induction through 5000rpm, 4 DEG C, centrifugal 10 minutes, abandon most supernatant, in bacterial sediment, add the BugBuster Protein Extraction agent (Novagen) of 5ml, after the precipitation that fully suspends, suspension is proceeded in centrifuge tube, be placed on shaking table, lysis at room temperature thalline 1 hour.By bacterial lysate in 4 DEG C, centrifugal 10 minutes of 10000rpm, gets supernatant and is stored in another clean centrifuge tube, and picking precipitates with the PBS of proper volume resuspended on a small quantity.The upper cleer and peaceful precipitation re-suspension liquid of taking out 5 μ l respectively does SDS-PAGE analysis, the solvability of qualification recombinant protein.Electrophoresis result (Fig. 3) shows recombinant protein and is mainly arranged in precipitation, forms inclusion body.

The purifying of embodiment 5 Schistosoma japonicum SjCTRL recombinant protein

The purifying of 5.1 recombinant proteins

1) recombinant protein thalline is expressed in a large amount of preparation

Get the fresh bacterium liquid of 30mL, be inoculated in the LB substratum of that resistance of 1L card, 37 DEG C, 220r/min shaking culture, to OD600 value about 0.6, adds the IPTG of 1mM, continues to cultivate 6h, 8000g, 4 centrifugal 30min, collects thalline.The lysate of the thalline 20mL after collection is resuspended, be placed in-80 DEG C frozen.

2) carrying out ultrasonic bacteria breaking is deposited-80 DEG C of refrigerators and is taken out thalline, and after thalline melts, piping and druming is even, ultrasonic disruption cell.Ultrasonic power is 160W, ultrasonicly opens 2s, ultrasonic pass 2s, ultrasonic number of times 150 times.Ultrasonic rear cellular lysate liquid 15000g, 4 DEG C of centrifugal 30min, get precipitation.

3) inclusion body process

In precipitation, add 80mL inclusion body washings, be placed on magnetic stirring apparatus, abundant agitator treating precipitation 2h, then 15000g, 30min, 4 DEG C of centrifugal collecting precipitations.Add 10mL extracting solution I and fully dissolve inclusion body, under room temperature, magnetic stirrer over night extremely dissolves completely, last 15000g, 30min, 4 DEG C of collected by centrifugation supernatants.Get supernatant with 1:10 ratio rapid dilution in the gradient dilution liquid of precooling, ice bath places 10min.Then 15000g, 30min, 4 DEG C of centrifugal collecting precipitations.Add magnetic agitation in extracting solution II and dissolve about 2h, 15000g, 30min, 10 DEG C of collected by centrifugation supernatants.

Protein purification uses HistrapFF prepacked column to carry out on AKTA protein purification instrument (GE company), after balance liquid (the same lysate of composition) balance nickel affinity column 5 column volumes, upper protein sample, foreigh protein removing is removed with balance liquid rinsing successively after end of the sample, after passing and can't detect albumen in liquid, use elutriant A, B, C or D instead and rinse successively, wait pass in liquid albumen detected time, sample should be collected immediately, its purity of SDS-PAGE electrophoresis detection.Pillar subsequent rinse removes remaining albumen, after 0.2MNaOH washing, use deionized water rinsing again, finally rinses with the ethanolic soln of 20% again, preserves for a long time for 4 DEG C.Purification result as shown in Figure 4.

The mensuration of 5.2 recombinant protein concentration

Utilize Bradford quantification of protein test kit (sky root) to measure the concentration of recombinant protein, operate to specifications.Key step comprises:

Coomassie Brillant Blue solution first balances to room temperature before use and gentleness puts upside down mixing, preheating spectrophotometer.0,5,10,15,20,25,30 μ l bovine serum albumin (BSA) standardized solution (1mg/ml) are joined in centrifuge tube respectively, adds PBS and complement to 75 μ l.The recombinant protein of proper volume purifying is joined in centrifuge tube, and supplies 75 μ l with PBS.In each centrifuge tube, add 1425 μ l Coomassie Brillant Blue solution, mixing, room temperature leaves standstill 10 minutes.With the light absorption value at spectrophotometric determination 595nm place, and record reading.Not contain the absorbance value of BSA sample as blank, drawing standard curve, calculate the protein concentration of testing sample.If the protein concentration obtained exceeds the scope of typical curve, according to circumstances redeterminate by diluted sample or after concentrating.After calculating purifying according to the extent of dilution of table 4 and recombinant protein, the concentration of SjCTRL recombinant protein is 1.215mg/ml.

