CN114214351A - Shigella polysaccharide expression plasmid and application thereof - Google Patents

Shigella polysaccharide expression plasmid and application thereof Download PDF

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CN114214351A
CN114214351A CN202210007956.9A CN202210007956A CN114214351A CN 114214351 A CN114214351 A CN 114214351A CN 202210007956 A CN202210007956 A CN 202210007956A CN 114214351 A CN114214351 A CN 114214351A
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polysaccharide
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余昊霖
陈靖萱
李艺
蔡沐含
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Nanchang University
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Abstract

The invention discloses a Shigella polysaccharide expression plasmid and application thereof, belonging to the technical field of biological engineering, wherein the Shigella polysaccharide expression plasmid comprises a copy protein RepA, wherein the 99 th leucine of the RepA is mutated into lysine, and a section of nucleotide for coding an O-type antigen expression product gene of the Shigella polysaccharide, and the Shigella polysaccharide expression plasmid is applied to recombinant expression protein and can improve the expression of the O-antigen polysaccharide of Shigella. The invention is based on a classical low-copy plasmid replicon pSC101, and obtains a novel plasmid replicon pSC101-RepA-L99K through mutation transformation. The pSC101-RepA-L99K-SCO recombinant expression plasmid constructed in the invention has strong expression capability, and lays a foundation for further developing related vaccines.

Description

Shigella polysaccharide expression plasmid and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a shigella polysaccharide expression plasmid and application thereof.
Background
Shigella (Shigella Castellani) is a gram-negative Bacillus pumilus, the most common pathogenic bacterium of human bacillary dysentery, is mainly prevalent in developing countries, is known as Shigella dysenteriae, is cold-resistant, can grow on a common agar medium for 24 hours, and forms translucent smooth colonies with a diameter of 2 mm.
The applicant in the 202010575533.8 patent indicated that vaccine research for bacillary dysentery has been carried out since 40, 50 s in the 20 th century, but there are still problems to be solved. In the existing research, some potential candidate vaccine targets are found, wherein the Lipopolysaccharide (Lipopolysaccharide) of Shigella can show better protective power, and the research proves that the complex of the Lipopolysaccharide and protein of Shigella can stimulate the high serum antibody titer of mice and can show certain protective power, and clinical experiments show that the O antigen polysaccharide of Fowler 2a (Shigellaflexneri 2a) can cause specific immune protection, but the most key problem of the Lipopolysaccharide is that the O antigen polysaccharide enters a host body and cannot effectively reach the immune system of the host to be recognized by the immune system, so that the immune failure is caused with certain probability. The defect greatly limits the application effect of the shigella vaccine in actual clinic, so that the development of a new vaccine is urgent, and how to develop a new vaccine can ensure that a host can be efficiently stimulated to generate immune response needs to be solved at present. Wherein, how to improve the expression of O antigen polysaccharide is the problem to be solved firstly.
Previous studies have shown that shigella O antigen polysaccharide synthesis gene cluster (16Kb) is more stable in low copy plasmids than in high copy plasmids, and how to stably and efficiently express O antigen polysaccharide in a recombinant expression system through plasmid modification is a technical problem currently faced. In view of this, the invention is particularly proposed.
Disclosure of Invention
Aiming at the defects and problems in the prior art, the invention aims to provide a Shigella polysaccharide expression plasmid and application thereof, which are high-copy recombinant expression plasmids and further aim to construct a high-copy recombinant expression plasmid for recombinant expression of Shigella polysaccharide O-type antigen genes.
The invention is realized by the following technical scheme:
the invention provides a construction method of a Shigella polysaccharide expression plasmid, which comprises the steps of taking a pSC101 plasmid as a vector, and mutating leucine at the 99 th site of a replication protein RepA on the pSC101 plasmid into lysine to obtain the Shigella polysaccharide expression plasmid, wherein the sequence of the replication protein RepA is shown as SEQ ID No. 1.
Further, the codon for lysine adopts AAG codon.
Further, the shigella polysaccharide expression plasmid further comprises nucleotides encoding shigella polysaccharide O-antigen genes.
