CN103352047B - Trichinella Spiralis larva ES antigen gene vaccine and preparation method - Google Patents

Abstract

The invention discloses a Trichinella Spiralis larva ES antigen gene vaccine and a preparation method thereof. Trichinella Spiralis larva 43ku and 45ku ES protein genes are respectively inserted to a pVAXI eukaryotic expression vector to obtain recombinant plasmids, and the recombinant plasmids are mixed according to a ratio of 1:1 to prepare a combined recombined DNA vaccine for preventing Trichinella Spiralis infection and improving the worm reduction rate of bodies. After the injection immunization of hindlimb muscles of mice for three weeks, 2000 worms are killed, the worm reduction rate can reach 76.65%, and the quantity of eggs at the tongue tissues of the mice is obviously reduced.

Description

Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine and preparation method thereof

Technical field

The invention belongs to biological technical field, Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine and preparation method thereof.

Background technology

Trichinella spiralis parasitizes in people and multiple vertebrate, is cause trichinous parasitic zoonoses.Trichinella spiralis can parasitize in people and 150 many animals bodies, and the major cause of infection takes in the raw or not yet done meat product with cultivation of larvae of Trichinella spiralis from muscle packing due to body and causes, if Diagnosis and Treat not in time, can cause death.According to estimates, nearly 1,100 ten thousand the infecteds in the current whole world, China is after 1975, in Jilin, the report that also has this disease to break out of Liaoning, Heilungkiang, Henan, the province, city and region such as Hubei.This disease not only serious harm human health, also can cause huge financial loss to pig industry and meat outlet, World Organization for Animal Health reports 10,000 first-born pigs for 1998 and infects, all there be the popular of pigs trichina disease in 26 provinces, cities and regions of China, the country fair of the vast Rural areas of China often has after privately butchering and sell without the pork of Trichinella spiralis quarantine or pork product, bring great potential threat to human health and animal health cultivation.But, there is no effective Trichinella Spiralis Vaccination at present.

The etap of Trichinella spiralis is adult stage, newborn larvae phase and muscle larvae phase three phases, and somatic antigen composition is various, is mainly surface antigen, somatic antigen, excretory-secretory antigen (ES antigen), rod particle related antigen.Research shows that Trichinella spiralis ES antigen all can produce three periods, be Trichinella spiralis metabolic secretion be directly exposed in the immunity system of host, being the topmost protective antigen of the anti-trichinzation of host, is also the main Trichinella Spiralis Antigens of current research.

Trichinella spiralis ES antigen is the antigenic substance of the secretion of its natural hole or excretion, and the polypide of different times can produce this antigenic substance, and main Secreting section is the staff cell secretion near polypide food meatus.ES antigen has multiple protein to become to be grouped into, and the SDS-PAGE such as Despommier finds there are 28 kinds of antigenic components in Trichinella spiralis ES antigen, and can find 37 kinds of antigenic components by IEF method, wherein glycoprotein is 22 kinds; Domestic scholars Zhu Xing congruence is is also researched and analysed by experiment and is confirmed that the composition of Encoding ES Antigen from Trichinella spiralis Muscle Larvae divides very complicated.Research finds, 43ku and 45ku ES antigen can induce body to produce main protection antibody.

Trichinella spiralis 43ku antigen is the major protein component of ES antigen, has the conservative property of height in Trichinella.Research shows the nutrition of 43ku ES antigen and host, cell is formed, parasite elimination reacts and protective immune response is relevant, and 43ku ES glycoprotein is by as immunodiagnosis and the good candidate antigen that develops vaccine.

