CN101275140B - Cysticercosis cellulosae recombination gene, vaccine and preparation thereof - Google Patents

Abstract

The present invention provides a taenia solium oncosphere recombination gene and the preparing method of the recombination gene, and the use of the recombination gene, especially an oral vaccine for preventing pig cysticercosis prepared by the recombination gene and a preparing method of the vaccine. The recombination gene of the invention removes the signal peptide bases in the original sequence of TsoL18 gene of taenia solium oncosphere, and mutates the partial bases. The vaccine of the invention is recombining attenuation Salmonella enterica which carries with the taenia solium oncosphere TsoL18 recombination gene, and the gene can be expressed in the immunologic effector cells parasitized in attenuated salmonella typhimurium and/or salmonella.

Description

Cysticercosis cellulosae recombination, vaccine and preparation method thereof

Technical field

The present invention relates to the preparation method of taeniasis suis oncosphere recombination, this recombination, the purposes of this recombination, particularly adopt the oral vaccine and the preparation method who prepares this vaccine that are used to prevent and treat pork measles of this recombination preparation.

Background technology

Taeniasis suis (Taeniasis solium) is a kind of universal Amphixenosis; Develop into the larva cysticercus (Cysticercus cell μ lose) of taeniasis suis after the worm's ovum oncosphere (oncosphere) of taeniasis suis is eaten by intermediate hosts such as pig or wild boars, the people eats behind the article that the pork that infects cysticercus or contact polluted by cysticercus very easily infected pigs's taeniasis by mistake.Therefore; Cysticercosis cellulosae not only causes financial loss to livestock industry, has a strong impact on the quality of meat, prior serious harm human beings'health; Cysticercosis has caused the common concern and the great attention of countries in the world governments, and how controlling and eliminate cysticercosis effectively is the focus of paying close attention to.

Control about cysticercosis does not at present also have sophisticated means; Several kinds of chemicalses once played vital role to the treatment of cysticercosis; Like benzene sulphur imidazoles, PRAZIQUANTEL BP 98 cysticercus there are good expeling and therapeutic action; But the residual harm that possibly cause human body of spinoff that the use that Along with people's is prolonged and repeated, pharmacological agent cause and food Chinese traditional medicine has demonstrated the limitation of drug alone treatment.Molecular biology and immunologic developing rapidly and the appearance of various new technologies and the research that application has greatly promoted new generation vaccine; Study new vaccine carrier, inquire into new vaccine form, with the focus that obtains safely, stable, effective new generation vaccine becomes people's concern.

Chinese invention patent application 200410065671.2 discloses a kind of as recombinant vaccine that prevents pork measles and preparation method thereof.This vaccine is that the core protein gene with the core protein of hepatitis B virus or hepatitis B virus is a carrier; On a plurality of positions between the 1st amino acids to the 183 amino acids of this carrier, insert the cysticercus cellulosae protective antigen, constitute the subunit protein vaccine or the nucleic acid vaccine of prevention pork measles with gene engineering method; Its preparation method is to be carrier with hepatitis B virus core albumen; The 1st amino acids to the at hepatitis B virus core albumen (HBe) gene: insert the cysticercus cellulosae protective antigen gene on a plurality of positions between 183 amino acids; Be cloned on prokaryotic expression plasmid or the eukaryon expression plasmid; Transform the host bacterium,, can obtain preventing the subunit protein vaccine or the nucleic acid vaccine of pork measles through inducing destination gene expression, obtaining target protein or column purification eukaryon expression plasmid DNA.

Summary of the invention

First purpose of the present invention provides a kind of taeniasis suis oncosphere recombination, expects that this recombination has positive effect to the prevention to pork measles with treatment; Second purpose of the present invention provides the preparation method of this taeniasis suis oncosphere recombination; The 3rd purpose of the present invention provides a kind ofly utilizes taeniasis suis oncosphere recombination disclosed by the invention preparation to be used to treat or prevent the medicine or the vaccine of pork measles, the particularly a kind of vaccine that can orally use, that better immunization is arranged and the preparation method of this vaccine.

Recombination of the present invention is that the 1st to the 48th signal peptide base in the TsoL18 gene original series of taeniasis suis oncosphere all removed, and the 12nd bit base a in the TsoL18 gene order of truncated signals peptide is sported t, the 333rd bit base a sports that c, the 335th bit base a sport c, the 336th bit base a sports t.

