CN109852632A - Use of spoIIIJ protein as anchor protein in Bacillus surface display system - Google Patents
Use of spoIIIJ protein as anchor protein in Bacillus surface display system Download PDFInfo
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Abstract
本发明公开了spoIIIJ蛋白作为芽孢杆菌表面展示系统中锚定蛋白的用途。本发明还提供一种芽孢杆菌表面展示系统，包括：含有融合基因的原核穿梭表达载体，宿主菌；所述的融合基因由锚定蛋白编码基因和外源目的蛋白编码基因融合而成；其中，所述的锚定蛋白是spoIIIJ蛋白，所述的宿主菌是芽孢杆菌。本发明进一步提供了一种将外源目的蛋白基因在芽孢杆菌的芽孢表面进行高效展示表达的方法。本发明所提供的枯草芽孢杆菌表面展示系统可以在芽孢表面高效展示大分子量的目的蛋白，能够应用于包括固定化酶的制备、生产抗原、多肽的高通量筛选、重金属的生物吸附以及构建生物传感器在内的多个领域。
The invention discloses the use of spoIIIJ protein as an anchor protein in the surface display system of Bacillus. The present invention also provides a Bacillus surface display system, comprising: a prokaryotic shuttle expression vector containing a fusion gene, and a host bacterium; the fusion gene is formed by fusion of an anchor protein encoding gene and an exogenous target protein encoding gene; wherein, The anchor protein is spoIIIJ protein, and the host bacteria is Bacillus. The present invention further provides a method for efficiently displaying and expressing the exogenous target protein gene on the spore surface of Bacillus. The Bacillus subtilis surface display system provided by the present invention can efficiently display the target protein of large molecular weight on the spore surface, and can be applied to the preparation of immobilized enzymes, the production of antigens, the high-throughput screening of polypeptides, the biological adsorption of heavy metals and the construction of biological sensors in many fields.
Description
Technical field
The present invention relates to the new applications of spoIIIJ albumen more particularly to spoIIIJ as Bacillus surface display systems
The purposes of middle anchorin belongs to the building of bacillus surface display system and application field.
Background technique
Bacillus subtillis surface display technologies are by external source target gene and brood cell's housing egg containing own promoter
Then this carrier is transformed into Bacillus host by white Gene Fusion, gene fusion construct expression vector, external source purpose
Gene will express under the starting of housing protein gene promoter, and by the effect with housing albumen coupling by foreign protein
It shows on the surface of thallus.
Bacillus subtilis surface display system as a kind of most common surface display system, advantage include: (1) outside
Source destination protein is not necessarily to wear membrane process during surface display, therefore improves the displaying efficiency of external source destination protein；(2)
It can the biggish foreign protein of display molecule amount；(3) polymer foreign protein can be shown；(4) the distinctive resistance of gemma increases
For the application range of the external source destination protein of displaying.
Bacillus subtilis surface display technologies can show the destination protein of macromolecule and destination protein is not necessarily to cross-film, because
This application range is extremely wide, comprising: production antigen, immobilised enzymes, the high flux screening of polypeptide, heavy metal biological adsorption with
And building biosensor etc..
The successful building of bacillus subtilis surface display system depends on three elements, it may be assumed that anchorin, destination protein
And host strain；Wherein, obtaining suitable anchorin is the key that the building of bacillus subtilis surface display system success
One of.
One successful anchorin has to meet four Xiang Yaoqiu, it may be assumed that (1) must have a structural domain that can make external source
Destination protein is shown on thallus outer capsid proteins surface；(2) a powerful anchoring domain must ensures external source purpose
Albumen is capable of fixing in cell surface without being separated；(3) anchorin and foreign protein must be compatible with forming fusion protein, no
Occur making anchorin unstable phenomenon occur due to the insertion of external source destination protein；(4) anchorin must have centainly
The ability for resisting protease in culture medium or periplasmic space.