Trichinella spiralis 45 ku antigen is a kind of secreting glycoprotein, is also one of major antigen composition of cultivation of larvae of Trichinella spiralis from muscle ES product.First Arasu etc. have cloned 45 ku antigen genes, and its recombinant protein can by the identification of Level of Mice Infected Trichinella Spiralis serum.It is serine protease multigene family that Trichinella spiralis 45 ku antigen is inferred, a kind of albumen of coding muscle larvae, but eliminates Mei Huohang center.Research also finds that this antigen not only has good immunogenicity, and has stronger immune protective for Trichinella spiralis challenge infection.Current research shows, this gene has expression in cultivation of larvae of Trichinella spiralis from muscle and adult, Trichinella spiralis 45 ku antigen comprises peptide epitopes and sugared epi-position, not only having can the common epitope of variance in form Trichinella spiralis kind, and there is the species specific epitope of Trichinella spiralis can be recogned by monoclonal antibody, in king, congruent people applies RT-PCR method and carries out cloning and expression to Trichinella spiralis 45 ku antigen gene, in increased 890 bases, there is the gene homology reported with American scholar to reach 99.6%, this shows that 45ku antigenic protein gene exists the conservative property of height, it is the good candidate gene of DNA vaccination development.

DNA vaccination refers to and is injected directly in animal body by the recombinant eukaryon expression vector of certain antigen of coding, foreign gene is expressed, the immunity system of the antigen activates body of generation, thus the humoral immunization of inducing specific and cellullar immunologic response in vivo.This vaccine had both had the advantage of attenuated vaccine, simultaneously again without the danger reversed, was therefore more and more subject to people's attention, was counted as the third generation of vaccine after traditional vaccine and genetic engineering subunit vaccine.

Summary of the invention

The object of this invention is to provide a kind of effective prevention trichinzation, the union and recombination DNA vaccination improving worm reduction rate and preparation method thereof.

The carrier for expression of eukaryon pVAX I of cultivation of larvae of Trichinella spiralis from muscle restructuring 43ku ES antigen gene- 43ku, it inserts in carrier for expression of eukaryon pVAX I 43kugene, described 43kuits base sequence is as shown in sequence table SEQ ID No.1;

The carrier for expression of eukaryon pVAX I of cultivation of larvae of Trichinella spiralis from muscle restructuring 45ku ES antigen gene- 45ku, it inserts in carrier for expression of eukaryon pVAX I 45kugene, described 45kuits base sequence is as shown in sequence table SEQ ID No.2;

Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine, it comprises: by recombinant plasmid pVAX I- 43kuwith pVAX I- 45ku;

Their weight ratio is 1: 1;

Described pVAX I- 43kuwith pVAX I- 45kuconcentration be 1 μ g/ μ L.

Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine preparation method, comprising:

(1) prepare cultivation of larvae of Trichinella spiralis from muscle total serum IgE, reverse transcription is cDNA, and is template with cDNA, with primer:

43ku -P1：5’-CGCGGTACCATGCGAATATACATTTTTCTTAG-3’

43ku -P2：5’-CGAGGATCCTTAGCTGTATGGGCAA-3’

45ku -P1：5’-CGCGGTACCATGAAACTCTTGCTTTTAACA-3’

45ku -P2：5’-GCGGATCCTTAGCCTTGCTTAGAGAG-3’

PCR method increases 43ku, 45ku ES antigen gene respectively, and its base sequence of 43ku, 45ku gene is as shown in sequence table SEQ ID No.1,2;

(2) 43ku, 45ku gene is inserted respectively in pVAX I carrier for expression of eukaryon, build recombinant eukaryon expression vector pVAX I- 43kuwith pVAX I- 43ku;

(3) two kinds of recombinant eukaryon expression vectors are proceeded to respectively e.coliin DH5 α, be inoculated in LB liquid nutrient medium, 37 DEG C, 150-180 r/min shaking table is cultivated.

(4) from above-mentioned cultivation e.colia large amount of preparation pVAX I in DH5 α- 43kuwith pVAX I- 45ku, and mix after both adjustment concentration.

