CN107365792A - The method for analyzing stability of taeniasis suis Recombinant Lactococcus lactis expression system - Google Patents
The method for analyzing stability of taeniasis suis Recombinant Lactococcus lactis expression system Download PDFInfo
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Abstract
Taeniasis suis of the present invention to be logged in GenBankTSOL18 gene orders are template, carry out gene optimization by host system of Lactococcus lactis MG1363, synthesize target geneTSOL18 and SPTSOL18, construction of expression vector and expression system.Enter performing PCR identification, SDS PAGE and Western blot analyses,TSOL18 detects that target protein is expressed in intracellular, extracellular to be not detected by target protein, and SPTSOL18 has target protein expression in intracellular with extracellular；pMG36e‑TSOL18 and pMG36e SPTSOL18 plasmids existL.lactisIn the generation of continuous passage 20 in MG1363, there is preferable genetic stability.Development and utilization for cysticercus cellulosae disease vaccine provides solid reference.
Description
Technical field
The present invention relates to taeniasis suis live recombined vaccines research field, specially taeniasis suis TSOL18 genetic recombination lactic acid
The method for analyzing stability of galactococcus expression system.
Background technology
Cysticercosis cellulosae (cysticercosis cellulosae) is commonly called as pork measles (bladder disease), is
Caused by the middle silk ribbon phase larva cysticercus cellulosae (cysticercus cellulosae) of taeniasis suis (Taenia solium, Ts)
A kind of serious harm Animal husbandry production and human health parasitic zoonoses, be health ministry planning preventing and treating weight
One of point parasitic disease.This disease is in worldwide distribution, the warp such as Major Epidemic is non-in, South Africa, Latin America, East Asia and South Asia
Ji, sanitary condition difference it is national and regional.China's Major Epidemic is in Heilungkiang, Jilin, Yunnan, Henan, Hebei, Shandong and Guangxi
Deng 31 provinces, cities and autonomous regions.According to generally investigating in recent years, the infection rate of China's taeniasis suis is 0.112%, and some areas are up to 0.66%
~6.00%, number of the infected 1,260,000, the infection rate of cysticercus is 0.14%~3.20%, number of the infected 3,000,000.Wherein 2.3%~
25.0% taeniasis suis patient is due to autoinfection and with the parasitism of cysticercus.Therefore safe and efficient vaccine control is researched and developed
The disease is significant.
TSOL18 genes are relatively conservative, are present in the TSO of activation, have good immunogenicity and antigenicity, recognized
To be the candidate vaccines gene of most development prospect.Lactococcus lactis (Lactococcus lactis, L.lactis) is in LAB
Most important pattern bacterium, application are closely related in food, field of medicaments.With the development of technique for gene engineering, selection tool
There is L.lactis that is efficient, having no toxic side effect as carrier, construct a system in fields such as bacterium, virus, tumour, parasites
Row recombinant vaccine.
For above-mentioned background technology field, there is not yet taeniasis suis restructuring L.lactis report.
The content of the invention
It is an object of the invention to provide the method for analyzing stability of taeniasis suis Recombinant Lactococcus lactis expression system.
To reach above-mentioned purpose, the technical scheme that uses for：
The method for analyzing stability of taeniasis suis Recombinant Lactococcus lactis expression system, according to taeniasis suis TSOL18 genes
Sequence, TSOL18 and SP-TSOL18 target gene is respectively synthesized, carries out recombinant plasmid pMG36e-TSOL18 and pMG36e-SP-
TSOL18 structure and taeniasis suis restructuring pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis
Structure；Analysis method comprises the steps of：
1) it is, that the plasmid containing target gene of acquisition is red mould with being free of in the GM17 fluid nutrient mediums containing erythromycin respectively
In the GM17 fluid nutrient mediums of element, after 30 DEG C of culture 24h, 4 DEG C stand overnight simultaneously conservation；
2) step 1) culture, is taken to carry out streak inoculation after GM17 flat boards, 30 DEG C of culture 24h respectively, in streak plate
Upper 100 single bacterium colonies of picking respectively are inoculated in accordingly culture medium of the culture medium containing erythromycin with being free of erythromycin, 30 degree of trainings
Growth bacterium colony is counted after supporting 24h；
3), repeat and reached for 20 generations；
4) the colony growth number of erythromycin flat board, is inoculated according to 100 single bacterium colonies, calculates its inheritance stability of its plasmid
Property.