A kind of preparation method of taeniasis suis oncosphere recombination of the present invention gets the terminal sophisticated gravid segment of taeniasis suis; Therefrom take out hatching and activated oncosphere; Extract total RNA of oncosphere; Total RNA with oncosphere is that template is carried out RT-PCR, preparation TsoL18 goal gene, and the upstream and downstream primer of employing is respectively:

Upstream primer P1:5 '-ccgg AattcGcagcggtgaccgtacattcgg-3 '

Downstream primer P2:5 '-acgc GtcgacCtacgaacggcggaccttcttgt-3 '

Taeniasis suis oncosphere recombination of the present invention can be used to prepare the medicine of preventing and treating cysticercosis or be used to prepare the vaccine of preventing and treating cysticercosis.An one of which concrete embodiment is the vaccine of preventing and treating cysticercosis that a kind of taking orally of preparation is used; This vaccine is the recombination attenuated salmonella typhimurium cell that contains aforesaid taeniasis suis oncosphere recombination; Wherein said recombination attenuated salmonella typhimurium carries taeniasis suis oncosphere TsoL18 recombination, and this gene can be expressed in attenuated salmonella typhimurium and/or in the immune effector cell of Salmonellas parasitism.

The preparation method of the above-mentioned oral vaccine of preventing and treating pork measles is connected taeniasis suis oncosphere recombination of the present invention to make up recombinant expression vector pYA3342-TsoL18 with Salmonellas expression vector pYA3342; Electricity transformed into escherichia coli X6212 obtains X6212 (pYA3342-TsoL18), extracts recombinant plasmid from it; The CaCl2 method transforms Salmonellas intermediate host X3730; Make recombinant plasmid obtain the methylation patterns of Salmonella typhimurtum, from the X3730 bacterium, separate recombinant plasmid again, electricity transforms final host's vaccine strain X4550; The bacterium X4550 (pYA3342-TsoL18) that obtains recombinating, vaccine promptly of the present invention.

Salmonella typhimurium (Salmonella typhimurium) is a kind of intra-cellular pathogens that produces part or systemic infection; Salmonellas through the gene engineering method attenuation is to the pathogenic remarkable reduction of host; But still keep good invasiveness, mucosal tissue there is very strong preferendum.Can be directly behind the Salmonellas live vector vaccine oral administration immune animal be transported to and carry out effective expression in the antigen presenting cell carrying the expression of exogenous gene carrier; The immune response of inducing specific; And; The liposome class material of attenuation salmonella, the unmethylated motif (motif) of DNA of bacteria and bacterium CpG sequence etc. can also be brought into play the effect of immunological adjuvant, strengthen the immunogenicity of vaccine greatly, can excite animal to produce powerful body fluid, cell and mucosal immunoreaction.

Utilize Salmonellas on the different animal model, to obtain the experiment proof at present and can produce efficient immune as vaccine live vector expression bacterium, virus and parasite antigen.For example; Attenuated salmonella typhimurium successfully has been applied in the research of listeria bacteria dna vaccination; The S.typhimurium of carrier for expression of eukaryon that carries coding listeria bacteria hemolysin gene can induce after with the oral route infecting mouse and produce high antihemolysin antibody of tiring, and can also induce CD ₈ ⁺And CD ₄ ⁺T cell responses, t helper cell also can produce high-caliber interferon-gamma (IFN-γ) and interleukin 4 (IL-4), and can resist the listeria bacteria attack of lethal dose; Darji etc. place CMV promotor downstream with the lacZ gene, are carrier oral route immune mouse with S.typhimurium transgenation strain, but inducing producing specificity antibody, T cell proliferation and ctl response; Shata etc. discover with the Salmonellas to be that carrier carries HIV-1gp140 gene DNA vaccine and direct intramuscular injection naked DNA can be induced the CD of equality strength ₈ ⁺T cell responses; Direct injection such as paglia codings finds that through the flow cytometer counting both have the GFP sign in the spleen BMDC number is close after the plasmid of green fluorescent protein (GFP) being arranged and being the immunity of carrier oral route with the Salmonellas; Marcela etc. successfully carry the segmental plasmid pTETnirl 5 of coding tetanus toxin C through the intranasal vaccination mouse with attenuated salmonella typhimurium disappearance strain CVD915 (guaBA lacks strain); Inducing mouse has produced IgG1, IgG2a and the IgG2b antibody to the segmental high titre of C, has also shown the generation of mouse boosting cell to Salmonella typhimurium and C segmental proliferative response and IL-2, IFN-γ simultaneously; Doggett etc. will cause that Suo Burui suis I/II type antigenic surface albumen (SpaA) gene clone of dental caries goes into attenuated salmonella typhimurium X4072 (pYA2905; Cya; Crp/asd) in; Use this bacterium oral immunity mouse then, find that the titre of anti-SpaA IgG in the mice serum is very high, and the titre of anti-SpaA IgG can detect also in the enteron aisle; Oscar Application of I type hemolysin (H1Yi) excretory system; Make attenuated salmonella typhimurium SL3261 and CVD908-htrA express exocytosis plasmodium falciparum sporozoite surface protein 2 (SSP-2); The immune mouse result shows in the intranasal; It can produce the immunne response (as producing the cell of IFN-γ) of specific cell mediation by excitating organism, and these all are to use attenuation salmonella development live vector vaccine to have established solid basis.

