CN107217056A - The structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system - Google Patents
The structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system Download PDFInfo
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Abstract
The invention discloses the structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system, gene chemical synthesis and design of primers, recombinant plasmid pMG36e SP TSOL18 structure and identification, taeniasis suis restructuring pMG36e SP TSOL18/L.lactis structure and the step such as identification and protein expression identification are included.Gene optimization is carried out by host system of Lactococcus lactis MG1363, synthesize target gene SP TSOL18, double digestion is connected to pMG36e carriers, build intracellular type expression vector pMG36e SP TSOL18, again by its Electroporation Transformation to L.lactis MG1363, taeniasis suis Lactococcus lactis expression system pMG36e SP TSOL18/L.lactis MG1363 are successfully built.Enter performing PCR identification, SDS PAGE and Western blot analysis SP TSOL18 has target protein expression in intracellular with extracellular, identified by AKTA protein purifications and stability analysis, pMG36e SP TSOL18 plasmids generation of continuous passage 20 in L.lactis MG1363, there is preferable genetic stability, the 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 structure and authentication method of galactococcus secretion type 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, and Major Epidemic is non-in, South Africa, Latin America, East Asia and South Asia etc. are passed through
The national and area of Ji, sanitary condition difference.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 due to autoinfection with cysticercus parasitism.Therefore safe and efficient vaccine control is researched and developed
The disease is significant.
TSOL18 genes are guarded relatively, are present in the TSO of activation, with good immunogenicity and antigenicity, are recognized
To be the candidate vaccines gene of most development prospect.During Lactococcus lactis (Lactococcus lactis, L.lactis) is LAB
Most important pattern bacterium, application is 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, having constructed one in fields such as bacterium, virus, tumour, parasites is
Row recombinant vaccine.
For above-mentioned background technology field, there is not yet taeniasis suis recombinate L.lactis report.
The content of the invention
It is an object of the invention to provide the structure of taeniasis suis Recombinant Lactococcus lactis secretion type expression system and identification
Method.
To reach above-mentioned purpose, the technical scheme used for：
The structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system, are comprised the steps of：
1), TSOL18 gene chemical synthesis and design of primers：According to taeniasis suis TSOL18 gene orders, using based on PAS's
Method, Sac I and Hind III digestion sites and protectiveness base are respectively devised at the two ends of primer, synthesize 749bp SP-
TSOL18 genes；
2), recombinant plasmid pMG36e-SP-TSOL18 structure and identification：The target gene and expression vector of synthesis
PMG36e is connected with Sac I/Hind III double digestions, obtains recombinant plasmid pMG36e-SP-TSOL18, conversion to Top clones
Bacterial strain, extracts positive plasmid sequencing identification；
3), taeniasis suis restructuring pMG36e-SP-TSOL18/L.lactis structure and identification：
3.1), prepared by the activation of galactococcus and competence：Extracting lactic acid coccus MG1363 bacterium solutions are inoculated in GM17 solid cultures
Base flat board, then collects thalline, with 1:Thalline is resuspended in 100 phosphoglycerol buffer solution；
3.2), recombinant plasmid pMG36e-SP-TSOL18 electricity conversion MG1363：By the pMG36e-SP-TSOL18 matter of acquisition
Grain is mixed with competence MG1363, carries out ice bath and electric shock, is added sucrose MRS culture mediums, is then coated erythromycin GM17
Flat board culture, chooses positive bacterium colony and carries out erythromycin fluid nutrient medium culture again；
3.3), positive clone identification：The culture bacterium solution of above-mentioned 3.2) step is taken, centrifugation is abandoned supernatant, washed successively, again
Outstanding, boiling water bath, ice bath, centrifugation, take supernatant stand-by as pcr template；Enter performing PCR using the primer of gene specific to identify, PCR
Product is identified with 1% agarose gel electrophoresis；
4), protein expression is identified：
4.1), SDS-PAGE is identified：Choose unconverted Lactococcus to be incubated in GM17 fluid nutrient mediums, picking step
3.3) bacterium solution of the positive is accredited as, precipitation and supernatant is collected by centrifugation；It will be deposited in ice bath and carry out ultrasonication, add isometric
2 × SDS sample-loading buffers, boiling water bath 4-8min takes loading after cooling, passes through the expression that SDS-PAGE detects supernatant intracellular
Situation；
4.2) TSOL18 albumen, is purified by AKTA, using the albumen of Anti-TSOL18 antibody tests after purification in 15D
Left and right whether there is purpose band；
4.3), Western blot are identified：Take AKTA to purify TSOL18 albumen and carry out Western blot identification and analysis.