1992, Errington etc. identified spoIIIJ base in bacillus subtilis (Bacillus subtilis)
Cause, the gene mutation cause the transcription of precursor spore specific gene to stop, to block gemma at phase III (stage III)
(sporulation) formation.Conjecture SpoIIIJ may relate to signal transduction pathway (Errington J et at that time
al.Structure and function of the spoIIIJ gene of Bacillus subtilis:a
vegetatively expressed gene that is essential for sigma G activity at an
intermediate stage of sporulation.J Gen Microbiol,1992,138:2609-2618)。
Summary of the invention
An object of the present invention provides a kind of new application of spoIIIJ albumen, i.e., as Bacillus surface exhibition
Show the anchorin of system；
The second object of the present invention is to spoIIIJ albumen is applied to building bacillus surface display system；
There is provided a kind of bacillus surface display systems for the third object of the present invention；
The fourth object of the present invention opens up external source destination protein gene on the surface of bacillus there is provided a kind of
The method shown；
Above-mentioned purpose of the invention is achieved through the following technical solutions:
The present invention passes through bioinformatics technique first, from the bacillus subtilis that genome sequencing is completed
In (Bacillus subtilis) 168 bacterial strains, it is predicted that 47 have 5 and the above transmembrane domain and the end amino acid C-
End is exposed to extracellular albumen.On this basis, the present invention designs specific primer according to the code sequence of each gene, from withered grass bud
168 strain gene group of spore bacillus expands to obtain specific fragment, and chooses green fluorescence protein gene egfp as reporter gene,
Fusion is obtained by overlap extension pcr, is sequenced after fusion after purification is connected to pUBC19 carrier
Analysis.Correct recombinant plasmid transformed will be sequenced into bacillus subtilis WB600 competent cell.By measuring in 484nm
And the absorption value under 509nm identifies spoIIIJ (GenBank:NP_391984.1) (SEQ ID No.1) in bacterium solution culture
There is stronger fluorescence signal (such as Fig. 1) after 16h.In addition thereto, it is carried in addition to GltT (Genbank:NP_388903.1) in low-copy
Also detected that relatively low fluorescence signal on body pHY300PLK after amalgamation and expression gfp, others prediction anchorins with
The recombinant plasmid transformed constructed after gfp fusion is not detected fluorescence to bacillus subtilis WB600 competent cell or fails
Success constructs and obtains recombinant plasmid.
Although showing that the albumen may have cell wall anchor active to the structure prediction of spoIIIJ, whether the albumen
It can be used as the transporter of surface display system, could especially act on other bacillus and still need to experimental verification.Therefore,
The present invention chooses green fluorescence protein gene egfp as reporter gene, and plasmid is arrived in fusion " spoIIIJ-egfp " recombination
PHY300plk2 is simultaneously imported in Bacillus subtillis recipient bacterium WB600, observes expression knot using super-resolution Laser Scanning Confocal Microscope
Fruit；Result viewing issues green fluorescence and fluorescence localization in cell surface to recombinant cell.Meanwhile the present invention is by fusion
" spoIIIJ-egfp " recombination is to plasmid pHY300plk and imports in bacillus licheniformis recipient bacterium BL10, utilizes super-resolution
Laser Scanning Confocal Microscope detects expression of results；Testing result shows that the expression realizes preferably expression effect.With BL10 and form
Type Promoter P43 directly initiates the bacterial strain of egfp expression as control；It can clearly observe that P43 directly initiates egfp expression
Bacterial strain have fluorescence signal in entire cell, and the bacterial strain fluorescence signal of anchorin spoIIIJ amalgamation and expression egfp is fixed
Positioned at cell surface.
Further to verify spoIIIJ in the presence of with other function albumen in the form of fusion protein, if still have
The organic phosphorus drop from Bacillus subtillis is chosen in transmembrane transport activity and the function or activity for keeping the protein, this experiment
Solution enzyme OPHC2 is analyzed as target protein.By external structure fusion spoIIIJ-ophc2 and import recipient bacterium
Expressed in BL10, to the full cell of recombinant bacterium spoIIIJ-ophc2-BL10, supernatant, precipitating and broken liquid enzyme activity respectively into
Measurement is gone；Measurement result shows that the recipient bacterium BL10 as negative control is not detected enzyme activity, and the full cell of recombinant bacterial strain
Enzyme activity increases with the increase of incubation time, and reaches 1.666U/mL in 32h, and enzyme activity expression in precipitating, will mainly sink
Broken rear enzyme activity of forming sediment is not improved, and further demonstrates the successful building of bacillus subtilis surface display system.