Further, the nucleotide sequence of the shigella polysaccharide O-type antigen gene is shown as SEQ NO: 2, respectively.
The invention also provides a Shigella polysaccharide expression plasmid constructed by the method.
The invention also provides the application of the Shigella polysaccharide expression plasmid in recombinant expression of protein.
Compared with the prior art, the invention is based on the classical low-copy plasmid replicon pSC101, and obtains a novel plasmid replicon pSC101-RepA-L99K through mutation transformation. The pSC101-RepA-L99K-SCO recombinant expression plasmid constructed in the invention has strong expression capability, and lays a foundation for further developing related vaccines.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the upstream fragment amplification of Shigella O antigen gene cluster.
FIG. 2 shows the downstream fragment amplification of Shigella O antigen gene cluster.
FIG. 3 is the PCR identification result of Shigella O antigen gene cluster expression plasmid.
FIG. 4 is the lipopolysaccharide map identification result of Shigella O antigen gene cluster expression plasmid, wherein 1: blank control; 2: pSC101-RepA-L99-SCO expression result; 3: pSC101-RepA-L99K-SCO expression results.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Unless otherwise indicated, the experimental methods, detection methods, preparation methods, and reagents disclosed herein all employ conventional techniques or reagents of the art, which are conventional in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1: construction of pSC101-RepA-L99K replicon in vitro
1) In the reported wild-type pSC101 replicon (Cohen et al 1973Proc Natl Acad Sci US A; cohen et al.1977J Bacteriol; based on the Vocke C and Bastia D.1983Proc Natl Acad SciUSA) sequence, plasmid pGENT1 (which carries pSC101-RepA wild-type replicon, Kan resistance and LacZ selection marker) with the wild-type replicon was synthesized, and a primer for L99K mutation of RepA protein was designed,
the F primer is: gtccactggaaaatnnnaaagcctttaaccaaaagattcctgatt；
The primer R is as follows: attttccagtggacaaactatgccaagttctcaagcgaaaaatta, Shanghai Producer company.
2) A mutation at position L99K of the pSC101 replicon RepA protein was constructed using a PCR amplification mutagenesis method. Wherein, the PCR system is as follows: LA Taq DNA polymerase: 0.5 mul; 10 × PCR buffer (Mg2 +): 2.5 mul; dNTP: 3 mu l of the solution; f primer: 0.25 ul; r primer: 0.25 ul; template plasmid: 2 ul. The PCR procedure was: 95 ℃ for 4 min; 95 ℃ for 30 cycles, 30s- >55 ℃, 30s- >72 ℃ for 4 min; 72 ℃ for 10 min.
3) The pGENT1 plasmid (pGENT 1-L99N for short) carrying the RepA-L99K mutation is recovered by tapping a DNA gel recovery kit (Tiangen organisms).