Further, the step 3.1) in, keep stuffy after bacterium solution inoculation, 30 DEG C of overnight incubations are 0.2- to OD values
0.8, then by culture ice bath 10min, it is collected by centrifugation thalline, and with cold phosphoglycerol buffer solution washing thalline, then carry out
Thalline is resuspended.
Further, the step 3.2) in electric shock electricity to turn parameter as follows：Voltage 2000V, electric capacity 25UF, the Ω of resistance 200.
Further, the step 3.3) in, it is using the primer of gene specific：Forward primer F (5'
ATGGTTTGTCGTTTTGCTT 3') and reverse primer R (5'TTATGAACGACGAACCTTTTTA3')；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 of extension 30min, cyclic amplification 30 times, last 72 DEG C of renaturation 10min.
Further, the step 4.1) in, 2 × SDS sample-loading buffers are：0.1mol/L Tris-HCl, pH6.8,10%
Two sulphur threoses, 4%SDS, 0.2% bromophenol blue and 20% glycerine.
Further, the step 4.3) be specially：Take step 4.2) albumen after purification, plus loading buffer boil it is cold
But 20ul loadings, deposition condition are taken afterwards：5% concentration glue：90V30min；10% separation gel：120V, 20min；Transferring film：Fold respectively
Put three metafiltration paper, SDS-PAGE glue and pvdf membrane well, pvdf membrane is needed methanol to activate 15s or so, carried out using transferring film instrument in advance
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 rinsings
5min；Primary antibody is incubated and dyed, and primary antibody is 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, Western blot identification and analysis is carried out.
The taeniasis suis TSOL18 gene orders that the present invention is logged in using in GenBank is templates, with Lactococcus lactis
MG1363 is that host system carries out gene optimization, synthesizes target gene SP-TSOL18, double digestion is connected to pMG36e carriers, structure
Intracellular type expression vector pMG36e-SP-TSOL18 is built, then its Electroporation Transformation is successfully built into pig to L.lactis MG1363
Band tapeworm Lactococcus lactis expression system pMG36e-SP-TSOL18/L.lactis MG1363.Enter performing PCR identification, SDS-PAGE
There is target protein expression with extracellular in intracellular with Western blot analysis SP-TSOL18, identified by AKTA protein purifications
And stability analysis, in pMG36e-SP-TSOL18 plasmids generation of continuous passage 20 in L.lactis MG1363, there is preferable heredity
Stability, the development and utilization for cysticercus cellulosae disease vaccine provides 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), analysis is carried out and excellent to target gene
Change.
2TSOL18 gene chemical synthesis and design of primers
Using the method based on PAS (PCR-based Accurate Synthesis), respectively devised at the two ends of primer
Sac I and Hind III digestion sites and protectiveness base, by Nanjing, bronze object biology laboratory is respectively synthesized TSOL18 and SP-
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, conversion 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, culture to OD values is 0.2-0.8, 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 carry out the first subpulse, immediately after
The sucrose MRS culture mediums of 900ul low temperature are added, 10min is placed on ice, during which should not be vibrated, 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, are placed in 1ml G/L-SGM17+5ug/ml erythromycin fluid nutrient mediums, and quiescent culture 72h at 30 DEG C treats molten
Liquid occurs stand-by after obvious muddiness.
4.3 positive clone identification
The bacterium solution of above-mentioned culture is taken, 10000rpm centrifugation 10min abandon supernatant, ddH2O is washed 3 times, 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 of extension 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.
5.2SDS-PAGE identification
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, ultrasound are carried out in ice bath broken
Broken, each 4s (300w) is spaced 8s, 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.
5.4Western blot are identified
After the albumen of Anti-TSOL18 antibody tests after purification, albumen after purification, plus loading buffer is taken to boil
20ul loadings are taken after boiling cooling.Deposition condition：5% concentration glue：90V30min；10% separation gel：120V, 20min.Transferring film：Point
Three metafiltration paper, SDS-PAGE glue and pvdf membrane (pvdf membrane needs methanol to activate 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 drifts
Wash 5min.Primary antibody is incubated and dyed, and primary antibody is 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, Western blot analyses are carried out.
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
Determine, continuously reached for 20 generations, 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, is inoculated in GM17+ (containing erythromycin) Liquid Culture respectively
In base and GM17 (being free of erythromycin) fluid nutrient medium, after 30 DEG C of culture 24h, 4 DEG C are stood overnight and conservation, and culture is taken respectively
Streak inoculation is carried out in 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, its plasmid is calculated
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 is as shown in figure 1, sequence code is as shown in Fig. 2 original G/C content is as shown in figure 3, optimization after optimization
G/C content is as shown in Figure 4 afterwards.