As a result, the present invention be determined spoIIIJ albumen can as the anchorin of bacillus surface display system,
By being expressed after merging with external source destination protein in host strain bacillus, show external source destination protein in cell table
Face.
The present invention provides a kind of bacillus surface display systems, comprising: the protokaryon shuttling expressing containing fusion
Carrier and host strain；The fusion is merged by anchorin and external source target gene；Wherein, the anchoring
Albumen is spoIIIJ albumen, and the nucleotides sequence of encoding gene is classified as shown in SEQ ID No.1, the promoter of the encoding gene
Sequence is shown in SEQ ID No.2；The host strain is preferably bacillus subtilis (Bacillus subtilis).
Invention further provides a kind of method for being shown external source target gene on the surface of bacillus, packets
It includes: obtaining fusion after spoIIIJ encoding gene is merged with external source destination protein encoding gene to be expressed；By the fusion
Gene with protokaryon shuttle vector is operable links together to obtain recombinant plasmid；Recombinant plasmid is transferred to bacillus in LB
It is cultivated in culture medium, external source destination protein is made to be shown expression on the surface of bacillus.
In order to further improve external source destination protein in the expression quantity of Bacillus surface, the present invention spoIIIJ with
The link peptide with αhelix is added between OPHC2, but expression of results has been found that, expressed OPHC2 enzyme activity
Only have and slightly improves.
It can make the fusion protein activity shown and stability enhancing to screen to obtain suitable link peptide.The present invention exists
A series of continuous molecular connection peptide genes of 6 same amino acid passwords have been merged between spoIIIJ and eGFP obtains a system
This series of fusion is inserted respectively into Escherichia coli B.subtilis shuttle vector by the fusion of column
PHY300plk2 constructs recombinant plasmid, these recombinant plasmids are transformed into respectively in bacillus subtilis WB600；As a result it sends out
It is existing, under the premise of cell growth is consistent, 6 × GGA (SEQ ID No.2), 6 × GCT (SEQ ID No.2) and 6 × AAT ammonia
The insertion of base acid residue linker peptide can increase the expression of GFP, higher 58.318% than original expression respectively when for 24 hours,
5.995%, 3.339%；In contrast, other four link peptides are not only without the expression efficiency of raising external source destination protein,
The expression efficiency for instead resulting in external source destination protein is in be decreased obviously.
Therefore, invention further provides a kind of bacillus surface display systems, comprising: the original containing fusion
Nuclear expression carrier and host strain；The fusion is melted by anchorin encoding gene and external source destination protein encoding gene
It closes；Wherein, there is a connection peptide gene in anchorin encoding gene and external source destination protein encoding gene；Wherein, institute
The anchorin stated is spoIIIJ albumen, and the nucleotides sequence of encoding gene is classified as shown in SEQ ID No.1, the encoding gene
Promoter sequence be SEQ ID No.2 shown in；The nucleotides sequence of the connection peptide gene is classified as SEQ ID No.3, SEQ
Any one nucleotide sequence shown in ID No.4 or SEQ ID No.5；The host strain is preferably bacillus subtilis
(Bacillus subtilis)。
External source target gene efficiently shown on the surface of bacillus invention still further provides a kind of
Method, comprising: spoIIIJ encoding gene is connect peptide gene by one with external source destination protein encoding gene to be expressed and is connected
It is connected together after fusion and obtains fusion, link together to obtain weight for the fusion and protokaryon shuttle vector are operable
Group plasmid；Recombinant plasmid is transferred to bacillus in LB culture medium to cultivate, makes external source destination protein in bacillus
Spore surface be shown expression；Wherein, the link peptide gene nucleotide series are SEQ ID No.3, SEQ ID
Any one nucleotide sequence shown in No.4 or SEQ ID No.5；The host strain is preferably bacillus subtilis
(Bacillus subtilis)。
Bacillus subtilis surface display system provided by the present invention can efficiently show macromolecule in spore surface
Destination protein, can be used in produce antigen, immobilised enzymes, the high flux screening of polypeptide, heavy metal biological adsorption and structure
Build the various aspects such as biosensor.
