CN115044598B - Efficient preparation method of bacterial ghost of enterobacteriaceae - Google Patents

Abstract

The invention belongs to the fields of microbiology and genetic engineering, and discloses a high-efficiency preparation method of bacterial ghost of enterobacteriaceae, which comprises the following steps: connecting phage ID52 cleavage gene E with a temperature control vector by adopting an RF cloning method to construct a temperature control recombinant plasmid; transferring the recombinant plasmid into competent cells of the escherichia coli, and screening by taking chloramphenicol as resistance to obtain a recombinant escherichia coli monoclonal; inoculating the obtained recombinant escherichia coli monoclonal into an LB liquid medium containing chloramphenicol for shake culture; when the bacterial liquid OD ₆₀₀ When the value reaches more than 0.8, inducing the expression of the cracking gene E, and finally collecting bacterial ghosts after the induction expression. The phage ID52-E cleavage protein has stronger cleavage activity and higher bacterial ghost formation rate. When the OD is induced initially ₆₀₀ When the value reaches 2.0, the inducer L-arabinose is added, and OD thereof is calculated ₆₀₀ The value can be reduced to 0.3-0.4, and the invention obviously improves the yield of bacterial ghost.

Description

Efficient preparation method of bacterial ghost of enterobacteriaceae

Technical Field

The invention belongs to the fields of microbiology and genetic engineering, and particularly relates to a method for preparing bacterial shadows by efficiently lysing enterobacteriaceae bacteria by using an escherichia coli phage ID52 lysate E.

Background

Bacterial ghost (Bacterial ghost) is an inactivated Bacterial empty shell without cell contents such as nucleic acid, protein and the like, maintains the original whole cell morphology and size of bacteria and structures such as immune antigen substances on the cell wall surface, and adjuvant effect components such as lipopolysaccharide, lipid A, peptidoglycan and the like contained on the surface of the Bacterial ghost can effectively stimulate the immune system of an organism so as to generate humoral immunity, cellular immunity and mucosal immunity, so that the Bacterial ghost can be used as a good vaccine and vaccine adjuvant. In addition, the hollow cavity structure, the periplasm gap and the inner and outer membranes with wide bacterial shadows can be loaded with antigens with single or composite components, such as various substances including medicines, nucleic acids, proteins and the like, can be applied to research and development of multivalent vaccines and combined vaccines, can be used as a good delivery carrier system, and has good application prospects.

Currently, bacterial ghosts are produced mainly by the induction of expression of phages in gram-negative bacteriaThe gene E is prepared and formed by way of cleavage. Studies have shown that phage->Belonging to the family of the micro-viruses, the genome is very small and is ssDNA. The cracking gene E codes membrane protein containing 91 amino acids, the cracking protein is integrated on the inner and outer membranes of cells through oligomerization and cotranslations, and finally the fusion of the inner and outer membranes of bacteria is induced to form a specific transmembrane channel of 40-200 nm. Due to the difference in osmotic pressure inside and outside the cell membrane, the cell contents such as nucleic acids, proteins, etc. are discharged through the transmembrane channel.

Most scholars control phage by different promotersThe expression of gene E was lysed to prepare bacterial ghosts. But only induce phage +.1 in the prophase of logarithmic growth (OD about 0.3-0.6)>The host bacteria can be effectively cracked only by expressing the cracking gene E, so that bacterial ghosts are formed. However, the initial induction cracking OD value is low, so that the yield of bacterial ghosts is low, and the method is unfavorable for mass production application.

And phageSimilarly, E.coli phage ID52 also belongs to the family of micro-viruses, which are obtained by protein homology alignment, the lytic protein E of phage ID52 is identical to phage +.>The homology of the cleavage protein E was 55.34%. Studies show that the lytic protein E of phage ID52 can also form a transmembrane pore canal on the cell membrane of a host bacterium to form a bacterial ghost. Furthermore, it is->Different lytic proteins E induce phage ID52E lytic protein expression in the late logarithmic growth phase, and can also effectively lyse host bacteria. The ghost formation efficiency of the lytic protein E of phage ID52 is significantly higher than that of phage +.>The protein is split, and can induce the expression of the protein at high initial OD, and the bacterial ghost yield is high.