The invention provides Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine and preparation method thereof, utilize the antigenicity of Trichinella spiralis 43ku, 45ku ES albumen, the union and recombination DNA vaccination that preparation can be used for preventing trichinzation, improves body worm reduction rate, through mouse hind leg intramuscular injection immunity after three weeks, attack worm 2000, worm reduction rate can reach 76.65%, and mouse tongue organizes worm's ovum number obviously to reduce.

The invention has the advantages that: 43ku, 45ku ES albumen that (1) is chosen is the protective antigen albumen that Trichinella has period, be beneficial to the polypide that body removes different times; (2) vaccine classes belongs to DNA vaccination, and structure is simple, and purifying technique is easy, and production cost is lower, is suitable for producing in enormous quantities; (3) DNA vaccination molecule stable, can be made into DNA vaccination freeze-dried vaccine, can recover original activity during use in salts solution, is convenient to transport and preserves; (4) DNA vaccination safety, elimination traditional vaccine easily reverses, the danger of anti-poison.

Accompanying drawing explanation

The pcr amplification of Fig. 1 .43ku and 45ku antigen gene; Wherein, A:43ku antigen gene pcr amplification; B:45ku antigen gene pcr amplification

Fig. 2. pMD18T- 43kuand pMD18T- 45kuenzyme cuts qualification; Wherein, A:pMD18T- 43kuenzyme cuts qualification; 2-B:pMD18T- 45kuenzyme cuts qualification;

Fig. 3. the preparation of eukaryon expression plasmid pVAX I;

Fig. 4. recombinant expression vector pVAX I- 43kuwith pVAX I- 45kuenzyme cut qualification; Wherein, A:pVAX I- 43kuenzyme cuts qualification; B:pVAX I- 45kuenzyme cuts qualification;

Fig. 5. pVAX I- 43kuwith pVAX I- 45kuthe indirect immunofluorescene assay of transfection MA104 cell; Wherein, A:pVAX I- 43kutransfection; B:pVAX I- 45kutransfection; C:pVAX I transfection (contrast); D pVAX I- 43kuimmune mouse attacks worm;

Fig. 6. mouse tongue histopathology result; Wherein, A: normal mouse does not attack worm; B PBS gavage mouse attacks worm; C pVAX I immune mouse attacks worm; E:pVAX I- 45kuimmune mouse attacks worm; F combined immunization mouse attacks worm.

embodiment:

the acquisition of embodiment 1 cultivation of larvae of Trichinella spiralis from muscle 43ku and 45ku antigen gene

(1) design of primers and synthesis

The cultivation of larvae of Trichinella spiralis from muscle 43ku logged according to GenBank and the gene order of 45ku ES antigen, adopt Primers 5.0 to design primer, and design respectively at the upstream and downstream primer of goal gene kpni He bamHi restriction enzyme site, primer sequence is synthesized by Shanghai Sheng Gong biotechnology company limited.Primer sequence is as follows

43ku upstream primer (43-P1): 5 '-CGC gGTACCaTGCGAATATACATTTTTCTTAG-3 '

43ku downstream primer (43-P2): 5 '-CGA gGATCCtTAGCTGTATGGGCAA-3 '

45ku upstream primer (45-P1): 5 '-CGC gGTACCaTGAAACTCTTGCTTTTAACA-3 '

45ku downstream primer (45-P2): 5 '-GC gGATCCtTAGCCTTGCTTAGAGAG-3 '

(2) pcr amplification of 43ku and 45ku antigen gene

(total serum IgE, reverse transcription is cDNA, and is template with cDNA, adopts above-mentioned primer to carry out pcr amplification to 43ku and 45ku antigen gene respectively for cultivation of larvae of Trichinella spiralis from muscle to adopt conventional Trizol legal system.Trichinella spiralis is international standard strain, and T.spiralis (strain ISS 534), is preserved by this laboratory.