4) pGENT1-L99N obtained in step 3 was transformed into E.coli Top10 strain, plated on LB (Kan) -resistant plates, and cultured overnight at 37 ℃. Meanwhile, the wild-type plasmid pGENT1 was transformed into E.coli Top10 strain, which was coated with LB (Kan) resistant plate and LB (Amp) resistant plate, respectively, and cultured overnight at 37 ℃ as the control for the next experiment. The sequence of RepA protein L99K SEQ ID NO.1 is as follows:
YQATKVYGTLFFQFPKVLLYSPTYKNLSAEAKLAYVILKDRLEYSLHNDWVDENGNI YFIFSNTELQQILNCSEPKVIKTKKELEQANKLFQKKMGFDPKMKRNNPNRLYLGDLNVSA TDVYKRENEASQSTISPATSGTKNSLARHKMPQSLATSGTKNSLARDKVPQSLATSGTENS LVYQYKDLETQARDNKETEKFDFSTDQYSPEIIRKQNQDLVRRAKDYLPESANGGLFLNKE GVELLGLWCRSPKQMRRFLGIILNAKKAVEREHEGTAIVLDDPRCQEMINKTMRRFFNVLR SDSKKINNVENYLFGAMKETLVAYWNKSLMTANGGDPDEF
example 2 Shigella polysaccharide O-type antigen Gene preparation
The genome DNA template of Shigella flexneri 2a is prepared by boiling lysis method, and single colony is picked up and cultured overnight at 37 ℃. 0.5 ml of bacterial liquid is taken for 12000r/min and centrifuged for 3min, the supernatant is discarded, and the thalli are collected, washed once by ultrapure water and resuspended. Boiling in boiling water for 10min, cooling, centrifuging at 12000r/min for 3min, and collecting supernatant as template. The genome of the gene is taken as a template, LA taq polymerase is used for amplification, the complete O antigen gene cluster is amplified through two pairs of primers 2a-1F/2a-1R and 2a-2F/2a-2R, the sizes of the primers are respectively about 8000bp, a vector fragment is amplified by taking a vector pSC101 as the template, the size of the vector fragment is about 6000bp, the fragment is recovered by using crystal violet agarose gel with the concentration of 0.8 percent to prevent the fragment from mutating or degrading under ultraviolet light, and the fragment is recovered for later use, and the result is shown in figure 1 and figure 2.
Name (R) Sequence of
2a-1F 5’CCGCCATTCTGAAATGGGCTATATGCAGGCGTTTGTGAA3’
2a-1R 5’AATGCATTTCTTACTCGATAATAAC3
2a-2F 5’CTGTGGGATTAACATACCAATTCAC3’
2a-2R 5’ATTGTCTCATGAGCGGTATAACCACGACTTTCGATGTTG3’
An amplification system:
Figure RE-GDA0003492627270000031
Figure RE-GDA0003492627270000041
the reaction procedure was as follows: pre-denaturation at 95 ℃ for 3 min; melting at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 8min, total 30 cycles, incubation at 72 ℃ for 10min, and final storage at 4 ℃.
Example 3: construction of pSC101-RepA-L99K-SCO plasmid
The fragments can be ligated into a complete plasmid by Gibson assembly enzyme through overlapping fragments by adding overlapping fragments to the fragments when designing primers using the Gibson assembly kit (NEB). The method comprises the following specific steps: the fragments were added to the reaction system in a total volume of 10. mu.l at the same molar ratio as follows:
Figure RE-GDA0003492627270000042
after the system is added, the system is acted for 1 hour at 50 ℃, 2 microliters of the connector is converted into TOP10 competent cells by an electric conversion method, the competent cells are coated on a Kan plate, the competent cells are placed at 37 ℃ and subjected to PCR identification by using corresponding primers after bacterial colonies grow out, the bacterial colonies which are identified as positive by the PCR are subjected to subsequent identification on the expressed O antigen, and the result is shown in figure 3.
Comparative example 1: construction of pSC101-RepA-L99-SCO plasmid
The method is described in example 3, except that primers are designed to different sequences (i.e., mutation at position L99 of the RepA protein).
Example 4: shigella O antigen expression identification
Overnight culturing 5ml of Escherichia coli TOP10 strain containing pSC101-RepA-L99K-SCO plasmid or pSC101-RepA-L99-SCO identified as positive by PCR, centrifuging to collect the thallus, taking 200 μ L of buffer solution A (the components are 0.5M Tris-Cl pH 6.8, 10% glycerol, 10% SDS and 5% Beta-mercaptoethanol) to re-suspend the thallus, boiling the mixture in boiling water for 10 minutes after the sample is fully mixed, centrifuging for 15 minutes after the sample is cooled to remove undissolved impurities, and after the centrifugation is finished, carrying out centrifugation on the supernatant according to the ratio of 1: 10 (10. mu.l to 90. mu.l) buffer B (composition: 0.5M Tris-Cl pH 6.8, 10% glycerol, 0.05% Bromophenol blue) was added and 1. mu.l proteinase K was added at a concentration of 20 mg/ml. After mixing well, the sample was placed at 37 ℃. After one hour, 15 μ l of SDS-PAGE gel at 15% concentration was run through the sample, and after running the gel, the gel was stained with silver ammonia, and if shigella O antigen was expressed, a band was visible, and if not expressed, the lipopolysaccharide structure of TOP10 appeared to be rough.