Due to conventional temperature controlThe bacterial ghost preparation system needs to culture the host bacteria at 30 ℃ or below to a certain OD ₆₀₀ After that, the host bacteria were transferred to 42℃to induce lysis. Most of host bacteria grow slowly due to lower culture temperature before induced lysis, which is unfavorable for mass propagation of host bacteria and has longer culture time. At the same time, when the temperature is raised to 42 ℃, the epitope structure on the bacterial surface may be damaged. In addition, compared with the induction of the bacteria to the chemical reagent, the bacteria are slower in temperature induction, so that the cracking is correspondingly slower, and the cracking time is longer.

Disclosure of Invention

The invention aims to establish a high-efficiency preparation system of bacterial ghost of enterobacteriaceae, and solves the defect of low yield of the traditional preparation method of bacterial ghost.

In order to achieve the above object, the technical scheme of the present invention is as follows:

a high-efficiency preparation method of bacterial ghost of enterobacteriaceae comprises the following steps:

(1) Phage cloning Using RF cloningThe lytic gene E and the phage ID52 lytic gene E are respectively connected with a temperature control vector to construct a temperature control recombinant plasmid;

(2) An arabinose induction type recombinant plasmid is constructed by adopting a seamless cloning homologous recombination method, and the process comprises the following steps: performing PCR amplification by taking the temperature-controlled plasmid pBV220-ID52-E constructed in the step (1) and the plasmid pKD46 as templates to obtain a pBV220-ID52-E linearization fragment and an arabinose inducible promoter araC-ParaBAD fragment which lack a temperature-sensitive repressor lambda cI857 gene, and performing recombination reaction on the two obtained fragments by adopting a seamless cloning homologous recombination method to construct an arabinose inducible ID52-E recombinant plasmid;

(3) Transferring the recombinant plasmid in the step (1) or the step (2) into competent cells of the escherichia coli, and screening by taking chloramphenicol as resistance to obtain a recombinant escherichia coli monoclonal;

(4) Inoculating the recombinant escherichia coli monoclonal obtained in the step (3) into an LB liquid medium containing chloramphenicol for shake culture; when the bacterial liquid OD ₆₀₀ When the value reaches more than 0.8, inducing the expression of the cracking gene E, sampling and measuring OD every 30min ₆₀₀ Finally, collecting bacterial shadows after induced expression;

(5) Centrifuging the collected bacterial shadow, washing with PBS, fixing with 2.5% glutaraldehyde, washing with deionized water, dehydrating with ethanol step by step, drying critical points, spraying gold, and observing with a field emission scanning electron microscope;

(6) The collected bacterial shadows are observed by a transmission electron microscope after the steps of centrifugation, PBS washing, 2.5% glutaraldehyde fixation, deionized water washing, phosphotungstic acid dyeing and the like.

The phage described in step (1)The gene sequence of the cleavage gene E is shown in SEQ ID No. 1.

Preferably, the phage ID52 lytic gene E of step (1) has a gene sequence shown in SEQ ID No. 2.

Preferably, the vector in the step (1) is pBV220, and the gene sequence of the vector is shown as SEQ ID No. 3.

Preferably, the gene sequence of the arabinose inducible promoter araC-ParaBAD fragment is shown in SEQ ID No. 4.

Preferably, the competent cells of E.coli in the step (3) are E.coli BL21, E.coli Nissle1917 or DH 5. Alpha. Competent cells, the transformation method of E.coli BL21 and DH 5. Alpha. Is a heat shock method, and the transformation method of E.coli Nissle1917 is an electric transformation method.

More preferably, the E.coli nissle1917 is knocked out with the arabinose consumption related genes araB, araA and araD using lambda Red recombination technology.

More preferably, the voltage of the electrotransformation is 2.0-3.0 kV.

Preferably, the induction condition of the expression of the lysate of the escherichia coli containing the temperature-controlled recombinant plasmid in the step (4) is that the temperature is raised to 37-42 ℃, and the induction condition of the expression of the lysate of the escherichia coli containing the arabinose-inducible recombinant plasmid is that the inducer L-arabinose is added.