The synthesis of 7.2 target gene：Using the TSOL18 genes and 749bp SP- of the method synthesis 576bp 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 is consistent with expected sequence, sees 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, 1% agarose gel electrophoresis is used, 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 M in Fig. 7,8, 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,
Positive clone molecule is selected, genomic DNA measure is carried out, confirms as positive colony, see M in Fig. 9, figure:DNAMarker:1-6:
TSOL18 positive clone molecule genomic DNAs, 7-12:SP-TSOL18 positive clone molecule genomic DNAs, 13:The strain of MG1363 empty bacteriums.
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 inoculation of the clone bacterium of the positive and the GM17 fluid nutrient mediums containing erythromycin 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 is detected in extracellular supernatant and intracellular precipitation
The expression of target protein, is shown in Figure 12,13.M in Figure 12:Protein Marker,1:CK cultivates 72 hours supernatants, 2:CK cultures 72
Hour precipitation, 3:PMG36e-TSOL18 converts MG1363 72 hours supernatants of strain culturing, 4:PMG36e-TSOL18 is converted
MG1363 strain culturings are precipitated for 72 hours；In Figure 13, M:Protein Marker,1：CK cultivates 72 hours supernatants, 2:CK is cultivated
Precipitate within 72 hours, 3:PMG36e-SP-TSOL18 converts 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, is shown in 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, under the conditions of containing Erythromycinresistant, and 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:TSOL18
10 generation plasmids, 6:After TSOL18 10 generation plasmid enzyme restrictions, 7:TSOL18 15 generation plasmids, 8:After TSOL18 15 generation plasmid enzyme restrictions, 9:
TSOL18 20 generation plasmids, 10:After TSOL18 20 generation plasmid enzyme restrictions；In Figure 19, M:DNA Marker,1:SP-TSOL18 is primary
Plasmid, 2:After the primary plasmid enzyme restrictions of SP-TSOL18,3:SP-TSOL18 5 generation plasmids, 4:After SP-TSOL18 5 generation plasmid enzyme restrictions,
5:SP-TSOL18 10 generation plasmids, 6:After SP-TSOL18 10 generation plasmid enzyme restrictions, 7:SP-TSOL18 15 generation plasmids, 8:SP-
After TSOL18 15 generation plasmid enzyme restrictions, 9:SP-TSOL18 20 generation plasmids, 10:After SP-TSOL18 20 generation plasmid enzyme restrictions.
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, with 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, 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,
It may influence the albumen after the synthesis and secretion of recombinant protein, and glycosylation that there is height heterogeneity, recombinant protein can be made
Function changes；Salmonella typhimurium belongs to gram-negative bacteria, and its lipopolysaccharides toxin for expressing generation 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 there is bacterial virulence
Return strong danger；Bifidobacterium obligate anaerobic Gram-positive bacillus, is unfavorable for operation, at present still can not be as Escherichia coli
Equally extensively and conventional, expression system is immature, and it is not also very thorough to be studied with regard to its genetic background and molecular biological characteristic etc.,
Moreover, finding that its protecting effect is less than satisfactory (74.85%) through the research of this seminar；BCG is used as one kind 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 results in infection knot
Core disease, it is dissatisfied as vaccine expression effect, and experimental period is long, there is risk, Difficulty in operation.Therefore, it is necessary to grind
Study carefully new taeniasis suis vaccine.