The staining procedure is briefly as follows:
1) fixing: placing the glue into the fixing solution for fixing overnight, and then rinsing with ultrapure water for three times, wherein each time lasts for 10 min;
2) sensitization: adding sensitizing solution into a dyeing disc, shaking for 10min, and washing with ultrapure water for three times, each time for 10 min;
3) silver coating: adding silver dye solution into a dyeing disc, shaking for 10min, and washing with ultrapure water for three times for 10min each time;
4) color development: adding color developing liquid into the dyeing disc, slightly oscillating, immediately replacing ultrapure water after strips appear, rinsing the glue until the glue does not change color, and performing photographic preservation analysis.
Western blot identification: separating the treated lipopolysaccharide sample by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and after electrophoresis is finished, transferring an O antigen band onto a nitrocellulose membrane (nitrocellulose membrane) by a transfer printer. After the transfer of the membrane was completed, blocking with Tris buffer containing 5% skim milk for 2 hours, and then adding rabbit antiserum (1: 100 dilution) specific for Shigella Fowler 2a O antigen and incubating at room temperature for 2 hours. After the incubation was complete, the goat anti-rabbit IgG secondary antibody labeled with alkaline phosphatase was mixed at a ratio of 1: incubation was performed at 10000 fold dilution and after two hours three times washing with TBS buffer. The immunoblotting strips were developed by adding BCIP developing solution. The reaction was terminated by washing the membrane in ultrapure water. As shown in FIG. 4, pSC101-RepA-L99K-SCO was able to increase the expression of Shigella multiple O antigen saccharide.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of Nanchang
<120> Shigella polysaccharide expression plasmid and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 341
<212> PRT
<213> replication protein RepA (pSC101-RepA)
<400> 1
Tyr Gln Ala Thr Lys Val Tyr Gly Thr Leu Phe Phe Gln Phe Pro Lys
1 5 10 15
Val Leu Leu Tyr Ser Pro Thr Tyr Lys Asn Leu Ser Ala Glu Ala Lys
20 25 30
Leu Ala Tyr Val Ile Leu Lys Asp Arg Leu Glu Tyr Ser Leu His Asn
35 40 45
Asp Trp Val Asp Glu Asn Gly Asn Ile Tyr Phe Ile Phe Ser Asn Thr
50 55 60
Glu Leu Gln Gln Ile Leu Asn Cys Ser Glu Pro Lys Val Ile Lys Thr
65 70 75 80
Lys Lys Glu Leu Glu Gln Ala Asn Lys Leu Phe Gln Lys Lys Met Gly
85 90 95
Phe Asp Pro Lys Met Lys Arg Asn Asn Pro Asn Arg Leu Tyr Leu Gly
100 105 110
Asp Leu Asn Val Ser Ala Thr Asp Val Tyr Lys Arg Glu Asn Glu Ala
115 120 125
Ser Gln Ser Thr Ile Ser Pro Ala Thr Ser Gly Thr Lys Asn Ser Leu
130 135 140