The present invention is using the taeniasis suis TSOL18 gene orders logged in GenBank as template, with Lactococcus lactis
(Lactococcus lactis, L.lactis) MG1363 is that host system carries out gene optimization, by way of gene chemical synthesis,
Target gene TSOL18 is synthesized, double digestion is connected to pMG36e carriers, builds secreted expression carrier pMG36e-SP- respectively
TSOL18 and intracellular type expression vector pMG36e-TSOL18, digestion and sequencing identification；Again by its Electroporation Transformation to L.lactis
MG1363, successfully build taeniasis suis Lactococcus lactis expression system pMG36e-SP-TSOL18/L.lactis MG1363 and
pMG36e-TSOL18/L.lactis MG1363.Enter performing PCR identification, SDS-PAGE and Western blot analyses TSOL18 exists
Intracellular detects that target protein is expressed, extracellular to be not detected by target protein, and SP-TSOL18 has target egg in intracellular with extracellular
White expression；Identified by AKTA protein purifications and stability analysis, pMG36e-TSOL18, pMG36e-SP-TSOL18 plasmid exist
In the generation of continuous passage 20 in L.lactis MG1363, there is preferable genetic stability.Exploitation and profit for cysticercus cellulosae disease vaccine
With providing solid reference.
Brief description of the drawings
Fig. 1 is the original series code of taeniasis suis TSOL18 gene orders；
Fig. 2 is sequence code after the optimization of taeniasis suis TSOL18 gene orders；
Fig. 3 is the original G/C content figure of taeniasis suis TSOL18 gene orders；
Fig. 4 is G/C content figure after the optimization of taeniasis suis TSOL18 gene orders；
Fig. 5 is TSOL18 Gene Partial sequence alignment result figures；
Fig. 6 is SP-TSOL18 Gene Partial sequence alignment result figures；
Fig. 7 is recombinant plasmid pMG36e-TSOL18 double digestion qualification figures；
Fig. 8 is recombinant plasmid pMG36e-SP-TSOL18 double digestion qualification figures；
Fig. 9 is that galactococcus MG1363 positive clone molecules extract genomic DNA measure figure；
Figure 10 is that PCR identifies TSOL18 positive colony subgraphs；
Figure 11 is that PCR identifies SP-TSOL18 positive colony subgraphs；
Figure 12 is TSOL18 bacterial strain expression identification analysis result figures；
Figure 13 is SP-TSOL18 bacterial strain expression identification analysis result figures；
Figure 14 is TSOL18 protein purification qualification figures；
Figure 15 is SP-TSOL18 protein purification qualification figures；
Figure 16 is the Western blot qualification figures of TSOL18 albumen after purification；
Figure 17 is the Western blot qualification figures of SP-TSOL18 albumen after purification；
Figure 18 is TSOL18 plasmid identification result figures；
Figure 19 is SP-TSOL18 plasmid identification result figures.
Embodiment
The present invention is further described with reference to embodiment and accompanying drawing, but the present invention is not limited only to following embodiments, can be with
Those skilled in the art are predicted in the case where combining prior art, there may be many variations for performance.
1 target gene is analyzed
According to taeniasis suis TSOL18 gene order (accession number：AF017788.1), target gene analyze and excellent
Change.
2 TSOL18 gene chemical synthesis and design of primers
Using the method based on PAS (PCR-based Accurate Synthesis), respectively devised at the both ends of primer
Sac I and Hind III digestion sites and protectiveness base, TSOL18 and SP- are respectively synthesized by Nanjing bronze object biology laboratory
TSOL18 target gene.
3 recombinant plasmid pMG36e-TSOL18, pMG36e-SP-TSOL18 structure and identification
The target gene of synthesis is connected with expression vector pMG36e with Sac I/Hind III double digestions, obtains restructuring matter
Grain pMG36e-TSOL18, pMG36e-SP-TSOL18, convert to Top clone strains, extract positive plasmid sequencing identification.