PCR operational conditions: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 57 DEG C of annealing 1min, 72 DEG C extend 1min, totally 35 circulations; 72 DEG C of total elongation 10min.After reaction terminates, get 3 μ l products and carry out 0.8% agarose gel electrophoresis qualification, result shows and successfully amplifies 43ku and 45ku antigen gene, as shown in Figure 1.

(3) connection of 43ku and 45ku antigen gene and pMD18T, conversion

Adopt Axygen company DNA gel to reclaim test kit, operate to specifications, amplifying target genes is reclaimed, reclaim fragment and be connected with cloning vector pMD18T.

Condition of contact: 16 DEG C of connections are spent the night.

Adopt conventional CaCl ₂method will connect product conversion e.colidH5 α competent cell, and screen recombinant clone.

(4) pMD18T- 43kuand pMD18T- 45kuqualification and sequential analysis

The sub-plasmid of the doubtful positive colony of screening is extracted by the operation of plasmid little extraction reagent kit specification sheets.Adopt kpni He bamHi restriction enzyme carries out enzyme to extracted plasmid vector and cuts qualification, and it is as follows that enzyme cuts system:

Reaction conditions: 37 DEG C of water-bath 2 h.After end, detect through 0.8% agarose gel electrophoresis and show, have successfully been obtained recombinant expression vector pMD18T- 43kuand pMD18T- 45ku, as shown in Figure 2.

By recombinant plasmid pMD18T- 43kuand pMD18T- 45kube sent to Beijing Hua Da Gene science limited-liability company to check order, sequencing result shows through Blast compare of analysis, and it is 97% that the sequence similarity that the 43ku ES antigen gene increased and GenBank log in reaches 100%, 45ku ES antigen gene.

embodiment 2 recombinant eukaryotic expression plasmid pVAX I- 43kuwith pVAX I- 45kupreparation

Carrier for expression of eukaryon pVAX I purchased from American Invitrogen company, by plasmid little extraction reagent kit specification sheets operation preparation pVAX I, as shown in Figure 3.

(1) recovery of goal gene, connection and conversion

Adopt kpni and restriction enzyme respectively to pMD8T- 43ku, pVAX I-45ku and pVAX I carries out double digestion, to obtain 43ku, 45ku ES antigen gene and pVAX I large fragment; Double digestion system is as follows:

Reaction conditions: 37 DEG C of water-bath 3 h.After end, reclaim the operation of test kit specification sheets by DNA gel, goal gene fragment is reclaimed.

43ku, 45ku ES antigen gene is connected with pVAX I carrier large fragment respectively.Linked system is as follows:

Reaction conditions: 21 DEG C connect 4 h, and 4 DEG C are spent the night; Next day conventional CaCl ₂method transforms e.colidH5 α competence, and screen recombinant clone.

(2) recombinant expression vector pVAX I- 43kuwith pVAX I- 45kuenzyme cut qualification

Adopt kpni He bamHi restriction enzyme carries out enzyme to extracted plasmid vector and cuts qualification, and it is as follows that enzyme cuts system:

Reaction conditions: 37 DEG C of water-bath 2 h.After end, detect through 0.8% agarose gel electrophoresis and show, successfully construct recombinant expression vector pVAX I- 43kuwith pVAX I- 45ku, as shown in Figure 4.By restructuring correct for qualification e.colidH5 α is inoculated in LB liquid nutrient medium, 37 DEG C, and 150-180 r/min shaking table is cultivated, and adopts the large extraction reagent kit of plasmid, prepares recombinant eukaryon expression vector in a large number.

embodiment 3 pVAX I- 43kuwith pVAX I- 45kutransfection MA104 cell and expression product qualification

According to Lipofectamine ^tM2000 working instructions operations, by pVAX I- 43ku, pVAX I- 45kutransfection MA-104 cell respectively, and make blank with pVAX I.