Each pipe solution reaction system for drawing standard curve is as shown in table 4 below:

Table 4

The animal protection experiment of embodiment 6 Schistosoma japonicum SjCTRL recombinant protein

The Immunoprotection test of 6.1 recombinant proteins

1) experiment grouping

4-6 week C57BL/6 female mice, is divided into A protein immunization group (6), B vehicle control group (6) two groups.

2) immunity

Mouse is divided into 3 immunity respectively at 0,2,4 week back or subcutaneous abdomen.

Before immunity: emulsification: ice-bath ultrasonic after adjuvant mixes with albumen, drips to emulsifying agent and become oil droplet shape not scatter in the water surface.

A recombinant protein immune group (6):

First time immunity (0d): recombinant protein (25 μ gin50 μ l8Murea)+50 μ l Freund's complete adjuvants

Second time immunity (14d): recombinant protein (25 μ gin50 μ l8Murea)+50 μ l Freund's incomplete adjuvants

Third time immunity (28d): recombinant protein (25 μ gin50 μ l8Murea)+50 μ lPBS

B vehicle control group (6):

First time immunity (0d): 50 μ l8Murea+50 μ l Freund's complete adjuvants

Second time immunity (14d): 50 μ l8Murea+50 μ l Freund's incomplete adjuvants

Third time immunity (28d): 50 μ l8Murea+50 μ lPBS

3) infect

After immune two weeks of third time, carry out miracidium infection attack to the mouse after immunity, miracidium infection amount is every mouse 40 (± 2) head.

4) adult, egg count

After miracidium infection attacks 35 days, cut open and kill the collection that mouse carries out Schistosoma japonicum.

Schistosoma japonicum adult counts: collect Schistosoma japonicum all in every mouse body, and carry out counting statistics.

Egg count in mouse liver: get every rat liver and weigh, adds 5ml physiological saline tissue homogenate, with 5ml10%KOH in 37 DEG C of digestion 6h, gets the whole worm's ovum number of microscopy after 20 μ l smears.Employing double-blind method counts, and everyone microscopy 3 smears, calculate mean value and the egg reduction rate of every gram of excrement worm's ovum number of every mouse.

Method of calculation

Worm reduction rate (per-cent)=[1-(and protein immunization group on average every mouse obtain adult number/vehicle control group on average every mouse obtain into borer population)] × 100%

Liver egg reduction rate (per-cent)=[1-(protein immunization group is the on average worm's ovum number contained by every gram of liver of worm's ovum number/vehicle control group contained by every gram of liver on average)] × 100%

Table 5 is result charts of Schistosoma japonicum SjCTRL recombinant protein animal protection test in embodiment 6.

Table 5 Schistosoma japonicum SjCTRL protein immunization mouse

Table1EffectofimmunizationwithSjCTRLinmice

* EPG is every gram of liver worm's ovum number.EPG:Numberofeggspergramliver.

Through the checking of the animal protection test of above-mentioned Schistosoma japonicum SjCTRL recombinant protein; Schistosoma japonicum SjCTRL recombinant antigen protein immune mouse of the present invention can reduce the one-tenth borer population and liver worm's ovum number that infect Schistosoma japonicum; particularly Be very effective in egg reduction rate; can be used as potential anti-schistosome vaccine target spot, for the preparation of anti schistosoma vaccine.

Claims (8)

1. a Schistosoma japonicum SjCTRL recombinant antigen protein, is characterized in that, the albumen with aminoacid sequence shown in SEQIDNO.2 or the albumen with identical function by this albumen, one or more amino acid whose displacement, disappearance or insertion occurring and formed; This Schistosoma japonicum chymotrypsin-like recombinant protein is adopted and is prepared with the following method:

1) amplification of Schistosoma japonicum SjCTRL gene order: designing the nucleotide sequence shown in SEQIDNO:3 or the nucleotide sequence shown in its complementary strand and SEQIDNO:4 or its complementary strand is primer, with Schistosoma japonicum cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;

2) structure of Schistosoma japonicum SjCTRL recombinant plasmid and qualification;

3) by this recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein of expressing;

4) recombinant protein of Ni-NTA agarose affinity column purifying expression.