The taeniasis suis oncosphere is through enteron aisle invasion host; The TsoL18 relevant with the invasion of oncosphere (Taeniasolium oncosphere 18) albumen is the important antigen molecule in oncosphere stage; Mainly be present in oncosphere and infect early stage behind the intermediate host; In the taeniasis suis oncosphere of different geographical isolates, the TsoL18 gene is quite conservative, and this shows that TsoL18 is vital in the etap of taeniasis suis oncosphere; And; TsoL18 antigen also has excellent protective to the host, and the immune serum of TsoL18 recombinant antigen can kill oncosphere in vitro tests, so TsoL18 antigen becomes one of important vaccine candidate antigen of prevention cysticercosis cellulosae.

In research in the past, TsoL18 antigen is many with the inclusion body formal representation in prokaryotic expression system, and the expression amount of soluble proteins is extremely low; Although the formation of inclusion body is highly beneficial to the purifying of expression product; But the albumen of TsoL18 inclusion body after sex change, renaturation is extremely unstable, and immune effect also receives very big influence, and protein purification technology is very complicated; Cost is higher, thereby has influenced the TsoL18 Application of Recombinant greatly.Discover that it is a lot of to influence eukaryotic gene factor of expression level in prokaryotic system, such as based composition of different carrier systems, bacterial classification, abduction delivering condition and goal gene itself or the like.Main determining factor is the based composition of goal gene after carrier system is confirmed; Wherein how much rare codon is that near the rare codon of the key factor that influences the exogenous protein expression amount, especially initiator codon is bigger to the expression amount influence of target protein.The sequence that the research proof is right after the initiator codon downstream plays a significant role in the translation initiation process; Near transcript 5 ' end, exist rare codon will influence translation efficiency; The gene of highly expressing shows codon preference greatly than low gene of expressing; Through improving the expression level of foreign gene in bacterium with codon replacement rare codon commonly used or with " rare " tRNA gene co-expressing; And, in the bacterium genetic expression poor to rare codon (AGA, the AGG) expression effect that shows no favouritism to, expression efficiency is lower when the AGA-AGG continuous sequence exists especially.Research proves that also the secretion signal of target gene sequences N end is very big to the stability influence of eukaryotic gene expression product in prokaryotic system in addition; Research such as Lightowler proof is when 17 amino acid signal peptides of taenia ovis 45W genetically deficient N end; The solubility of expressing protein rolls up, and the stability of recombinant antigen also improves greatly.

The Salmonellas live vector vaccine is as the research focus of a new generation vaccine, and application development is very rapid, but utilizes the research of the oral live vector vaccine of attenuated salmonella typhimurium development cysticercus cellulosae not appear in the newspapers as yet.The object of the invention is through genetic engineering modified taeniasis suis oncosphere TsoL18 gene; And be connected to the attenuated salmonella typhimurium expression vector; Structure carries the attenuated salmonella typhimurium vaccine strain of TsoL18 gene, thereby the oral live vector vaccine of characteristics such as a kind of easy, long-acting, comprehensive induction of immunity reaction is provided.

Advantage of the present invention is:

1, recombination of the present invention is clipped 16 amino acid whose signal peptide sequences of TsoL18 gene N end through pcr amplification; And four bacterial expression rare codons of the 12nd (a sports t) of clipping the TsoL18 gene order, 333 (a sports c), 335 (a sports c), 336 (a sports t) position have been carried out rite-directed mutagenesis; Near the TsoL18 gene start codon the rare codon that influences bacterial expression is sported the bacterial expression preference codon, do not form but do not change its amino acid.These transformations have increased the solubility expression of TsoL18 gene in Salmonellas, and the proteic stability of TsoL18 also improves a lot.

2, the attenuated salmonella typhimurium carrier that utilizes among the present invention can directly be expressed target protein at the intestines wall as engineering bacteria; Expressed proteins can infect full-time antigen presenting cells (APC) such as BMDC through intestines wall microfold cell target, effectively avoids the degraded of target protein; Oncosphere is through enteron aisle invasion host, and the oral antigenic recombinant salmonella live vector vaccine of TsoL18 that carries can be blocked oncosphere through mucous membrane of small intestine invasion host, thereby reaches protection host's purpose.