4 taeniasis suis restructuring pMG36e-TSOL18/L.lactis, pMG36e-SP-TSOL18/L.lactis structure and
Identification
It is prepared by the activation of 4.1 galactococcuses and competence
Extracting lactic acid coccus MG1363 bacterium solutions are inoculated in GM17 solid medium flat boards, under the conditions of nonventilated, 30 degree of trainings
Support overnight, it is 0.2-0.8 to cultivate to OD values, cultivates culture ice bath 10min after terminating, thalline is collected by centrifugation, with ice-cold
Phosphoglycerol buffer solution washing thalline, then with 1:Thalline is resuspended in 100 phosphoglycerol buffer solution, it is seen that tiny circular white
Opaque colony occurs, and colony edge is in obvious rough shape, is coincide from form with Bacillus acidi lactici culture.
4.2 recombinant plasmid pMG36e-TSOL18, pMG36e-SP-TSOL18 electricity conversions MG1363
PMG36e-TSOL18, pMG36e-SP-TSOL18 plasmid of acquisition are mixed with competence MG1363 respectively, ice bath
10min, electric shock.It is as follows that electricity turns parameter：Voltage 2000V, electric capacity 25UF, the Ω of resistance 200, the first subpulse is carried out, immediately after
The sucrose MRS culture mediums of 900ul low temperature are added, 10min is placed on ice, during which not vibrate, 30 DEG C of recovery culture 2-3h, by bacterium
Concentrated in after liquid 3000g centrifugation supernatant discardings in 100ul sucrose MRS culture mediums, be coated on 10ug/ml erythromycin GM17
On flat board, 2-3d is cultivated.Period keeps relative closed environment, observes colony growth situation, one week or so, is formed successively tiny
Circular white opaque colony.Choose different positive bacterium colonies, respectively on each positive bacterium colony using sterilizing toothpick picking 2~
3 single bacterium colonies, it is placed in 1ml G/L-SGM17+5ug/ml erythromycin fluid nutrient mediums, quiescent culture 72h at 30 DEG C, treats molten
It is stand-by after the obvious muddiness of liquid appearance.
4.3 positive clone identification
The bacterium solution of above-mentioned culture is taken, 10000rpm centrifugation 10min, abandons supernatant, ddH2O is washed 3 times, and 10000rpm centrifugations are abandoned
Supernatant；Add 30ul dd H2O, boiling water bath 10min after resuspension；Ice bath 2min, 10000rpm centrifugation 10min, draws supernatant and makees
It is stand-by for pcr template.Enter performing PCR using the primer of gene specific to identify, primer is：Forward primer F (5'
ATGGTTTGTCGTTTTGCTT 3') and reverse primer R (5'TTATGAACGACGAACCTTTTTA3'), primer is by Nanjing bronze object
Biotech firm synthesizes.Reaction system is：With each 1.0ul of forward and reverse primer, 1ul genomes are template, archaeal dna polymerase
0.5ul, dNTP2.5ul, 10 × PCR buffer5.0ul, UP to ddw 50ul；PCR amplification conditions：95 DEG C of pre-degenerations
5min, 95 DEG C of denaturation 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30min, cyclic amplification 30 times, last 72 DEG C of renaturation 10min.PCR
Product is identified with 1% agarose gel electrophoresis.
5 protein expressions are identified
5.1 analysis of protein
Protein transmembrane and close and distant water analysis, protein signal peptide analysis.
5.2 SDS-PAGE are identified
Choose unconverted Lactococcus and be named as CK and be incubated in GM17 fluid nutrient mediums, picking is accredited as the positive respectively
#1, be inoculated in the GM17 fluid nutrient mediums containing erythromycin；After 30 DEG C of quiescent culture 72h, 6000rpm, 4 DEG C of centrifugations
The 15min hearts collect precipitation and supernatant is standby, precipitation are resuspended in the PBS of precooling, ultrasonication, and it is broken that ultrasound is carried out in ice bath
It is broken, each 4s (300w), 8s is spaced, ultrasound about 20min, adds isometric 2 × SDS sample-loading buffers (0.1mol/L
Tris-HCl, pH6.8,10% 2 sulphur threose, 4%SDS, 0.2% bromophenol blue, 20% glycerine), boiling water bath 4-8min.After cooling
Take 20ul loadings；The expression of supernatant intracellular is detected by SDS-PAGE respectively.