Detailed description of the invention
Fig. 1 predicts the measurement of the bacillus subtilis strain fluorescent value of dockerin fusion expression egfp；
The imaging of Fig. 2 super-resolution Laser Scanning Confocal Microscope observation bacillus licheniformis recombinant bacterium；
The imaging of Fig. 3 super-resolution Laser Scanning Confocal Microscope observation bacillus licheniformis recombinant bacterium；
The enzyme activity determination of Fig. 4 spoIIIJ amalgamation and expression organic phosphorus degrading enzyme ophc2.
Specific embodiment
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that can modify without departing from the spirit and scope of the invention to details and form of the invention or
Replacement, but these modifications and replacement are fallen within the protection scope of the present invention.
The building of 1 anchorin recombinant plasmid of experimental example and screening experiment
Through bioinformatics technique, from 168 bacterial strain of bacillus subtilis that genome sequencing is completed, it is predicted that
47 have 5 and the above transmembrane domain and amino acid C-terminal is exposed to extracellular albumen.According to the volume of each gene
Code sequence designs specific primer, expands to obtain specific fragment from 168 strain gene group of bacillus subtilis, and choose green fluorescence
Protein gene egfp obtains fusion as reporter gene, by overlap extension pcr, and fusion after purification is connected
Sequencing analysis is carried out after being connected to pUBC19 carrier.Correct recombinant plasmid transformed will be sequenced to experience to bacillus subtilis WB600
In state cell.SpoIIIJ (GenBank:NP_391984.1) is identified by measuring the absorption value at 484nm and 509nm
(SEQ ID No.1) has stronger fluorescence signal (such as Fig. 1) after bacterium solution culture 16h.
GltT (Genbank:NP_388903.1) is also detected that after amalgamation and expression gfp on low copy carrier pHY300PLK
Relatively low fluorescence signal, all prediction anchorins are as shown in table 1.
The selection result of the prediction anchorin of table 1
The positioning analysis of cell surface display system fusion protein of the experimental example 2 using spoIIIJ as transporter is tested
Although showing that the albumen may have cell wall anchor active to the structure prediction of spoIIIJ, whether the albumen
It can be used as the transporter of surface display system, could especially act on other bacillus and still need to experimental verification.Therefore,
This experiment chooses green fluorescence protein gene egfp as reporter gene, and plasmid is arrived in fusion " spoIIIJ-egfp " recombination
PHY300plk2, and import in Bacillus subtillis recipient bacterium WB600.Expression knot is observed using super-resolution Laser Scanning Confocal Microscope
Fruit；The green fluorescence and fluorescence localization that result viewing is issued to recombinant cell are in cell surface (Fig. 2).
Meanwhile fusion " spoIIIJ-egfp " is recombinated to plasmid pHY300plk and is imported lichens brood cell by this experiment
In bacillus recipient bacterium BL10, expression of results is detected using super-resolution Laser Scanning Confocal Microscope；Testing result shows that the expression is realized
Preferably expression effect (Fig. 3).
Using BL10 and constitutive promoter P43 directly initiate egfp expression bacterial strain as compare；It can apparent biology
The bacterial strain for observing that P43 directly initiates egfp expression has fluorescence signal (Fig. 3 B) in entire cell, and anchorin
The bacterial strain fluorescence signal of spoIIIJ amalgamation and expression egfp is positioned at cell surface (Fig. 3 C).