3, the attenuated salmonella typhimurium of utilization of the present invention is a kind of invasive enteron aisle born of the same parents endoparasitism bacillus; Carry and expression alien gene as carrier; After immunity, not only induce body to produce to the antigenic immunne response of purpose; And can produce immunne response to carrier itself, increased the immune scope of vaccine.

4, the unmethylated motif (motif) of the liposome class material of living vaccine carrier attenuated salmonella typhimurium of the present invention, DNA of bacteria and bacterium CpG sequence etc. can also be brought into play the effect of immunological adjuvant, have strengthened the immunogenicity of vaccine greatly.

5, the oral living vaccine of making carrier with attenuated salmonella typhimurium among the present invention can be induced Th1 and Th2 mixed immunity answer-mode; And pig immunne response pattern in the not same period of infecting cysticercus is different; Preponderate with the Th1 cell in early days; Preponderate with the Th2 cell in the middle and later periods, live vector vaccine inductive immunization type of the present invention like this and pig infect the autoimmune response type that excites behind the cysticercus and are consistent, and can better bring into play the immune effect of this vaccine.

6, behind the living vaccine carrier attenuated salmonella typhimurium invasion host of the present invention, can be in for some time in host cell endoparasitism and limited propagation, the foreign protein of its expression continues to stimulate to the host, can save the booster immunization of repeated multiple times.

7, the expressed target antigen of vaccine of the present invention does not need in vivoexpression, separation, purifying and renaturation, can directly be used for immunoprotection, has removed the complicated procedures of forming of protein aftertreatment from, and its preparation is cheap, is easy to produce, stores and transportation.

8, vaccine strains of the present invention through experiment proof can be stable carry expression vector going down to posterity of continous-stable in vivo and in vitro; Expressed proteins has immunogenicity; And can inducing mouse produce corresponding antibodies, for a new approach has been opened up in the control of cysticercosis cellulosae and the research of vaccine for cysticercus cellulosae.

Description of drawings

Fig. 1 is the structure flow process and the final structure synoptic diagram of recombinant expression vector among the present invention.

Fig. 2 is the gel electrophoresis figure of the TsoL18 target gene PCR that arrives involved in the present invention, and among the figure: 1 is DNAMarker DL2000; 2 is the product of TsoL18 behind PCR.

Fig. 3 is that the gel of the TsoL18 goal gene that arrives involved in the present invention reclaims electrophorogram, and among the figure: 1 is the product that TsoL18 reclaims through PCR; 2 is DNAMarker DL2000.

Fig. 4 be involved in the present invention to the enzyme of recombinant expression plasmid pYA3342-TsoL18 cut evaluation figure, among the figure: 1 is DNAMarker λ-EcoT14I digest; 2 is recombinant plasmid pYA3342-TsoL18; 3 is recombinant plasmid pYA3342-TsoL18/Sal I; 4 is recombinant plasmid pYA3342-TsoL18/EcoR I; 5 is recombinant plasmid pYA3342-TsoL 18/Sal I+EcoR I; 6 is DNA Marker DL2000.

Fig. 5 is urea-SDS-PAGE figure as a result of X4550 that arrives involved in the present invention (pYA3342-TsoL18) and X4550 (pYA3342), and among the figure: 1 is X4550 (pYA3342-TsoL18); 2 is ProteinMolec μ lar Weight Marker; 3 is X4550 (pYA3342).

Fig. 6 is the Western-blot figure as a result of X4550 that arrives involved in the present invention (pYA3342-TsoL18) and X4550 (pYA3342), 1:Protein Molec μ lar Weight Marker among the figure; 2:X4550 (pYA3342-TsoL18); 3:X4550 (pYA3342).

Fig. 7 be involved in the present invention to the stability test of reorganization bacterium in 100 generations of continuous passage after transfer the incubation growth situation map of 117 strains reorganization bacterium at random.

Fig. 8 is the growth curve chart of the reorganization bacterium that arrives involved in the present invention.

Fig. 9 is the separate groups of mice antibody horizontal variation diagram that arrives involved in the present invention.

Embodiment

Embodiments of the invention below are provided:

1. bacterial classification used in the present invention and plasmid

Intestinal bacteria X6212 (Δ asd) (hereinafter to be referred as X6212), Salmonellas intermediate host X3730 (Δ asd, Tc ^s, Str ^r, Δ Gal) and (hereinafter to be referred as X3730), Salmonellas final host X4550 (Δ asd, Δ crp, Nal ^r, Δ cya) and (hereinafter to be referred as X4550) and Salmonellas expression vector pYA3342 (asd ⁺Expression vector, asd ^-/ asd ⁺).Above bacterial classification is disclosed in U.S. Pat 6872547.