5.3 protein purifications are identified
Pass through AKTA purifying proteins.
5.4 Western blot are identified
After the albumen of Anti-TSOL18 antibody tests after purification, albumen after purification is taken, adds loading buffer to boil
20ul loadings are taken after boiling cooling.Deposition condition：5% concentration glue：90V30min；10% separation gel：120V, 20min.Transferring film：Point
Three layers of filter paper, SDS-PAGE glue and pvdf membrane (pvdf membrane needs methanol activation 15s or so in advance) are not stacked, use transferring film instrument
Carry out wet method transferring film, constant pressure 100V, 60min.Closing：5% skimmed milk power PBST solution close membranes, 37 DEG C of shaking table, 2h, PBS drift
Wash 5min.Primary antibody is incubated and dyeing, primary antibody are incubated：4 DEG C overnight；PBST rinses 5min × 3 at 37 DEG C, and secondary antibody is incubated：Antibody 1:
1500 dilutions, 37 DEG C of 1h；PBST rinses 5min × 4 at 37 DEG C.2min is exposed, carries out Western blot analyses.
The stability analysis of 6 restructuring TSOL18 and SP-TSOL18 albumen
Using the characteristic of the erythromycin resistance gene contained in pMG36e plasmids, by the plasmid containing target gene of acquisition
Continuous passage is carried out respectively in without erythromycin and the fluid nutrient medium containing erythromycin, and a plasmid stabilisation rate is done every 5 generations
Measure, continuously reached for 20 generations, and plasmid stabilisation rate=choose 100 single bacterium colonies from the flat board without erythromycin is inoculated in containing red mould
The clump count finally grown on the flat board of element.
Detailed process is as follows：Single bacterium colony on picking MG1363 flat boards, GM17+ (containing erythromycin) Liquid Culture is inoculated in respectively
In base and GM17 (being free of erythromycin) fluid nutrient medium, after 30 DEG C of culture 24h, 4 DEG C stand overnight simultaneously conservation, take culture respectively
Streak inoculation is carried out after GM17 flat boards, 30 DEG C of culture 24h, 100 single bacterium colonies of picking are inoculated in phase respectively on streak plate
It should contain in culture medium of the culture medium of erythromycin with being free of erythromycin, growth bacterium colony is counted after 30 degree of culture 24h, repeat to grasp above
Reached for 20 generations, then stop passage.The colony growth number of erythromycin flat board is inoculated according to 100 single bacterium colonies, calculates its plasmid
Its genetic stability.
7 results
7.1 objective gene sequence analysis and optimizations：
Wildtype gene sequence：
ATGGTGTGTCGGTTTGCTCTCATCTTCTTGGTGGCCGTCGTTTTGGCGAGCGGTGACCGAACATTCGGC
GACGATATTTTCGTGCCATACCTTCGCTGCTTCGCCCTTAGCGCTACCGAAATTGGGGTGTTTTGGGATGCTGGAGA
GATGGTTGGCCATGGCGTAGAGGAGATCAAAGTGAAAGTAGAAAAAGCAATACACCCATACAAGATCTGGAATGCAA
CAGTCAGCGCGAACAATGGAAAAGTCATCATCAGAGACTTGAAGGCGAAGACAATTTACAGAGTGGACGTAGACGGT
TATCGAAACGAAATCATGGTGTTTGGTTCGCAGCGTTTCGCGACAACACTTCCGAAAAAGCAGATCAAGCACAAGAA
GGTCCGAAGATCGTAG
Gene order after optimization：
ATGGTTTGTCGTTTTGCTTTAATTTTTTTAGTTGCTGTTGTTTTAGCTTCAGGTGATCGTACTTTTGGT
GATGATATTTTTGTTCCATATTTACGTTGTTTTGCTTTATCAGCTACTGAAATTGGTGTTTTTTGGGATGCTGGTGA
AATGGTTGGTCATGGTGTTGAAGAAATTAAAGTTAAAGTTGAAAAGGCTATTCATCCATATAAAATTTGGAATGCTA
CTGTTTCAGCTAATAATGGTAAGGTTATTATTCGTGATTTAAAAGCTAAAACTATTTATCGTGTTGATGTTGATGGT
TATCGTAATGAAATTATGGTTTTTGGTTCACAACGTTTTGCTACTACTTTACCAAAAAAGCAAATTAAGCATAAAAA
GGTTCGTCGTTCATAA
Its original series code as shown in figure 1, optimization after sequence code as shown in Fig. 2 original G/C content as shown in figure 3, optimization
G/C content is as shown in Figure 4 afterwards.
