CN103290033B - Clostridium difficile exotoxin B carboxyl-terminal protein gene highly expressing in Escherichia coli - Google Patents
Clostridium difficile exotoxin B carboxyl-terminal protein gene highly expressing in Escherichia coli Download PDFInfo
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- 241000193163 Clostridioides difficile Species 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 title abstract description 16
- 241000588724 Escherichia coli Species 0.000 title abstract description 11
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 title abstract 4
- 239000002095 exotoxin Substances 0.000 title abstract 4
- 231100000776 exotoxin Toxicity 0.000 title abstract 4
- 230000014509 gene expression Effects 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 11
- 101710182223 Toxin B Proteins 0.000 claims description 21
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 10
- 230000000968 intestinal effect Effects 0.000 claims description 8
- 101710192606 Latent membrane protein 2 Proteins 0.000 claims description 5
- 101710109576 Terminal protein Proteins 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
- 239000013600 plasmid vector Substances 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 2
- 238000012637 gene transfection Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 2
- 238000005457 optimization Methods 0.000 abstract 1
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- 208000015181 infectious disease Diseases 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
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- 239000003053 toxin Substances 0.000 description 2
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- 108700012359 toxins Proteins 0.000 description 2
- 238000012549 training Methods 0.000 description 2
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- 108700022831 Clostridium difficile toxB Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 102000000340 Glucosyltransferases Human genes 0.000 description 1
- 108010055629 Glucosyltransferases Proteins 0.000 description 1
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Abstract
The invention discloses a Clostridium difficile exotoxin B carboxyl-terminal protein gene highly expressing in Escherichia coli, and its high expression method in Escherichia coli. The optimization of the Clostridium difficile exotoxin B carboxyl-terminal protein gene in the invention improves the influences of a plurality of duplicate blocks of an original sequence to the chemical gene synthesis and reduces the synthesis cost, the gene can highly express in Escherichia coli, and the expression level can reach 70% of total mycoproteins, and is far higher than that of present Clostridium difficile exotoxin B carboxyl-terminal protein genes.
Description
Technical field
The invention belongs to biomedicine technical field, relate to E. coli clostridium difficile extracellular toxin B carboxyl end groups because of and high-efficiency expression method in intestinal bacteria.
Background technology
Clostridium difficile is a kind of Gram-positive, the bacillus fusiformis producing gemma, obligate anaerobic, is the main pathogenic infected in Hospitals at Present.The infection of clostridium difficile is that contact is propagated, by mouth-excrement approach field planting at colon, and by directly contacting with patient, contaminated equipment or article (such as bedpan, bathtub and electronic rectal thermometers), medical personnel's indirect contact, through in a few days to the latent period of 8 weeks, C. difficile infection in hospital is caused to break out.The difficult point of clostridium difficile treatment is can tolerate multiple sterilizing agent as the gemma of dormancy form, makes it can exist for a long time in hospital environment, thus once hospital's generation C. difficile infection is broken out, is difficult to eradicate at short notice.
Clostridium difficile toxicity mainly comes from its two kinds of extracellular toxins of expressing, toxin A and toxin B.These two kinds of toxin all belong to glucosyltransferase, can make the GTP enzyme deactivation of Rho protein family.Toxin B is a kind of cytotoxin, is the important virulence factor of clostridium difficile.There are some researches prove that the antibody of anti-Clostridium difficile toxin B carboxyl terminal can effectively neutralize its cytotoxicity, thus active effect has been played to the treatment of clostridium difficile.So clostridium difficile extracellular toxin B carboxyl terminal is the ideal targets of antibody exploitation, and is preparation and the basis detecting its antibody by suitable method acquisition extracellular toxin B carboxyl-terminal fragment, can also be used for the preparation of anti-C. difficile vaccine.
But, existing clostridium difficile extracellular toxin B carboxyl end groups because of the expression amount in intestinal bacteria on the low side, affect clostridium difficile extracellular toxin B carboxyl end groups because of production.
Summary of the invention
An object of the present invention is when ensureing that carboxy terminal amino acid sequence is constant, provide a kind of clostridium difficile extracellular toxin B carboxyl end groups at E. coli because of.In another object of the present invention, also provide a kind of clostridium difficile extracellular toxin B carboxyl end groups because obtaining the method for clostridium difficile extracellular toxin B carboxyl terminal albumen at E. coli.