The recombinant bacterium cell enzyme activity of the surface display organic phosphorus degrading enzyme in Bacillus subtillis of experimental example 3 is tested
Further to verify spoIIIJ in the presence of with other function albumen in the form of fusion protein, if still have
Having from pseudomonad false pain alkali bacillus is chosen in transmembrane transport activity and the function or activity for keeping the protein, this experiment
Machine phosphorus degrading enzyme OPHC2 is analyzed as target protein.
Pass through external structure fusion spoIIIJ-ophc2 and import in recipient bacterium BL10 and expressed, to recombinant bacterium
Full cell, supernatant, precipitating and the broken liquid enzyme activity of spoIIIJ-ophc2-BL10 is determined respectively；Measurement result display is made
Be not detected enzyme activity for the recipient bacterium BL10 of negative control, and the enzyme activity of the full cell of recombinant bacterial strain with the increase of incubation time and
Increase, and reaches 1.666U/mL (Fig. 4) in 32h.Mainly in precipitating, the broken rear enzyme activity of precipitating is not obtained for enzyme activity expression
To raising, the successful building of Bacillus surface display systems is further demonstrated.From expression of results as it can be seen that spoIIIJ with
After OPHC2 adds the link peptide with αhelix, enzyme activity, which only has, slightly to be improved.
Experimental example 4 merges the sieve that different link peptides improves the expression efficiency of fusion protein between spoIIIJ and eGFP
Choosing experiment
The previous research of the present inventor show that suitable link peptide can be such that the fusion protein activity shown and stability increases
By force.The present invention has merged a series of continuous molecular connection peptidyls of 6 same amino acid passwords between spoIIIJ and eGFP
Required gene is inserted into Escherichia coli B.subtilis shuttle vector pHY300plk2, constructs recombinant plasmid by cause.Matter will be recombinated
Grain is transformed into bacillus subtilis WB600.The result shows that cell growth it is consistent under the premise of, 6 × GGA, 6 × GCT and
The insertion of 6 × AAT amino acid residue linker peptide will increase the expression of GFP, higher 58.318% than original expression respectively when for 24 hours,
5.995%, 3.339% (table 2).Other four link peptides also make not only without the expression efficiency of raising external source destination protein
The expression efficiency of high external source destination protein is decreased obviously.
The influence that 2 joint peptide of table expresses SPOIIIJ
SEQUENCE LISTING
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>purposes of the spoIIIJ albumen as anchorin in Bacillus surface display systems
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Cited By (4)
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| WO2021179892A1 (en) * | 2020-03-09 | 2021-09-16 | Taipei University Of Technology | Methods of detection of compound, antibody or protein using recombinant endospores or bacteria as sensing element |
| CN114438007A (en) * | 2022-03-25 | 2022-05-06 | 两山生态科技(山东)有限公司 | Recombinant bacillus subtilis capable of efficiently adsorbing multiple heavy metals and preparation method and application thereof |
| CN117363632A (en) * | 2023-09-22 | 2024-01-09 | 南京林业大学 | Biomembrane dispersion regulating gene spoIIIJ and application thereof |
| CN121136885A (en) * | 2025-11-18 | 2025-12-16 | 中国科学院合肥物质科学研究院 | Bacillus licheniformis and application thereof, and method for recycling uranium ions in sewage |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021179892A1 (en) * | 2020-03-09 | 2021-09-16 | Taipei University Of Technology | Methods of detection of compound, antibody or protein using recombinant endospores or bacteria as sensing element |
| CN114438007A (en) * | 2022-03-25 | 2022-05-06 | 两山生态科技(山东)有限公司 | Recombinant bacillus subtilis capable of efficiently adsorbing multiple heavy metals and preparation method and application thereof |
| CN117363632A (en) * | 2023-09-22 | 2024-01-09 | 南京林业大学 | Biomembrane dispersion regulating gene spoIIIJ and application thereof |
| CN121136885A (en) * | 2025-11-18 | 2025-12-16 | 中国科学院合肥物质科学研究院 | Bacillus licheniformis and application thereof, and method for recycling uranium ions in sewage |
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