The synthesis of 7.2 target gene：Using method synthesis 576bp TSOL18 genes and 749bp SP- based on PAS
TSOL18 genes, are consistent with expected results.
TSOL18 genes：
SP-TSOL18 genes：
The sequencing identification of 7.3 recombinant plasmids：The TSOL18 genes and SP-TSOL18 genes of synthesis, double digestion to pMG36e
The Sac I and Hind III sites of carrier, sequencing result are consistent with expected sequence, see Fig. 5,6.
The digestion identification of 7.4 recombinant plasmids：Recombinant plasmid pMG36e-TSOL18 and pMG36e-SP-TSOL18 is through Sac I
With Hind III double digestions, with 1% agarose gel electrophoresis, it was demonstrated that the TSOL18 genes and SP-TSOL18 genes of acquisition are correct
Be inserted between Sac I and the Hind III of pMG36e carriers, be consistent with expected results, see Fig. 7,8, M in figure:DNA
Marker；1:Plasmid before digestion；2:Plasmid after digestion.
The positive clone identification of recombinant plasmid in 7.5 galactococcus MG1363：Recombinant plasmid is extracted from galactococcus MG1363,
Select positive clone molecule, carry out genomic DNA measure, confirm as positive colony, see Fig. 9, M in figure:DNA Marker:1-6:
TSOL18 positive clone molecule genomic DNAs, 7-12:SP-TSOL18 positive clone molecule genomic DNAs, 13:MG1363 empty bacterium strains.
The PCR identifications of recombinant plasmid in 7.6 galactococcuses：With the recombinant plasmid pMG36e- extracted in galactococcus MG1363
TSOL18 and pMG36e-SP-TSOL18 is template, and entering performing PCR using gene-specific primer expands, and as a result shows 1-6 swimming lanes
For the PCR primer of TSOL18 positive clone molecules, Figure 10,11 are seen；M in Figure 10:DNA Marker；1-6:Positive clone molecule
TSOL18PCR identifications, 7:CK (feminine gender-MG1363)；M in Figure 11:DNA Marker；1-6:Positive clone molecule SP-TSOL18PCR
Identification, 7：CK (feminine gender-MG1363)；Wherein 1,2,4,5,6 swimming lanes be SP-TSOL18 positive clone molecules PCR primer, can obtain
393bp TSOL18 genetic fragments, are consistent with expected results.
7.7 restructuring pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis expression products
SDS-PAGE is analyzed：Picking is accredited as in the clone bacterium inoculation and the GM17 fluid nutrient mediums containing erythromycin of the positive respectively, with
Unconverted MG1363 (CK) is incubated in GM17 fluid nutrient mediums, after 30 DEG C of quiescent culture 72h, carries out SDS-PAGE electrophoresis,
The albumen of target is all not detected by CK (MG1363 bacterial strains) intracellular supernatants and precipitation；TSOL18 is not detected by mesh in extracellular supernatant
Albumen is marked, but has the expression for detecting target protein in intracellular, SP-TSOL18 detects in extracellular supernatant and intracellular precipitation
The expression of target protein, see Figure 12,13.M in Figure 12:Protein Marker,1:CK 72 hours supernatants of culture, 2:CK cultures 72
Hour precipitation, 3:PMG36e-TSOL18 conversion MG1363 72 hours supernatants of strain culturing, 4:PMG36e-TSOL18 is converted
MG1363 strain culturings precipitate for 72 hours；In Figure 13, M:Protein Marker,1：CK 72 hours supernatants of culture, 2:CK is cultivated
Precipitate within 72 hours, 3:PMG36e-SP-TSOL18 conversion MG1363 72 hours supernatants of strain culturing, 4:pMG36e-SP-TSOL18
MG1363 strain culturings are converted to precipitate within 72 hours.