2.TsoL18 the preparation of goal gene

1) hatching of taeniasis suis worm's ovum and activation

Smash filling clothes teniasis patient to pieces with the Semen Cucurbitae that fries, take spissated betel nut juice expelling parasite behind the 30min, with the integrity of assurance polypide and the vigor of worm's ovum.Select the terminal sophisticated gravid segment of taeniasis suis according to nodal plate length; Behind the normal saline flushing several; Shred nodal plate with eye scissors in the horizontalization ware; And crush and grind discharges worm's ovum fully, removes by filter the body wall fragment with 100 order copper mesh, the filtrating of collection with the centrifugal rinsing of saline water 3000r/min 3 times to the microscopy worm's ovum clean fully till.With the saline water worm's ovum that suspends, add equivalent Youxiaolin (NaClO) back mixing rapidly, put immediately microscopically observe treat most of worm's ovum broken shells after, add the 0.2mol/L Sulfothiorine (NaS of equivalent rapidly ₂O ₃) mixing is with termination reaction.The 3000r/min centrifuge washing is removed the worm's ovum fragment, with the saline water deposition that suspends again, adds the hatching activation liquid that contains pancreatin and Fel Sus domestica; 37 ℃ of effect 45min, every at a distance from 10min piping and druming mixing 1min, the centrifugal supernatant of abandoning of 3000rpm; Deposition is hatching and activated oncosphere, with its liquid nitrogen cryopreservation.

2) extraction of the total RNA of oncosphere

It is following to press Promega company SV Total RNA isolation system description operation:

1. from liquid nitrogen, take out the frozen oncosphere of activation, place miniature homogenizer, in ice bath, grind rapidly;

2. every pipe adds 175 μ l SV RNA lysis buffers, puts upside down to mix;

3. be transferred in the eppendorf pipe that the DEPC of a sterilization handles, add 350 μ l SV RNA dilution buffer liquid, put upside down and mix 3-4 time, 70 ℃ of water-baths 3 minutes;

4. 13000g, 4 ℃ centrifugal 10 minutes, supernatant is gone in the fresh centrifuge tube, add 200 μ l absolute ethyl alcohols (95%), piping and druming mixes 3-4 time;

5. shift mixture to Spin Column, 13000g, 4 ℃ centrifugal 1 minute, pour out the liquid in the collection tube;

6. the SV RNA lavation buffer solution that on spin column, adds 600 μ l alcohol dilutions, 13000g, 4 ℃ are centrifugal 1 minute;

7. add 50 μ l DNase incubation mixtures and (contain 5 μ l DNase I, 5 μ l 0.09M MnCl ₂) cover on the cylinder, 20-25 ℃ hatch 15 minutes after, add 200 μ l DNase stop buffers again, centrifugal 1 minute of 13000g;

8. add 600 μ l, 250 μ l SV RNA washingss respectively, 13000g, centrifuge washing was twice in 1 minute;

9. change the eppendorf pipe of a new no RNase,, put-70 ℃ of preservations with 100 μ l nuclease free water elution RNA.

3) Tso18 Gene RT-PCR amplification

1. primer design is with synthetic

Taeniasis suis TsoL18 gene order according to the Genbank login; Design upstream and downstream primer P1, P2; Upstream primer P1 contains EcoR I restriction enzyme site; Downstream primer P2 contains Sal I restriction enzyme site, clips 16 amino acid whose signal peptide sequences of TsoL18 gene N end when increasing through designed primer RT-PCR; Utilize 5 ' and 3 ' end of the TsoL18 gene order of Graphical Codon Usage Analyser 2.0 software analysis truncated signals peptides in bacterial expression, to belong to four bacterial expression rare codons of the base of rare codon and the 12nd (a sports t) through this gene of design of primers rite-directed mutagenesis, 333 (a sports c), 335 (a sports c), 336 (a sports t), but do not change its coded albumen.

Upstream primer P1:5 '-ccggaattcgcagcggtgaccgtacattcgg-3 '

Downstream primer P2:5 '-acgcgtcgacctacgaacggcggaccttcttgt-3 '

2. RT-PCR amplification

Reverse transcription is carried out in the following solution of adding and the agent of having a try in the PCR of 0.5ml reaction tubes:

Total RNA suspension 10 μ l

Oligo(dT) 1μl

The rearmounted 75 ℃ of preparatory sex change 5min of water-bath of abundant mixing in the cold but 3min that goes of ice bath, add following reagent rapidly then:

5 * AMV buffer (damping fluid), 4 μ l

2.5mmol?dNTP?Mixture 1μl

RNase?inhibitor(50u/μl) 0.5μl

Fowl source ThermoScript II (AMV, 10U/ μ l) 1 μ l

DEPC water 2 μ l

After putting the centrifugal mixing of Eppendorf centrifuge, 42 ℃ of water-baths 1 hour, 65 ℃ of 5min deactivation ThermoScript II.