Ala Arg His Lys Met Pro Gln Ser Leu Ala Thr Ser Gly Thr Lys Asn
145 150 155 160
Ser Leu Ala Arg Asp Lys Val Pro Gln Ser Leu Ala Thr Ser Gly Thr
165 170 175
Glu Asn Ser Leu Val Tyr Gln Tyr Lys Asp Leu Glu Thr Gln Ala Arg
180 185 190
Asp Asn Lys Glu Thr Glu Lys Phe Asp Phe Ser Thr Asp Gln Tyr Ser
195 200 205
Pro Glu Ile Ile Arg Lys Gln Asn Gln Asp Leu Val Arg Arg Ala Lys
210 215 220
Asp Tyr Leu Pro Glu Ser Ala Asn Gly Gly Leu Phe Leu Asn Lys Glu
225 230 235 240
Gly Val Glu Leu Leu Gly Leu Trp Cys Arg Ser Pro Lys Gln Met Arg
245 250 255
Arg Phe Leu Gly Ile Ile Leu Asn Ala Lys Lys Ala Val Glu Arg Glu
260 265 270
His Glu Gly Thr Ala Ile Val Leu Asp Asp Pro Arg Cys Gln Glu Met
275 280 285
Ile Asn Lys Thr Met Arg Arg Phe Phe Asn Val Leu Arg Ser Asp Ser
290 295 300
Lys Lys Ile Asn Asn Val Glu Asn Tyr Leu Phe Gly Ala Met Lys Glu
305 310 315 320
Thr Leu Val Ala Tyr Trp Asn Lys Ser Leu Met Thr Ala Asn Gly Gly
325 330 335
Asp Pro Asp Glu Phe
340
<210> 2
<211> 7583
<212> DNA
<213> Shigella polysaccharide O type antigen gene (Shigella polysaccharide type O antigen gene)
<400> 2
tgaagatact tgttactggt ggcgcaggat ttattggttc tgctgtagtt cgtcacatta 60
taaataatac gcaggatagt gttgttaatg tcgataaatt aacgtacgcc ggaaacctgg 120
agtcacttgc tgatgtttct gactctaaac gctatgtttt tgaacatgcg gatatttgcg 180
atgctgctgc aatggcgcgg atttttgctc agcatcagcc ggatgcagtg atgcacctgg 240
ctgctgaaag ccatgtggat cgttcaatta caggccctgc ggcatttatt gaaaccaata 300
ttgttggtac ttatgtcctt ttggaagcgg ctcgcaatta ctggtctgct cttgatggcg 360
acaagaaaaa tagcttccgt tttcatcata tttctactga cgaagtctat ggtgatttgc 420
ctcatcctga cgaagtaaat aataaagaac aattacccct ctttactgag acgacagctt 480
acgcgcctag tagtccttat tccgcatcaa aagcatccag cgatcattta gtccgtgcgt 540
ggaaacgtac ctatggttta ccgaccattg tgactaactg ttcgaataac tacggtcctt 600
atcactttcc ggaaaaattg attccactag taattcttaa tgctctggaa ggtaaggcat 660
tacctattta tggcaaaggg gatcaaattc gtgactggct gtatgttgaa gatcatgcgc 720
gtgcgttata tatcgtcgta accgaaggta aagcgggtga aacttataac attggtggac 780
acaacgaaaa gaaaaacatc gatgtagtgc tcactatttg tgatttgttg gatgagattg 840
taccgaaaga gaaatcttac cgcgagcaaa ttacttatgt tgccgatcgc ccgggacacg 900
atcgccgtta tgcgattgat gcagagaaga ttagccgcga attgggctgg aaaccgcagg 960
aaacgtttga gagcgggatt cgtaaaacgg tgggatggta cctctccaat acaaaatggg 1020
ttgataatgt aaaaagtggt gcctatcaat cgtggattga acagaactat gagggccgcc 1080
agtaatgaat atcctccttt tcggcaaaac agggcaggta ggttgggaac tacagcgtgc 1140
tctggcacct ctgggtaatt tgattgctct tgatgttcac tccactgatt actgtggtga 1200
ttttagtaat cctgaaggtg tagctgaaac cgtaagaagc attcggcctg atattattgt 1260
caacgcagcc gctcacaccg cagtagacaa agcagaatca gaaccggagt ttgcacaatt 1320
acttaacgcg acgagtgtcg aagcgatcgc gaaagcagcc aatgaagtcg gcgcctgggt 1380
tattcactac tctactgact acgtatttcc ggggaccggt gaaataccat ggcaggaggc 1440