7.8TSOL18 expression pattern analysis and product purification with SP-TSOL18 albumen：By AKTA purify TSOL18 and
SP-TSOL18 albumen, using the albumen of Anti-TSOL18 antibody tests after purification, two kinds of albumen are in the purposeful bars of 15D or so
Band, SP-TSOL18 albumen intracellular and it is extracellular there is specific chromogenic band, show that SP-TSOL18 protein antigenicities are better than
TSOL18 albumen, see Figure 14,15,16,17.M in Figure 14:Protein Marker,1:purified；M in Figure 15:Protein
Marker,1:purified；M in Figure 16:Protein Marker,1:CK,2:TSOL18 supernatants, 3:TSOL18 is precipitated；Figure 17
Middle M:Protein Marker,1:CK,2:SP-TSOL18 supernatants, 3:SP-TSOL18 is precipitated.
7.9 stability experiment：Plasmid pMG36e-TOSL18 containing target gene can be seen that by table 1,2,3,4,
PMG36e-SP-TOSL18 is in MG1363 Lactococcus during 20 generation of continuous passage, and under the conditions of without Erythromycinresistant, plasmid is steady
It is 100% to determine rate, and under the conditions of containing Erythromycinresistant, plasmid stabilisation rate is 99%.By the plasmid of each generation using limitation
Property restriction endonuclease Sac I/HindIII carry out double digestion identification, agarose gel electrophoresis shows that size is correct, continuously reaches for 20 generations
When, it is consistent with primary plasmid.Prompting pMG36E-TSOL18, pMG36E-SP-TSOL18 plasmid connects in Lactococcus MG1363
When resuming 20 generation of generation, there is preferable genetic stability, see Figure 18,19.In Figure 18, M:DNA Marker,1:TSOL18 is primary
Plasmid, 2:After the primary plasmid enzyme restrictions of TSOL18,3:TSOL18 5 generation plasmids, 4:After TSOL18 5 generation plasmid enzyme restrictions, 5:
TSOL1810 is for plasmid, and 6:After TSOL18 10 generation plasmid enzyme restrictions, 7:TSOL18 15 generation plasmids, 8:TSOL18 15 generation plasmid enzymes
After cutting, 9:TSOL18 20 generation plasmids, 10:After TSOL18 20 generation plasmid enzyme restrictions；In Figure 19, M:DNA Marker,1:SP-
The primary plasmids of TSOL18,2:After the primary plasmid enzyme restrictions of SP-TSOL18,3:SP-TSOL18 5 generation plasmids, 4:The generations of SP-TSOL18 5
After plasmid enzyme restriction, 5:SP-TSOL18 10 generation plasmids, 6:After SP-TSOL18 10 generation plasmid enzyme restrictions, 7:SP-TSOL18 15 generation matter
Grain, 8:After SP-TSOL18 15 generation plasmid enzyme restrictions, 9:SP-TSOL18 20 generation plasmids, 10:SP-TSOL18 20 generation plasmid enzyme restrictions
Afterwards.
Table 1：Inheritance stability rate of the pMG36e-TOSL18 plasmids under without erythromycin selection pressure in MG1363.
Table 2：Inheritance stability rate of the pMG36e-TOSL18 plasmids in the case where there is erythromycin selection pressure in MG1363.
Table 3：Inheritance stability rate of the pMG36e-SP-TOSL18 plasmids under without erythromycin selection pressure in MG1363.
Table 4：Inheritance stability rate of the pMG36e-SP-TOSL18 plasmids in the case where there is erythromycin selection pressure in MG1363.