MA-104 cell after to be transfected covers with cover glass and takes out, dry rear PBS(pH7.2) clean 1 time, 10-15 min is fixed with 10% acetone, PBS(pH7.2) clean 3 times, after blotting edge moisture, add mouse source 43ku and 45ku ES antigen monoclonal antibody respectively, put 37 DEG C of incubator effect 2 h, take out cover glass PBS(pH7.2) clean 3 times, add the goat anti-mouse IgG antibodies of FITC mark, put 37 DEG C of incubator effect 2 h, take out cover glass PBS(pH7.2) clean 3 times, dye with Yi Wensilan after blotting edge moisture, fluorescence microscopy Microscopic observation, MA-104 cell visible green fluorescence after recombinant expression vector transfection, result shows, the recombinant expression vector pVAX I built- 43ku, pVAX I- 45kuexpression can be obtained, as shown in Figure 5 in mammalian cell.

embodiment 4 pVAX I- 43kuwith pVAX I- 45kucombined immunization and effect detection

(1) immune programme for children

15 4-5 BALB/C mice in age in week are divided into 5 groups at random, and often organize 5, experiment mice grouping and process are in table 1.

The grouping of table 1 experiment mice and process

The inoculum concentration of B, C, D tri-groups is 1 μ g/ μ L, E group be 1 μ g/ μ L pVAX I- 43kuwith the pVAX I of 1 μ g/ μ L- 45kuequal-volume mixture.

Immunization route is mouse hind leg intramuscular injection, 1 time/week, continuous three weeks, and row next day of last injection attacks worm process, and dosage is 2000/, adopts a gavage mode.

(2) immune effect detects

After attacking worm surrounding, slaughter mouse, get whole muscle, ordinary method detects worm reduction rate; Calculation formula:

Worm reduction rate=control group muscle larvae number-recombinant plasmid group muscle larvae number/control group muscle larvae number × 100%.

Result shows, pVAX I- 43kuwith pVAX I- 45kuafter combined DNA vaccine immunity, worm reduction rate can reach 76.65%, as shown in table 2.

Table 2. is group polypide quantity and worm reduction rate respectively

Getting tongue organizes row HE to dye, and carry out histopathology, result shows, E group mouse tongue organizes worm's ovum number to be obviously less than all the other 4 groups, as shown in Figure 6.

110> Jilin Agriculture University

<120> Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine and preparation method thereof

<140>2013100406699

<160> 2

<210> 1

<211> 1035

<212> cDNA

<213> T.spiralis (strain ISS 534)

<400> 1

atgcgaatat acatttttct tagtgctttc tgggtcatct tgcacaactg tttgcaaatt 60

catgcagcta actgtacatg cagaactgct acagatgata cagaatggtt tttacttttt 120

aaacctgtag gtctattaaa ggctaagata atttctccag ctaatgctgg ttgggcaaat 180

gatggagcaa atatgaacac cgattccggc cacgctttgg ttcaaacgct tgctgaatgg 240

atggggccaa tacttgatga catgacagct cttggctata gcaacacgcc tccaaaatct 300

acgattacat ctcagactac ttcatctaaa ggtattttaa tgtttggaaa tgaaactacg 360

gatggatttt ggttactgca cacttttgaa cgagcatttc caaacagcgt tgcctggtca 420

tggccctcaa agtttacttc agaaggtcac atggctcttt gtttaagcat atctgaagat 480

aatgtgccac taatagttcc tgcacttcaa tatcaggaag tagtaattta ttttggtcaa 540

gtctcatcag aaaaggcaac ggaatttgct gatttaacat cattgattga tgggagcctg 600

ccaacaataa caccaccact ctggaaccag caaactatta caaccctaaa ttctgcactc 660

tcaacagttg tatattccaa aacatcttca tcccgattag aaatgtatgg tagcttcctc 720

gctaaagtta tggtagtcaa tatgcgcatc tgggctgtaa cagataatac actccaaaca 780

acatgtggtg ggaaaattgg tttcgtcaaa gttgtcaaaa gcccagtaac cattgatggc 840

acccaaaatg atagaagcaa agacaaatct caatgggcag ttatagatga caaacctgtg 900

ttctgcttta caacaaatgg ttactctact aaacagagaa cagtagctgg aagtgctaca 960

tgcattactc aacaagtagt cagcaatttg tttgctactt ctgctgcaaa ttttattcct 1020

tgcccataca gctaa 1035

<210> 2

<211> 858

<212> cDNA

<213> T.spiralis (strain ISS 534)