One aspect of the present invention relates to a kind of clostridium difficile extracellular toxin B carboxyl terminal protein gene, and described gene order is SEQIDNO.1 or its complementary sequence.
The present invention also relates to the plasmid vector containing above-mentioned clostridium difficile extracellular toxin B carboxyl terminal protein gene on the other hand.
The present invention also relates to the expression method of above-mentioned plasmid vector in intestinal bacteria on the other hand, described carrier is transfected in intestinal bacteria by the method for gene transfection, then be inoculated in LB substratum, 30 DEG C are cultured to that to add IPTG to final concentration when OD value is about 0.6 be 0.4mM, and be about 6h with same CMC model, extract the albumen in intestinal bacteria, clostridium difficile extracellular toxin B carboxyl terminal albumen can reach more than 70%.
The present invention passes through clostridium difficile extracellular toxin B carboxyl end groups because being optimized, improve the many iterons of original series to the impact of chemical gene chemical synthesis, reduce synthesis cost, can at E. coli, expression amount can reach 70% of total tropina, far above existing clostridium difficile extracellular toxin B carboxyl end groups because of.
Accompanying drawing explanation
The construction process schematic diagram of Fig. 1: recombinant plasmid vector pET32b (+)-TcdB-C.
Embodiment
Further illustrate the present invention by embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, or according to the condition that manufacturer advises." room temperature " described in embodiment refers to the temperature of carrying out the operation room tested, and is generally about 25 DEG C.
The codon optimized clostridium difficile extracellular toxin B carboxyl end groups of embodiment 1 is because of sequences Design and synthesis
1, the nucleotide sequence (toxB-C) designing and synthesizing SEQIDNO.1 carries out full genome synthetic and checks order, and confirms to test for Prokaryotic expression vector construction correctly;
2, goal gene fragment obtains: after synthetic gene toxB-C, subclone is in pUCE plasmid (being purchased from genome company), form subclone plasmid pUCE-toxB-C to increase, then EcoRV and XhoI double digestion subclone plasmid pUCE-toxB-C is used, gel electrophoresis is reclaimed and is obtained aim sequence fragment, and molecular weight is about 1770bp.
3, prokaryotic vector pET32b (+) obtains: design PCR primer:
Forward primer: AAGCTTGCGGCCGCACTCGAGCACCA
Reverse primer: CATACCAGAACCGCGTGGCACCAGA
The partial sequence of amplification pET32b (+), wherein XhoI restriction enzyme site is introduced in one end.PCR primer purification kit reclaims the reacted product of PCR, then through the carrier of XhoI single endonuclease digestion release containing sticky end.
4, ligation: the carrier that above-mentioned steps obtains and goal gene are under T4DNALigase effect, and carrier spends the night with goal gene and is connected, and temperature is set as 16 DEG C.Carrier in linked system: the mol ratio of goal gene is set as 1:7;
5, transform and identify: Calcium Chloride Method transformation of E. coli DH5 α, be spread evenly across the LB agar plate containing Amp resistance, 37 DEG C of overnight incubation, picking next day 8 single bacterium colonies carry out Tube propagation, and extract plasmid, screening positive plasmid, called after pET32b (+)-TcdB-C after order-checking is correct, recombinant plasmid size 7567bp, see Fig. 1.
6, abduction delivering and qualification: by pET32b (+)-TcdB-C plasmid Calcium Chloride Method transformation of E. coli Host Strains BL21 (DE3) after qualification, get single bacterium colony 37 DEG C of incubated overnight in the LB substratum containing 100ug/mlAmp with aseptic rifle choicest, speed setting is 220rpm.M seq is inoculated in fresh LB substratum by 1:10,37 DEG C, and 220rpm is cultured to and adds IPTG when OD value is about 0.6 and be 0.8mM to final concentration and be about 3h with same CMC model, and the SDS-PAGE electrophoresis detection of 12% is carried out in sampling.Electrophoresis showed target protein is between Marker first band and the second band, and target protein molecular weight is about 79kd, and target protein expression amount is about 30% (and original password be not suitable for directly at expression in escherichia coli, expression amount is on the low side).Preserve bacterial strain and called after pET32b (+)-TcdB-C/BL21 (DE3).