TSOL18 genes are a cDNA fragments by clones such as Gauci, and its length is 579bp, wherein ORFs
For 390bp, the protein of 130 amino acid of codified, theoretical relative molecular mass (Mr) it is 14.7kD.The gene is present in sharp
In TSO living, there is good immunogenicity and antigenicity, it is considered to be the most candidate vaccines gene of development prospect, by
The extensive concern of domestic and foreign scholars, it is the focus of current taeniasis suis vaccine research.In recent years, TSOL18 bases have successively been carried out
The recombinant protein vaccine of cause, DNA vaccination, recombination yeast vaccine, restructuring salmonella typhimurium vaccine, recombined bifidobacteria epidemic disease
Seedling and recombinant BCG vaccine, above-mentioned vaccine obtain preferable immune effect in animal model.With going deep into for vaccine research,
It is found that recombinant protein vaccine is needed by complex processes such as gene cloning, expression and protein purifications, technical difficulty is big, cost
It is higher, while it can only induction body fluid be immune and Th cell responses, it is impossible to inducing cytotoxic T lymphocyte responses；DNA vaccination
It is simple to operate, can induction body fluid is immune simultaneously, Th cells and CTL responses, but DNA can be incorporated into the genome of host cell,
There is the danger for causing Protooncogene Activated and suppressor to inactivate；In recombinant yeast pichia pastoris there is excessive glycosylation in expressing protein,
The synthesis and secretion of recombinant protein may be influenceed, and the albumen after glycosylation has height heterogeneity, can make recombinant protein
Function changes；Salmonella typhimurium belongs to gram-negative bacteria, and it expresses caused lipopolysaccharides toxin in vivo may be to place
Chief cell is toxic, may interfere with correct expression and synthesis of the encoding gene in host cell, and bacterial virulence be present
Return strong danger；Bifidobacterium obligate anaerobic Gram-positive bacillus, is unfavorable for operating, at present still can not be as Escherichia coli
Equally extensively and conventional, expression system is immature, be not also with regard to researchs such as its genetic background and molecular biological characteristics it is very thorough,
Find that its protecting effect is less than satisfactory (74.85%) moreover, being studied through this seminar；BCG is as a kind of attenuation ox type point
Branch bacillus, still suffers from toxicity action, there is the possibility for returning virulence, may produce toxic side effect to host, even result in infection knot
Core disease, is unsatisfied with as vaccine expression effect, and experimental period is grown, and operation has risk, Difficulty.Therefore, it is necessary to grind
Study carefully new taeniasis suis vaccine.
Live vector vaccine is that the encoding gene of protective antigens is inserted into virus or bacteria carrier using technique for gene engineering
In, become the recombinant virus for expressing exogenous antigen or bacterium.The sense in a manner of natural in body after its immune animal
Dye and process antigen, induction body produces comprehensive immune response, so as to play immanoprotection action.Currently used for this kind of vaccine
Carrier have salmonella, vaccinia virus, BCG etc., and relative to these viruses and bacteria carrier, lactic acid bacteria (Lactic acid
Bacteria, LAB) it is then safer for human body.
Lactococcus lactis (Lactococcus lactis, L.lactis) is most important pattern bacterium, application in LAB
It is closely related in food, field of medicaments.Its physio-biochemical characteristics is simple, and genetic background understands, has solely as vaccine delivery vehicle
Special advantage：(1) it is food-grade microorganisms, the Main Ingredients and Appearance of Dairy fermentation is safe；(2) secretory protein itself is less, subtracts
Interference of the carrier microorganisms oneself protein to external source secretory protein is lacked, and has not produced any extracellular protease, will not cause
Extracellular degraded occurs for secretory protein；(3) there is mucosal adjuvants characteristic；(4) it is basic with Bacillus acidi lactici difference, L.lactis
It is not colonized in intestines and stomach, normal flora in enteron aisle will not be endangered and cause potential gene contamination and avoid what long-term field planting was brought
Tolerance.Genetic engineering restructuring L.lactis is using L.lactis as engineering bacteria, is imported foreign gene using DNA recombinant techniques
In L.lactis, by L.lactis in the duplication of host, secreting, expressing exogenous antigen, to induce the specificity humoral to disease
And cellular immunity, improve the immunological effect that L.lactis is excited.
Van de Guchte in 1989 etc. are using the transcription and translation signal of L.lactis subsp. cremoris protease genes as base
Plinth, plasmid vector pMG36e is built, its size about 3.6kb, is easy to Host Strains to carry, is widely used in the expression of multiple protein.