<400> 2

atgaaactct tgcttttaac attcctttac tttgtcgatg cagtaagttc agaatgtggg 60

gaaaatgcaa ctgagactct tgcattagta tacaaaccgg tgcagcaagg ttcaaaacgt 120

gtacttggca ttgcatgtca gggtacaatt gtgccaggaa aacataaaaa tcacactgat 180

actgttttgg tatcgtcgta ctgcattatt gaagatcctc cggaaggtta tgttgtcagt 240

gtcggttctt ctgatcctca tggagatctt caatcaagtg cacaacaatt cagggcccaa 300

cgtattctaa attttccatt tgagcaacat ccagttggaa ttttgaaaac tccacaacca 360

atcatgtaca gtgatacagt tcaacctatg tgcatagcaa gcgttccttt accagatgaa 420

catgcatgca taatgggagt tgtaacaaag ggaggtttaa tgacattgcg tcatgtgcaa 480

atgttatatg aaagtgattg tgaaccactt gcggaaggtt tgagttcata tttgtgtgca 540

aaagtgaagg aaattgatgc agaagtaggt gaaactcttg gtctagatcc atcaatggat 600

atatatccat tttctgctcc acttgatttt gatatcaatg gtgtaaaagc tggaagtttg 660

gaaaatccac tattctgtct taccaatgag catccaacat ggtcagttta cggatttgca 720

ttaaatgcat ataatgtaac agatccagaa agtcctattt tgttttctga tgtatcaagt 780

gatctgacag ccataaagga acacagtgac atcagttatc aacaatgggt acaacgtatg 840

ctctctaagc aaggctaa 858

Claims (2)

1. Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine, it comprises: recombinant plasmid pVAX I- 43kuwith pVAX I- 45ku, pVAX I- 43kuwith pVAX I- 45kuit is the gene inserted in carrier for expression of eukaryon pVAX I respectively as shown in sequence table SEQ ID No.1 and 2; Recombinant plasmid pVAX I- 43kuwith pVAX I- 45kuweight ratio be 1: 1; Concentration is 1 μ g/ μ L.

2. Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine preparation method, comprising:

(1) prepare cultivation of larvae of Trichinella spiralis from muscle total serum IgE, reverse transcription is cDNA, and is template with cDNA, with primer:

43ku -P1：5’-CGCGGTACCATGCGAATATACATTTTTCTTAG-3’

43ku -P2：5’-CGAGGATCCTTAGCTGTATGGGCAA-3’

45ku -P1：5’-CGCGGTACCATGAAACTCTTGCTTTTAACA-3’

45ku -P2：5’-GCGGATCCTTAGCCTTGCTTAGAGAG-3’

PCR method increases 43ku, 45ku ES antigen gene respectively, and its base sequence of 43ku, 45ku gene is as shown in sequence table SEQ ID No.1,2;

(2) will 43ku, 45kugene inserts in pVAX I carrier for expression of eukaryon respectively, build recombinant eukaryon expression vector pVAX I- 43kuwith pVAX I- 43ku;

(3) two kinds of recombinant eukaryon expression vectors are proceeded to respectively e.coliin DH5 α, be inoculated in LB liquid nutrient medium, 37 DEG C, 150-180 r/min shaking table is cultivated;

(4) from above-mentioned cultivation e.colia large amount of preparation pVAX I in DH5 α- 43kuwith pVAX I- 45ku, and after both adjusting, concentration is 1 μ g/ μ L, by weight 1: 1 mixing.