The double-stranded cDNA of pcr amplification, the following reaction system of preparation in the PCR reaction tubes:

Reverse transcription product 10 μ l

10×PCR?buffer(Mg ²⁺free) 5μl

2.5mmol/L?dNTP?Mixture 4μl

Each 0.5 μ l of P1 and P2 primer (50pmol/ μ l)

25mmol/L?MgCl ₂ 4μl

Sterilization vaal water 26 μ l

In the rearmounted pcr amplification appearance of mixing, 95 ℃ of preparatory sex change added 0.5 μ l (5U/ μ l) Taq archaeal dna polymerase after 5 minutes, circulated as follows:

94℃ 50s

56℃ 50s

72℃ 1min

35 circulations, last 72 ℃ were extended 10 minutes.After amplification is accomplished, get PCR product 5 μ l, add 1 μ l, 6 * DNA sample-loading buffer, with 1.0% sepharose (contain 0.5 μ g/ml ethidium bromide, EB) electrophoresis observation, the result shows that the size of purpose band and expection is identical, is about 350bp, referring to Fig. 2.

3. the recovery of PCR product and purifying

DNA glue recovery test kit operation instructions by TaKaRa company is carried out, and gets PCR and reclaims product 5 μ l, adds 1 μ l, 6 * DNA sample-loading buffer, and (contain 0.5 μ g/ml ethidium bromide, EB) electrophoresis observation result is referring to Fig. 3 with 1.0% sepharose.

3.pYA3342-TsoL18 the structure of recombinant plasmid vector and evaluation

PCR product and plasmid vector pYA3342 that sepharose reclaims carry out EcoR I, Sal I double digestion 4h respectively; Glue reclaims the PCR product of about 350bp and the plasmid vector enzyme of 2.7Kb is cut the purpose segment; T4 DNA Ligase connects linked system: purpose fragment, 6 μ l; Plasmid vector, 2 μ l; T4 DNAUgase, 1 μ l; T4 DNA Ligase Buffer, 1 μ l (purpose fragment and plasmid vector mol ratio are about 5: 1); 16 ℃ connect 16h.The single bacterium colony of X6212 on the LB agar of picking (containing DAP 50 μ g/ml and the NA 20 μ g/ml) flat board, jolting overnight cultures in containing the LB liquid of DAP, the preparation electricity changes competence X6212.The product electricity be will connect and X6212, electric conversion condition: voltage 2500V, 25uF electric capacity transformed; 200 Ω resistance, 2mm electricity revolving cup, discharge time 3～5ms; 37 ℃ of 140rpm/min joltings of the X6212 bacterium liquid 1h that transforms, the centrifugal 10min of 4000rpm/min, bacterial sediment are coated with LB (containing 20 μ g/ml NA) agar plate after being dissolved in 100 μ l LB nutrient solutions; Cultivate 20～24h for 37 ℃; About 0.4～0.5mm the size of 15 diameters colony growths are arranged on the LB flat board, and 4 single bacterium colony joltings of picking are spent the night at random, behind the extracting plasmid with behind EcoR I, Sal I single endonuclease digestion and the EcoR I/Sal I double digestion; 1.0% agarose gel electrophoresis obtains about 350bp and 2.7kb two bands (Fig. 4), conforms to expection.Enzyme is cut the correct bacterium colony of evaluation send the order-checking of TaKaRa company, the homology of the taeniasis suis TsoL18 gene order of TsoL18 sequencing result and Genbank login is 97%, and amino acid identity is 100%.

The TsoL18 of the present invention's reorganization checks order row as follows: (adding the bold Italic base in the sequence table is the base of undergoing mutation)

agcggtgacc?g

acattcgg?cgacgatatt?ttcgtgccat?accttcgctg 50

cttcgccctt?agcgctaccg?aaattggggt?gttttgggat?gctggagaga 100

tggttggcca?tggcgtagag?gagatcaaag?tgaaagtaga?aaaagcaata 150

cacccataca?agatctggaa?tgcaacagtc?agcgcgaaca?atggaaaagt 200

catcatcaga?gacttgaagg?cgaagacaat?ttacagagtg?gacgtagacg 250

gttatcgaaa?cgaaatcatg?gtgtttggtt?cgcagcgttt?cgcgacaaca 300

cttccgaaaa?agcagatcaa?gcccctgaag?gtccgaagat?cgtag 345

Taeniasis suis oncosphere TsoL18 gene (AF017788) sequence of the last login of GenBank is following:

(wherein use and add sequence that bold Italic representes 16 amino acid whose signal peptide sequences) as the N end

ag 50

cggtgaccga?acattcggcg?acgatatttt?cgtgccatac?cttcgctgct 100

tcgcccttag?cgctaccgaa?attggggtgt?tttgggatgc?tggagagatg 150

gttggccatg?gcgtagagga?gatcaaagtg?aaagtagaaa?aagcaataca 200

cccatacaag?atctggaatg?caacagtcag?cgcgaacaat?ggaaaagtca 250

tcatcagaga?cttgaaggcg?aagacaattt?acagagtgga?cgtagacggt 300

tatcgaaacg?aaatcatggt?gtttggttcg?cagcgtttcg?cgacaacact 350

tccgaaaaag?cagatcaagc?acaagaaggt?ccgaagatcg?tag 393

4. the preparation of vaccine and detection

4.1 recombinant plasmid transformed final host's attenuated salmonella typhimurium X4550

Extracting recombinant plasmid from intestinal bacteria X6212 (pYA3342-TsoL18), CaCl ₂Method transforms Salmonellas intermediate host X3730; Make recombinant plasmid obtain the methylation patterns of Salmonella typhimurtum, from the X3730 bacterium, separate recombinant plasmid again, electricity transforms final host's vaccine strain X4550; Bacterium X4550 (pYA3342-TsoL18) obtains recombinating; Be vaccine of the present invention, its structure flow process and final structure are referring to Fig. 1, and the electric conversion condition in its process is with the above.Make up empty plasmid bacterium X4550 (pYA3342) as blank with method.

4.2TsoL18 expression of gene and Westren blotting analyze

Reorganization bacterium X4550 (pYA3342-TsoL18) and empty plasmid bacterium X4550 (pYA3342) are after IPTG (final concentration 1mmol/L) induces 6h; Centrifugal collection thalline adds 8M urea soln solvency action half a hour, adds appearance Buffer on 2 times the urea glue; Boiled 5 minutes, and carried out urea-SDS-PAGE.With above-mentioned urea-SDS-PAGE gel electrotransfer to nitrocellulose filter (NC film), albumen Marker puts in the amino black staining fluid and contaminates 3～5min, takes out the back with the rinsing liquid decolouring, takes off to the greatest extent until the background blueness; All the other NC films with PBST rinsing three times after; With 37 ℃ of 2% bovine serum albumins sealing 1h post rinsing three times, add after 4 ℃ of the anti-TsoL18 serum of rabbit spend the night, rinsing adds goat anti-rabbit igg for three times and combines 1h for 37 ℃; PBST rinsing three times; The NC film is transferred in the substrate solution, and room temperature lucifuge jog 3min observes the colour developing situation, changes room temperature preservation in the PBST damping fluid when waiting band to occur immediately over to.

The TsoL18 target protein that urea-SDS-PAGE electrophoresis result demonstration molecular weight is about 13ku has higher expression in X4550, consistent with the molecular weight of albumen of expection, referring to Fig. 5.

Western blot result shows that corresponding colour developing band (Fig. 6) is arranged on about 13ku position.Explain that the TsoL18 antigen protein of expressing among the X4550 (pYA3342-TsoL18) has antigenicity, can with anti-TsoL18 antibody response.

4.3 the vitro stability of reorganization bacterium X4550 (pYA3342/TsoL18) test

The thalline that 37 ℃ of shaking culture are spent the night is inoculated in the LB substratum that contains DAP by 10%; Continue to cultivate 12h; Above-mentioned culture is inoculated in the LB liquid that contains DAP and cultivates 12h by 10% amount again, and so cultured continuously four times is to 50h (being equivalent to thalline went down to posterity for 100 generations), with culture dilution 10 ⁶Doubly, get the 100ul diluent and be coated with the LB agar plate that contains DAP, after the incubated overnight, 100 single bacterium colonies of random choose change and plant at DAP ^-The LB agar plate on, if plasmid loss, bacterium can be at DAP ^-The LB agar plate on grow, measure the stability of recombinant plasmid with this.20 bacterium colonies of picking carry out antigenic expression at random, with the stability of confirming that the TsoL18 gene is expressed in the attenuated salmonella typhimurium host.

The result shows that reorganization bacterium X4550 (pYA3342-TsoL18) is not having under the situation of selective pressure 100 generations of continuous passage, and 117 colony lifts of random choose are to DAP ^-Flat board, all bacterial strains can both be grown, referring to Fig. 7.20 of picking plasmids carry out abduction delivering at random, and the purpose antigen presentation is all arranged, and show in the recombinant plasmid pYA3342-TsoL 18 ability stable existence attenuation host bacterium X4550.