In recent years, domestic and foreign scholars are transformed, and successfully construct food grade expression vector, for food, pharmaceutical production and food
Grade vaccine development is laid a good foundation.Therefore, food grade expression vector pMG36e is probably structure restructuring L.lactis vaccines
Ideal carrier.And with regard to parasite field, there is not yet the report of taeniasis suis restructuring L.lactis vaccines.This research uses full genome
Synthetic method builds TSOL18 and SP-TSOL18 antigen encoding genes, directs it and is cloned into food grade expression vector pMG36e
In, taeniasis suis recombinant plasmid pMG36e-TSOL18 and SP-TSOL18 are built, through double digestion, PCR and sequencing identification, it was demonstrated that
TSOL18 and SP-TSOL18 genes are successively inserted into pMG36e, then the recombinant plasmid electroporation successfully constructed is transferred to
L.lactis MG1363, structure taeniasis suis restructuring pMG36e-TSOL18/L.lactis, pMG36e-SP-TSOL18/
L.lactis, positive bacterium solution is selected from the GM17 fluid nutrient mediums containing Erythromycinresistant and enters performing PCR identification for template, it was demonstrated that pig
Band tapeworm restructuring pMG36e-TSOL18/L.lactis, pMG36e-SP-TSOL18/L.lactis are successfully constructed.And pass through SDS-
PAGE analyses show that TSOL18 is not detected by target protein in extracellular supernatant, but have the expression for detecting target protein in intracellular,
And SP-TSOL18 has the expression for detecting target protein in extracellular supernatant and intracellular precipitation, Western blot displays use
TSOL18 and SP-TSOL18 albumen of the Anti-TSOL18 antibody tests by AKTA after purification, two kinds of albumen are in 15D or so
Purposeful band, SP-TSOL18 albumen intracellular and it is extracellular there is specific chromogenic band, show that TSOL18 genes can be
Expressed in L.lactis, and the albumen expressed has special antigenicity, SP-TSOL18 protein antigenicities are better than TSOL18 eggs
In vain.Plasmid pMG36e-TOSL18, the pMG36e-SP-TOSL18 continuous passage in MG1363 Lactococcus containing target gene
During 20 generation, under the conditions of without Erythromycinresistant, plasmid stabilisation rate is 100%, under the conditions of containing Erythromycinresistant, plasmid stabilisation
Rate is 99%.The plasmid of each generation is subjected to double digestion identification using restriction enzyme Sac I/HindIII, agarose coagulates
Gel electrophoresis show that size is correct, when continuously reaching for 20 generation, are consistent with primary plasmid.pMG36E-TSOL18、pMG36E-SP-
TSOL18 plasmids during 20 generation of continuous passage, there is preferable genetic stability in Lactococcus MG1363.For cysticercosis cellulosae
The development and utilization of vaccine provides solid reference.
Claims (1)
1. the method for analyzing stability of taeniasis suis Recombinant Lactococcus lactis expression system, according to taeniasis suis TSOL18 gene sequences
Row, TSOL18 and SP-TSOL18 target gene is respectively synthesized, carries out recombinant plasmid pMG36e-TSOL18 and pMG36e-SP-
TSOL18 structure and taeniasis suis restructuring pMG36e-TSOL18/L.lactis and pMG36e-SP-TSOL18/L.lactis
Structure；It is characterized in that analysis method comprises the steps of：
1), by the plasmid containing target gene of acquisition respectively in the GM17 fluid nutrient mediums containing erythromycin with being free of erythromycin
In GM17 fluid nutrient mediums, after 30 DEG C of culture 24h, 4 DEG C stand overnight simultaneously conservation；
2), take step 1) culture to carry out streak inoculation after GM17 flat boards, 30 DEG C of culture 24h respectively, divide on streak plate
Other 100 single bacterium colonies of picking are inoculated in accordingly culture medium of the culture medium containing erythromycin with being free of erythromycin, 30 degree of culture 24h
Growth bacterium colony is counted afterwards；
3), repeat and reached for 20 generations；
4) the colony growth number of erythromycin flat board, is inoculated according to 100 single bacterium colonies, calculates its genetic stability of its plasmid.
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