Preferably, the inducing OD of step (4) ₆₀₀ The value reaches more than 2.0.

Preferably, the E.coli harboring the temperature-controlled recombinant plasmid in step (4) is cultured overnight at 28-30℃at 200-220rpm under the shaking culture condition of 28-37℃at 200-220 rpm.

Compared with the existing bacterial ghost preparation technology, the invention establishes an efficient bacterial ghost preparation system of enterobacteriaceae. And phageCompared with the splitting protein E, the splitting activity of the splitting protein of the phage ID52-E is stronger, and the bacterial ghost formation rate is higher. According to the efficient preparation system for the bacterial ghost of the enterobacteriaceae, E.coli BL21, E.coli nissle1917, salmonella enteritidis and other enterobacteriaceae bacteria are used as host bacteria, and the host bacteria are cultured at 37 ℃ before and after induction, so that the time consumed by bacterial culture before cracking is shortened; l-arabinose is used as an inducer, and because the bacteria sense a chemical reagent faster and receive signals faster, the bacteria start to crack after the L-arabinose is added, and the cost of the arabinose is lower than that of the inducer such as IPTG, and in addition, the L-arabinose is an edible substance and has high safety; when the OD is induced initially ₆₀₀ When the value reaches 2.0, the inducer L-arabinose is added, and OD thereof is calculated ₆₀₀ The value can be reduced to 0.3-0.4. The invention obviously improves the yield of the bacterial ghost, is the mass production of the bacterial ghostAnd applications provide a technical basis.

Drawings

FIG. 1 is a schematic diagram showing a construction process of a temperature-controlled recombinant plasmid and agarose gel electrophoresis, wherein A, B is a temperature-controlled plasmid respectivelyAnd construction of pBV220-ID52-E, C is the cleavage Gene +.>And PCR amplification electrophoretogram of the cleavage gene ID52-E, D is colony PCR electrophoretogram of the recombinant plasmid containing the cleavage gene, and M is DL 2000DNA marker.

FIG. 2 shows the construction of an arabinose-inducible ID52-E recombinant plasmid and agarose gel electrophoresis, wherein A is the construction of the arabinose-inducible ID52-E recombinant plasmid, B is the PCR amplification electrophoresis, lane 1 is the pBV220-ID52-E linearization fragment lacking the temperature-sensitive repressor lambda cI857 gene, lane 2 is the arabinose-inducible promoter araC-ParaBAD fragment, and C is the colony PCR electrophoresis of the arabinose-inducible ID52-E recombinant plasmid, wherein M is DL 5000DNA Maker.

FIG. 3 shows plasmids each containing a temperature controlE.coli BL21 cleavage Curve assay for pBV220-ID52-E and arabinose inducible ID52E recombinant plasmid araC-ParaBAD-ID52-E and OD thereof ₆₀₀ The degree of decrease was compared, wherein A-B, C-D, E-F, G-H are: the initial induction OD was 0.8, 1.2, 1.6, 2.0.

FIG. 4 shows the inactivation efficiency of different lytic plasmids in E.coli BL21 (DE 3).

FIG. 5 is a representation of E.coli BL21 (DE 3) genome and protein leakage containing different lytic plasmids, A: residual genome of the cells before and after lysis; b: degree of genome leakage; c: intracellular protein leakage.

FIG. 6 is a scanning electron microscope image of E.coli BL21, A is untreated morphologically intact wild-type E.coli BL21; b is a lytic plasmidForming bacterial shadows; c is a bacterial ghost formed by cleaving plasmid pBV220-ID52-E; d is a bacterial ghost formed by lysing the plasmid araC-ParaBAD-ID52-E; the arrow indicates the cleavage tunnel.

Fig. 7 is an e.coli BL21 transmission electron microscope image: a is untreated, morphologically intact wild type e.coli BL21; b is a lytic plasmidForming bacterial shadows; c is a bacterial ghost formed by cleaving plasmid pBV220-ID52-E; d is a bacterial ghost formed by lysing the plasmid araC-ParaBAD-ID 52-E.

FIG. 8 shows the cleavage curves of plasmid araC-ParaBAD-ID52-E in E.coli nissle1917. DELTA. AraBAD:: FRT under different concentrations of arabinose-inducing conditions.