Live vector vaccine is the encoding gene insertion virus or bacteria carrier by protective antigens using technique for gene engineering
In, become recombinant virus or the bacterium of expression exogenous antigen.With natural mode sense in body after its immune animal
Dye and process antigen, induction body produce 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) with Bacillus acidi lactici difference, L.lactis is basic
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, as engineering bacteria, to be imported L.lactis 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, builds plasmid vector pMG36e, 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 to build restructuring L.lactis vaccines
Ideal carrier.And with regard to parasite field, there is not yet taeniasis suis recombinate the report of 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, build 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, which are shown, to be used
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 of expression 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-
TOSL18 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 (6)
1. the structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system, it is characterized in that, comprising following
Step：
1), TSOL18 gene chemical synthesis and design of primers：According to taeniasis suis TSOL18 gene orders, using the method based on PAS,
Sac I and Hind III digestion sites and protectiveness base are respectively devised at the two ends of primer, 749bp SP-TSOL18 is synthesized
Gene；
2), recombinant plasmid pMG36e-SP-TSOL18 structure and identification：The target gene of synthesis is used with expression vector pMG36e
SacI/Hind III double digestions are connected, and obtain recombinant plasmid pMG36e-SP-TSOL18, conversion to Top clone strains, extracting
Positive plasmid sequencing identification；
3), taeniasis suis restructuring pMG36e-SP-TSOL18/L.lactis structure and identification：
3.1), prepared by the activation of galactococcus and competence：Extracting lactic acid coccus MG1363 bacterium solutions are inoculated in GM17 solid mediums and put down
Plate, then collects thalline, with 1:Thalline is resuspended in 100 phosphoglycerol buffer solution；
3.2), recombinant plasmid pMG36e-SP-TSOL18 electricity conversion MG1363：By the pMG36e-SP-TSOL18 plasmids of acquisition with
Competence MG1363 is mixed, and carries out ice bath and electric shock, is added sucrose MRS culture mediums, is then coated erythromycin GM17 flat boards
Culture, chooses positive bacterium colony and carries out erythromycin fluid nutrient medium culture again；
3.3), positive clone identification：The culture bacterium solution of above-mentioned 3.2) step is taken, supernatant is abandoned in centrifugation, then is washed successively, again
Outstanding, boiling water bath, ice bath, centrifugation, take supernatant stand-by as pcr template；Enter performing PCR using the primer of gene specific to identify, PCR
Product is identified with 1% agarose gel electrophoresis；
4), protein expression is identified：
4.1), SDS-PAGE is identified：Choose unconverted Lactococcus to be incubated in GM17 fluid nutrient mediums, picking step 3.3)
The bacterium solution of the positive is accredited as, precipitation and supernatant is collected by centrifugation；It will be deposited in ice bath and carry out ultrasonication, and add isometric 2
× SDS sample-loading buffers, boiling water bath 4-8min, loading after cooling detects the protein expression of supernatant intracellular by SDS-PAGE
Situation；
4.2) TSOL18 albumen, is purified by AKTA, using the albumen of Anti-TSOL18 antibody tests after purification in 15D or so
Whether there is purpose band；
4.3), Western blot are identified：Take AKTA to purify TSOL18 albumen and carry out Western blot identification and analysis.
2. structure and the identification side of taeniasis suis Recombinant Lactococcus lactis secretion type expression system according to claim 1
Method, it is characterized in that：The step 3.1) in, keep stuffy after bacterium solution inoculation, 30 DEG C of overnight incubations are 0.2-0.8 to OD values,
Then by culture ice bath 10min, it is collected by centrifugation thalline, and with cold phosphoglycerol buffer solution washing thalline, then be resuspended
Thalline.
3. structure and the identification side of taeniasis suis Recombinant Lactococcus lactis secretion type expression system according to claim 1
Method, it is characterized in that：The step 3.2) in electric shock electricity to turn parameter as follows：Voltage 2000V, electric capacity 25UF, the Ω of resistance 200.
4. structure and the identification side of taeniasis suis Recombinant Lactococcus lactis secretion type expression system according to claim 1
Method, it is characterized in that：The step 3.3) in, it is using the primer of gene specific：Forward primer F (5'
) and reverse primer R (5'TTATGAACGACGAACCTTTTTA 3') ATGGTTTGTCGTTTTGCTT3'；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 ddw50ul；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 of extension 30min, cyclic amplification 30 times, last 72 DEG C of renaturation 10min.
5. structure and the identification side of taeniasis suis Recombinant Lactococcus lactis secretion type expression system according to claim 1
Method, it is characterized in that：The step 4.1) in, 2 × SDS sample-loading buffers are：0.1mol/L Tris-HCl, pH6.8,10% 2
Sulphur threose, 4%SDS, 0.2% bromophenol blue and 20% glycerine.
6. structure and the identification side of taeniasis suis Recombinant Lactococcus lactis secretion type expression system according to claim 1
Method, it is characterized in that：The step 4.3) be specially：Take step 4.2) albumen after purification, plus loading buffer boil cooling
After take 20ul loadings, deposition condition：5% concentration glue：90V30min；10% separation gel：120V, 20min；Transferring film：Stack respectively
Three metafiltration paper, SDS-PAGE glue and pvdf membrane, pvdf membrane need methanol to activate 15s or so in advance, are carried out using transferring film instrument 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 rinsings 5min；
Primary antibody is incubated and dyed, and primary antibody is incubated：4 DEG C overnight；PBST rinses 5min × 3 at 37 DEG C, and secondary antibody is incubated：Antibody 1:1500 is dilute
Release, 37 DEG C of 1h；PBST rinses 5min × 4 at 37 DEG C；2min is exposed, Westernblot identification and analysis is carried out.
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