7, training systern: carry out training systern, from culture temperature, inducer concentrations and induction time three aspect consider, find temperature 30 DEG C, IPTG concentration 0.4mM induces about 6h, target protein expression amount can reach 70%, utilizes zymoplasm enzyme to cut obtained albumen and can obtain Clostridium difficile toxin B carboxyl terminal albumen further.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (1)
1. the expression method of plasmid vector in intestinal bacteria containing clostridium difficile extracellular toxin B carboxyl terminal protein gene, carrier is pET32b (+), described carrier is transfected in intestinal bacteria by the method for gene transfection, then be inoculated in LB substratum, 30 DEG C are cultured to that to add IPTG to final concentration when OD value is 0.6 be 0.4mM, and with same CMC model 6h, extract the albumen in intestinal bacteria, clostridium difficile extracellular toxin B carboxyl terminal albumen can reach more than 70%; Described clostridium difficile extracellular toxin B carboxyl terminal protein gene sequence is SEQ ID NO.1 or its complementary sequence.
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| CN103690948B (en) * | 2013-12-13 | 2016-01-20 | 山东国际生物科技园发展有限公司 | Neutralize the oral colon location preparation of anti-clostridium difficile toxin IgY |
| CN103665141B (en) * | 2013-12-31 | 2015-09-16 | 北京大学 | Albumen interactional with G. difficile cytotoxin B |
| CN103772509B (en) * | 2014-01-20 | 2017-01-11 | 山东国际生物科技园发展有限公司 | Fusion protein with clostridium difficile toxins A/B and encoding gene and application of fusion protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007013535A1 (en) * | 2005-07-27 | 2007-02-01 | Toyota Jidosha Kabushiki Kaisha | Body structure of vehicle having side-collision detection sensor |
| CN102181457A (en) * | 2011-03-21 | 2011-09-14 | 王世霞 | Clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B |
| CN102199611A (en) * | 2010-03-23 | 2011-09-28 | 王世霞 | Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007013535A1 (en) * | 2005-07-27 | 2007-02-01 | Toyota Jidosha Kabushiki Kaisha | Body structure of vehicle having side-collision detection sensor |
| CN102199611A (en) * | 2010-03-23 | 2011-09-28 | 王世霞 | Clostridium difficile exotoxin A carboxy-terminal gene sequence with optimized codon and nucleic acid vaccine thereof |
| CN102181457A (en) * | 2011-03-21 | 2011-09-14 | 王世霞 | Clostridium difficile exotoxin B amino-terminal gene sequence with optimized codon and nucleic vaccine of clostridium difficile exotoxin B |
Non-Patent Citations (3)
| Title |
|---|
| New method to generate enzymatically deficient clostridium toxin B as an antigen for immunization.;Genth H等;《Infect Immun》;20001231;第68卷(第3期);1094-1101 * |
| 艰难梭菌细胞毒素B功能区的表达;刘红升等;《第四军医大学学报》;20071231;第28卷(第6期);575-576 * |
| 艰难梭菌细胞毒素B羧基末端功能区片段的表达及纯化;刘红升等;《中国生物制品学杂志》;20070131;第20卷(第1期);29-32 * |
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Inventor after: Feng Dongxiao Inventor after: Zhang Shumin Inventor after: Liu Hong Inventor after: Dong Chuangchuang Inventor after: Xing Pingping Inventor after: Zhao Zhiwei Inventor after: Li Min Inventor after: Sun Junbo Inventor before: Feng Dongxiao Inventor before: Liu Hong Inventor before: Dong Chuangchuang Inventor before: Xing Pingping Inventor before: Zhao Zhiwei Inventor before: Li Min Inventor before: Sun Junbo |
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Free format text: CORRECT: INVENTOR; FROM: FENG DONGXIAO LIU HONG DONG CHUANGCHUANG XING PINGPING ZHAO ZHIWEI LI MIN SUN JUNBO TO: FENG DONGXIAO ZHANG SHUMIN LIU HONG DONG CHUANGCHUANG XING PINGPING ZHAO ZHIWEI LI MIN SUN JUNBO |
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Application publication date: 20130911 Assignee: YANTAI LANCHUANG BIOTECHNOLOGY CO.,LTD. Assignor: SHANDONG INTERNATIONAL BIOTECHNOLOGY PARK DEVELOPMENT Co.,Ltd. Contract record no.: X2023980049654 Denomination of invention: Efficient expression of the carboxyl terminal protein gene of Clostridium difficile exotoxin B in Escherichia coli Granted publication date: 20150624 License type: Exclusive License Record date: 20231204 |