4.4 the mensuration of the growth curve of reorganization bacterium

Picking X4550 (pYA3342) and X4550 (pYA3342-TsoL18) mono-clonal bacterial strain; Be inoculated in respectively in the LB liquid nutrient medium, 37 ℃ of incubated overnight are got 50 μ l again and are inoculated in the 5ml LB liquid nutrient medium from nutrient solution; 37 ℃ of joltings were cultivated, and whenever measured OD at a distance from 1 hour ₆₀₀Value (table one) is drawn and is measured the bacteria growing curve, referring to Fig. 8.As can beappreciated from fig. 8 the growth conditions basically identical of various bacterium representes that promptly the positive carrier of reorganization changes in the attenuation salmonella, does not influence metabolism and the growth of this bacterium.

Table 1 X4550 (pYA3342) and X4550 (pYA3342-TsoL18)

Cultivate the OD of different time ₆₀₀Value

4.5 the safety experiment of reorganization bacterium (being vaccine strain)

Reorganization bacterium through the oral various dose of BALB/c mouse is confirmed; Mouse elder generation fasting 12 hours; Prohibited water 4 hours, irritate every mouse of 30min before the stomach earlier with 10% sodium hydrogencarbonate 100 μ l irritate stomach with in and hydrochloric acid in gastric juice, the bacterium of will recombinate then filling stomach is immune; To mouse water and food are provided again behind the 30min, experimental result is seen table two.The result shows oral recombinant bacterial strain X4550 (pYA3342-TsoL18) 2.0 * 10 ¹²Behind the cfu 30 days, survival rate still is 100%.And according to the oral wild strain X3181 1 * 10 of existing research report C57BL/6 mouse ⁷Behind the cfu, all dead in 5 days, this proves that also recombinant bacterial strain of the present invention is safe.

Survival rate behind the reorganization Salmonella typhimurium of the oral various dose of table 2 mouse

Bacterial strain	The genes involved type	Oral dosage (c.f.u)	Observing time (my god)	Survival rate
					X4550(pYA3342-TsoL18)	?Δcrp，Δcya， TsoL18 +?	1.0×10 7 1.0×10 9 1.0×10 10 2.0×10 12	30 30 30 30	5/5 5/5 5/5 5/5

The experiment of 5 mouse immunes

30 of 18～20g BALB/c mouses are divided into three groups at random, are respectively X4550 (pYA3342-TsoL18) immune group, the immune control group of X4550 (pYA3342) and blank group, each immune secondary, and immunity is 2 weeks at interval, and 1.9～2.5 * 10 ⁹Bacterial strain/inferior the stomach of irritating as stated above, before each immunity and two exempt from two weeks of back, the ELISA of docking blood sampling all around measures antibody horizontal.

Respectively organize the antibody horizontal statistic analysis result behind the mouse oral immunity and see table three; Fig. 9 shows that X4550 (pYA3342-TsoL18) immune group antibody horizontal has tangible rising; And the antibody horizontal of immune control group of X4550 (pYA3342) and non-immune control group does not have obvious variation; Proof X4550 (pYA3342-TsoL18) vaccine strain can induce body to produce purpose antibody, has immune effect.

The mensuration result of antibody horizontal in the serum behind the oral live recombinant vectors vaccine of table 3 mouse

Annotate: mark letter not on the same group, difference is (P＜0.01) extremely significantly; Mark is alphabetical mutually on the same group, and difference is remarkable (P＞0.05) not.

More than test shows that the present invention more adapts in Salmonellas it and expresses through taeniasis suis oncosphere TsoL18 gene is transformed; Vaccine strain of the present invention can be stable carry the continuous passage in vivo and in vitro of attenuated salmonella typhimurium expression vector; Expressed proteins has good immunogenicity; And can produce purpose antibody by inducing mouse, for the control and the vaccine production of cysticercosis cellulosae have been opened up a new approach.

Claims (2)

1. taeniasis suis oncosphere recombination; It is characterized in that the 1st to the 48th bit base (signal peptide) in the taeniasis suis oncosphere TsoL18 gene original series is all removed, and the 12nd bit base a in the last gene order is sported t, the 333rd bit base a sport that c, the 335th bit base a sport c, the 336th bit base a sports t.

2. the preparation method of the described taeniasis suis oncosphere of claim 1 recombination; It is characterized in that getting the terminal sophisticated gravid segment of taeniasis suis; Therefrom take out hatching and activated oncosphere; Extracting total RNA of oncosphere again, is that template is carried out RT-PCR with total RNA of oncosphere, and the upstream and downstream primer of employing is respectively:

Upstream primer P1:5 '-ccggaattcgcagcggtgaccgtacattcgg-3 '

Downstream primer P2:5 '-acgcgtcgacctacgaacggcggaccttcttgt-3 '.

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