ggatgcaacc gcaccgctaa atgtttacgg tgaaaccaag ttagctggag aaaaagcatt 1500
acaagagcat tgtgcgaagc acctaatttt ccgtacaagc tgggtctatg caggtaaagg 1560
aaataacttc gccaaaacga tgttgcgtct gggaaaagag cgtgaagaat tagccgttat 1620
taatgatcag tttggtgcgc caacaggtgc tgaactgctg gctgattgta cggcacatgc 1680
aattcgtgtg gcactgaata aaccagaagt cgcaggcttg taccatctgg tagccactgg 1740
taccacaacc tggcacgatt atgctgcgct ggtttttgaa gaggcacgaa aagcaggtat 1800
tccccttgca ctcaacaagc tcaacgcagt accaacaaca gcttatccta caccagctcg 1860
tcgtccacat aactctcgcc ttaatacaga aaaatttcag caaaattttg cgcttgtttt 1920
gcctgactgg caggttggcg tgaaacgaat gctcaacgaa ttatttacga ctacagcaat 1980
ttaatagttt ttgcatcttg ttcgtgatga tggagcaaga tgaattaaaa ggaatgatgt 2040
aatgaaaacg cgtaaaggta ttattttagc gggtggctct ggtactcgtc tttatcctgt 2100
gactatggct gtcagtaaac agctattacc tatttatgat aagccgatga tctattaccc 2160
gctctctaca ctgatgttgg cgggtattcg cgatattctg attattagta cgccacagga 2220
tactcctcgt tttcaacaac tcctgggtga tggtagccag tgggggttaa atcttcagta 2280
caaagtgcaa ccgagtccag atggtcttgc gcaggcattt atcatcggtg aagagtttat 2340
cggtggtgat gattgtgctc tggttctcgg tgataatatc ttctacggtc atgatctgcc 2400
gaagttaatg gatgtcgctg tcaacaaaga aagtggtgca acggtatttg cctatcacgt 2460
taatgatcct gaacgctacg gtgttgttga gtttgataaa aacggtacgg caatcagcct 2520
ggaagaaaaa ccgctacaac caaaaagtaa ttatgcggta accgggcttt atttctatga 2580
taacgacgtt gtcgaaatgg cgaaaaacct taagccttct gcccgtggtg aactggaaat 2640
taccgatatt aaccgtattt atatggagca ggggcgttta tccgttgcca tgatgggacg 2700
tggttatgca tggctggaca cggggacaca tcaaagtctt attgaagcaa gcaacttcat 2760
tgcaacaatt gaagagcgcc aagggttaaa ggtatcttgc ctggaagaga ttgcttatcg 2820
taaaggcttt attgacgcag agcaggttaa tgtattagcc gaaccgctaa agaaaaatgc 2880
ttatggtcag tatctgttga aaatgattaa aggttattaa aaatgaatgt aattaaaact 2940
gaaattccag atgtattaat tttcgagccg aaagtttttg gtgatgaacg tggttttttt 3000
atggaaagct ttaaccagaa agttttcgaa gaggctgtag ggcggaaggt tgaatttgtt 3060
caggataacc attctaaatc aactaagggt gtgttacgcg gactgcacta tcagttggaa 3120
ccttatgctc aaggtaaatt agttcgttgt gttgtcggtg aagtttttga tgtagcagtt 3180
gatattcgta aatcgtcacc tacatttggg aaatggattg gggtgaattt gtctgctgag 3240
aataagcgtc agttgtggat acctgaagga tttgcgcatg gatttttggt gctgagtgaa 3300
acggctgagt ttgtttataa aacaacaaac tattacaatc caagttttga aaaaagtatt 3360
tcatactcag atcctaccat taaaattcag tggcccaatt tacaggatat gcattttaaa 3420
ttatcaaata aggatttgaa tgctaagaac ttttttaata acaatagttt aatgcaatga 3480
agaaaaatat attgctcttg ttcttagtac atggggcaaa ttatttgttc ccgtttatag 3540
ttcttccata tcaaactcga atattaagca tcgagacatt cgcagatgta gcaaaaattc 3600
aagccgctgt gatgctttta tctttaatcg taaattatgg atataactta tcaagtacaa 3660
gagctatagc tagggccgta tctcaagcag aaataaataa gatctatagt gagactctta 3720
ttgtaaaatt attattggca accatttgtc ttgcacttgg ttgcgtacat ttgatgtatg 3780
tcaaagagta ctcattgata tatcctttta taatcagttc gatatatctt tatggtagtg 3840