FIG. 9 is a diagram of E.coli nissle1917. DELTA. AraBAD:: FRT scanning electron microscopy, A is a diagram of E.coli nissle1917. DELTA. AraBAD::: FRT with intact morphology; b is E.coliNissle1917 delta araBAD after pyrolysis, FRT bacterial shadows are shown by arrows, and the pyrolysis pore canal is shown by arrows.

FIG. 10 is a transmission electron microscope image of E.coli nissle1917 before and after lysis, A is the morphological integrity E.coli nissle 1917. DELTA. AraBAD:: FRT, cells appear uniformly black due to their cell content; B. c is E.coliNissle1917 delta araBAD after lysis, wherein FRT is in bacterial shadows with different amplification factors, and the color of a bacterial cytoplasmic cavity part is lighter due to outflow of cell contents.

Detailed Description

The present invention will be further elucidated with reference to the drawings and the detailed description below, in order to make the core technology of the present invention more clear to a person skilled in the art. In the following methods, unless otherwise specified, the methods are conventional methods.

Experimental materials: e.coli DH 5. Alpha. And E.coli BL21 competence are purchased from Tiangen Biochemical technologies (Beijing) limited, E.coli nissle1917 is purchased from Ardeypharm limited liability company, germany, and plasmid pKD46 is provided by Youbao organism and the product number is VT1692.

The main reagent comprises: DL 2000DNA Marker and DL 5000DNA Marker were purchased from Takara corporation; protein markers were purchased from sameira company; plasmid extraction kits, universal DNA purification recovery kits, and bacterial genome extraction kits were purchased from the root biochemical technology (beijing) limited company; chloramphenicol, L-arabinose, etc. are purchased from the division of the biological organisms (Shanghai); LB broth was purchased from Beijing land bridge Biotechnology Limited.

Example 1: preparation of E.coli BL21 recombinant strain

1.1 temperature control recombinant plasmidAnd construction of pBV220-ID52-E

According to the plasmid pBV220-sGFP-Chl,And pET29a-ID52-E (plasmid map, see FIG. 1), and the upstream and downstream primers were designed using the RF cloning method, the primer sequences were as follows:

TABLE 1 cleavage genesAnd cleavage Gene ID52E PCR amplification primer

Preparing a PCR system toAnd pET29a-ID52-E are respectively used as templates for PCR amplification to synthesize +.>The gene (SEQ ID No. 1) and the ID52-E gene (SEQ ID No. 2) were subjected to the following PCR system and procedure:

TABLE 2 cleavage genesAnd ID52-E PCR ampliconThe reaction procedure

Taking 4 mu L of PCR amplified products for agarose gel electrophoresis analysis; purifying and recovering to obtain target geneGenes and ID52-E genes;

the method of linear RF amplification is adopted, pBV220-sGFP-Chl plasmid is used as a template, the recovered PCR product is used as a primer for linear amplification, and a PCR reaction system is as follows:

table 3 RF Linear amplification reaction System and reaction procedure

After the linear amplification reaction was completed, the unmethylated template plasmid in the system was digested with DpnI enzyme, and the procedure was as follows:

TABLE 4 DpnI cleavage System

After the system preparation is completed, enzyme cutting is carried out for 1h at 37 ℃;

converting the enzyme cutting product into E.coli DH5 alpha competence, and screening with chloramphenicol as resistance; the following day, the monoclonal was picked up and primed with specific primerColony PCR was performed with ID52E-F/ID52E-R, and positive clones with the correct band size were sequenced; positive clone plasmid with correct sequencing is successfully constructed recombinant plasmid, which is named asAnd pBV220-ID52-E;

1.2 construction of recombinant plasmid araC-ParaBAD-ID52-E

According to the sequences of the constructed plasmid pBV220-ID52-E and plasmid pKD46 (plasmid map, see FIG. 2), the upstream and downstream primers were designed by using the method of seamless cloning homologous recombination, and the sequences of the primers are as follows:

TABLE 5 PCR amplification primers for linearized pBV220-ID52-E fragment and arabinose inducible promoter araC-ParaBAD fragment