2. a pair Auele Specific Primer for the amplification of Schistosoma japonicum SjCTRL gene PCR, its sequence is:

Upstream: 5'- cG aATTTAGAATATCGTATACAAAATGGTT-3'(is as shown in SEQIDNO:3) or its complementary strand;

Downstream: 5'- cC tCAACCACTGCCTGCTATTG-3'(is as shown in SEQIDNO:4) or its complementary strand.

3. a preparation method for Schistosoma japonicum SjCTRL recombinant antigen protein as claimed in claim 1, is characterized in that, comprise the steps:

1) amplification of Schistosoma japonicum SjCTRL gene order: the nucleotide sequence of design shown in SEQIDNO:3 or the nucleotide sequence shown in its complementary strand and SEQIDNO:4 or its complementary strand are primer, with containing Schistosoma japonicum cDNA for template carries out pcr amplification; Gained PCR primer through agarose gel electrophoresis, and reclaims purifying;

2) structure of Schistosoma japonicum SjCTRL recombinant plasmid and qualification;

3) by this recombinant plasmid transformed in host cell, and to express in host cell, obtain the recombinant protein of expressing;

4) recombinant protein of Ni-NTA agarose affinity column purifying expression.

4. the preparation method of Schistosoma japonicum SjCTRL recombinant antigen protein as claimed in claim 3, is characterized in that, step 1) in, the reaction conditions of described pcr amplification is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1min, 35 circulations, and 72 DEG C extend 10min; Finally be stored in 4 DEG C.

5. the preparation method of Schistosoma japonicum SjCTRL recombinant antigen protein as claimed in claim 3, it is characterized in that, step 2) be specially: by step 1) the PCR primer fragment of gained and plasmid vector pET-28a (+) carry out double digestion with restriction enzyme BamH I and XhoI respectively, and reclaim purifying; Be connected with the concentration of carrier endonuclease bamhi according to the goal gene fragment after purifying, be built into recombinant plasmid pET-28a (+)-SjCTRL of SjCTRL encoding gene prokaryotic expression; This recombinant plasmid is passed through CaCl ₂method is transformed into bacillus coli DH 5 alpha competent cell, and coat after cultivation containing on kantlex LB agar plate, the bacterium colony on the above-mentioned flat board of random picking, collects thalline after cultivation, contained recombinant plasmid in extracting thalline, carries out PCR qualification, order-checking qualification.

6. the preparation method of Schistosoma japonicum SjCTRL recombinant antigen protein as claimed in claim 3, it is characterized in that, step 3) be specially: transformed step 2 by calcium) check order and identify that errorless pET-28a (+)-SjCTRL recombinant plasmid transformed enters in e. coli bl21 (DE3), coat on the LB agar plate containing kantlex after cultivation; Bacterium colony on the above-mentioned flat board of random picking, cultivates intestinal bacteria to logarithmic phase, adds inductor and continues to cultivate; SDS-PAGE electroresis appraisal abduction delivering result, directly with whole cell electrophoresis showed.

7. the preparation method of Schistosoma japonicum SjCTRL recombinant antigen protein as claimed in claim 3, it is characterized in that, step 4) be specially: get the higher frozen bacterial classification of expression Schistosoma japonicum recombinant protein SjCTRL amount and line on the LB agar plate containing kantlex, after overnight incubation, colony inoculation on the above-mentioned flat board of random picking is to the same liquid nutrient medium containing kantlex, cultivate bacterium liquid to logarithmic phase, add inductor and continue to cultivate, finally collect thalline; In above-mentioned thalline, add Protein Extraction agent cracking bacterium, centrifugal rear reservation supernatant liquor and precipitation, by SDS-PAGE electroresis appraisal result; According to the above results, by denaturing agent cracking gained precipitation, carry out purifying with Ni-NTA agarose affinity column, SDS-PAGE checks purification result.

8. Schistosoma japonicum SjCTRL recombinant antigen protein is preparing the application in anti schistosoma vaccine, the albumen that described Schistosoma japonicum SjCTRL recombinant antigen protein has aminoacid sequence shown in SEQIDNO.2 or the albumen with identical function by this albumen, one or more amino acid whose displacement, disappearance or insertion occurring and formed.