cattatttgc tacttggtta ttccaaggac ttgagaaaat gaaagcggtc gttatagcaa 3900
caacaatcgc taaactgact ggtgtgatac ttacttttat tttagttaag tctccaaatg 3960
atatagttgc agctcttttt acacaaaaca ttgggatgtt tataagtggt ataatatcta 4020
tttatttggt aaggaaaaac aaatatgcaa ccgtaatatg ttttcgactt aaaaatatta 4080
ttgtaagctt aaaagaagcg tggccgtttt ttttatcatt agctgcaaca agtgtatata 4140
catattttaa tgtgatttta ttatcttttt atgctggcga ctatgttgtg gcaaatttta 4200
atgctgctga taaattaaga atggctgctc aagggttact tattccaata ggacaggctg 4260
ttttcccacg attatctaaa ctagagggct atgaatatag ttctaaactt aaaatttatg 4320
caataaggta tgctattttt ggtgtttgca ttagtgcggg acttgtattt ttaggtccca 4380
tgttaactac tatttattta ggcaaagaat attcgttgtc aggagaatat cttcaaagta 4440
tgtttttact acctgccact atttcaatat cgactatact gagtcaatgg atgttgatac 4500
ctcaaggcaa agaaaaaata ttaagcagaa tctatattct aggcgccatt gtccatttat 4560
tatatgcatt tcctttagtt tactattatg gggcttgggg catggtaata tcaattttat 4620
ttactgaagt cttaattgta ttatttatgc ttaaggctgt gaaatgactt actttactgg 4680
ttttatttta atattgtttg ctattataat taaaagatta actccaagtc aaagcaagaa 4740
aaatattgtc ttaatagcta atgcgttttg gggaatattg ttggtaggtt atgctttcaa 4800
tgaacaatat ttcgtaccat taagtgcaac aaccttgttt tttatacttg cattcttatt 4860
tttctttagt atgacttata ttttaattgc taggagtgga agggttgttt tttctttcgg 4920
tactggtttt atagaaagca aatatattta ctggtttgct gggatgatta atattattag 4980
tatctgcttt ggcattatcc ttttatataa taatcatttt tctttaaaag taatgagaga 5040
aggaatttta gatggttcta ttagtgggtt tggattgggg ataagtttgc cactttcctt 5100
ctgctgtatg tatttagcaa gacatgagaa taaaaaaaat tatttctatt gttttacact 5160
actttcattc ttgcttgcgg tgttatcaac ttcaaagatc ttcttaatat tattccttgt 5220
atatattgtt ggaataaata gttatgtaag caaaaagaaa ttgcttattt atggagtgtt 5280
tgtatttgga ctgttcgctt tatcaagtat tatcttgggt aagttctctt cagaccctga 5340
aggcaagatt atttcagcaa tatttgatac gttaagggtt tatcttttct cgggattggc 5400
agcctttaat ctttatgttg aaaagaatgc cacgctcccc gaaaatttac ttttgtatcc 5460
atttaaggag gtttggggga cgacaaaaga tattcccaaa actgatattt tgccttggat 5520
caacattggt gtatgggaca cgaatgtata tacagctttt gcaccatggt atcagtcatt 5580
gggattatat gcagctataa ttattggtat tctcttaggg ttttattacg ggatatggtt 5640
tagctttcgt caaaatttag ctgtgggttt ttatcaaaca tttttgtgtt ttcctctttt 5700
aatgttgttt ttccaggagc attatttgtt gtcatggaaa atgcatttta tttatttttt 5760
atgtgcaatt ttattagcga tgagaaaagc attagagtat gaataaatat tgtatcttag 5820
tactatttaa tccagatata agtgttttta ttgataatgt caaaaagatt ttatctttgg 5880
atgtaagttt atttgtatat gacaattcag caaataaaca tgcattcctt gctctatcct 5940
cacaagagca aacaaagata aattactttt cgatatgtga aaatatcgga ttgtcgaaag 6000
cttataatga gacactaagg catattcttg aatttaataa gaatgtgaaa aataaaagca 6060
ttaatgatag tgtgcttttt ctcgaccaag actctgaagt tgatttaaat tccatcaata 6120
ttttgtttga aactatatca gcagcagagt ctaatgtgat gatagtcgcg gggaatccca 6180
taaggagaga tggactaccg tatatagatt acccccacac tgtaaacaat gtaaaatttg 6240
taattagtag ttatgctgtg tatcgcttag acgcatttag aaacatcggc ttgtttcaag 6300
aagatttttt tatagatcat atcgatagtg atttttgttc aaggctgata aaaagcaatt 6360
accaaattct ccttagaaaa gatgcctttt tttatcaacc aataggaata aaaccattca 6420
atctctgtgg tagatattta ttccctatcc catcacaaca ccgaacatat tttcaaatta 6480
gaaatgcttt tttaagttac aggcgcaatg gtgttacatt taatttttta tttagggaaa 6540
ttgtaaatag attgattatg agtatattct caggccttaa cgagaaagac ttattgaaac 6600
gattgcattt atatttaaaa ggaataaaag atggtcttaa aatgtaattc ttggctagaa 6660
gtgggggcgt tgtgattaaa aaaaaagtgg cggcgataat tataacatat aatccagatc 6720
taacaattct gcgagaaagt tatacgagtc tatataagca agtcgataaa ataattctta 6780
ttgataacaa ctctacaaac tatcaagaac ttaagaagtt attcgaaaaa aaagaaaaaa 6840
taaaaatagt gcccttgagt gataatatag gactagcagc agctcaaaat ttaggtttga 6900
acttagctat taaaaataac tatacttatg ctattttatt cgatcaggat agcgtcttac 6960
aagacaatgg aattaacagt ttcttttttg aatttgagaa attagttagt gaagaaaaat 7020
taaatatagt tgccattggg ccaagttttt ttgacgaaaa gacaggaaga cgctttcggc 7080
ctacaaaatt tatcggtccc tttttatatc cctttcgtaa aataaccaca aaaaatcctc 7140
taacagaagt tgacttcttg attgcttctg gttgtttcat aaaattggag tgtattaaat 7200
cagccggaat gatgactgaa tcgttattca tcgattatat tgatgttgaa tggtcatatc 7260
gtatgcgttc gtatggctat aagctatata ttcataatga tattcacatg agtcatttag 7320
tgggagaatc tcgagttaat ttaggattga aaactatttc tttacatggg ccgctaagac 7380
gatattactt atttaggaat tatatttcaa ttttaaaagt gagatatata ccgttaggat 7440
ataaaatacg tgagggtttt tttaatatcg gaagattttt ggtaagtatg attataacta 7500
aaaatagaaa aactttaatt ttatacacta taaaagcaat taaggacgga ataaataatg 7560
aaatggggaa atataaaggc taa 7583

Claims (5)

1. A shigella polysaccharide expression plasmid, characterized by: and (2) mutating 99 th leucine of a replication protein RepA on the pSC101 plasmid into lysine by taking the pSC101 plasmid as a vector to obtain the Shigella polysaccharide expression plasmid, wherein the sequence of the replication protein RepA is shown as SEQ ID No. 1.
2. The shigella polysaccharide expression plasmid of claim 1, wherein: the codon of the lysine adopts AAG codon.
3. The shigella polysaccharide expression plasmid of claim 1, wherein: the shigella polysaccharide expression plasmid also comprises nucleotide encoding shigella polysaccharide O-type antigen gene.
4. The method for constructing Shigella polysaccharide expression plasmid of claim 3, wherein the method comprises the following steps: the nucleotide sequence of the shigella polysaccharide O-type antigen gene is shown as SEQ NO: 2, respectively.
5. Use of the shigella polysaccharide expression plasmid of any one of claims 1-4 for recombinant protein expression.
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