Preparing a PCR system, and respectively carrying out PCR amplification by taking plasmids pBV220-ID52-E and pKD46 as templates to synthesize a linearized pBV220-ID52-E fragment and an arabinose inducible promoter araC-ParaBAD fragment (SEQ ID No. 4), wherein the PCR system and the procedure are as follows:

TABLE 6 PCR amplification System and reaction procedure for linearizing pBV220-ID52E-Strep-Chl fragment and arabinose-inducible promoter araC-ParaBAD fragment

Taking 4 mu L of PCR amplified products for agarose gel electrophoresis analysis; purifying and recovering to obtain a linearized pBV220-ID52E fragment and an arabinose-inducible promoter araC-ParaBAD fragment;

the obtained fragment linearized pBV220-ID52-E fragment and the arabinose induction promoter araC-ParaBAD fragment are subjected to recombination reaction by adopting a method of seamless cloning homologous recombination, and the reaction system is as follows:

TABLE 7 recombinant ligation reaction System of linearized pBV220-ID52-E fragment and arabinose-inducible promoter araC-ParaBAD fragment

The ligation was carried out at 50℃for 15min.

Transforming the recombinant connection product into E.coli DH5 alpha competence, and screening by using chloramphenicol as resistance; the next day, selecting a monoclonal, carrying out colony PCR by using a specific primer, and sequencing positive clones with correct strip sizes; the positive cloning plasmid with correct sequencing is the successfully constructed recombinant plasmid, which is named araC-ParaBAD-ID52-E;

1.3 recombinant plasmidsTransformation of pBV220-ID52-E and araC-ParaBAD-ID52-E

Recombinant plasmidThe pBV220-ID52-E and the araC-ParaBAD-ID52-E are respectively transferred into E.coli BL21 competent cells by a heat shock method, chloramphenicol is used as resistance for screening, and recombinant E.coli BL21 monoclonal is obtained;

example 2:cleavage Effect comparison of pBV220-ID52-E and araC-ParaBAD-ID52-E

The recombinant E.coli BL21 obtained in example 1 was inoculated into LB liquid medium containing 25. Mu.g/mL chloramphenicol, containing recombinant plasmidE.coli BL21 containing recombinant plasmid pBV220-ID52-E, were cultured overnight at 30℃and 220 rpm; transferring the culture medium into fresh LB liquid culture medium according to the volume ratio of 1:50 in the next day, and culturing at 30 ℃ and 220rpm until OD ₆₀₀ About 0.8, 1.2, 1.6, 2.0, raising the culture temperature to 42℃and inducing the cleavage protein +.>And ID52-E expression, sampled every 30min, assayedOD ₆₀₀ Value, drawing a cracking curve;

e.coli BL21 containing recombinant plasmid araC-ParaBAD-ID52-E was cultured overnight at 37℃and 220 rpm; transferring the culture medium into fresh LB liquid culture medium according to the volume ratio of 1:50 in the next day, and culturing at 37 ℃ and 220rpm until OD ₆₀₀ About 0.8, 1.2, 1.6, and 2.0, adding inducer 0.5mg/mL L-arabinose, inducing expression of lysate ID52E, sampling every 30min, and determining OD ₆₀₀ Value, drawing a cracking curve;

in addition, 100. Mu.L of OD before induction and after induction of lysis were taken, respectively ₆₀₀ The minimal bacterial solution is subjected to gradient dilution, respectively coated on LB agar plates containing 25 mug/mL chloramphenicol, cultured overnight in a 30 ℃/37 ℃ incubator, and counted to obtain OD before induction and after induction and lysis ₆₀₀ The number of viable bacteria at the lowest point was calculated as the bacterial inactivation solution rate, bacterial inactivation efficiency= (pre-induction CFU-post-induction CFU)/pre-induction cfu×100%, and the results are shown in table 8 and fig. 4, with the initial induction of OD ₆₀₀ Elevated, temperature controlled cleavage proteinThe inactivation efficiency of the temperature control cracking protein ID52-E and the arabinose induction ID52-E is gradually improved, and no obvious difference exists between the inactivation efficiency and the inactivation efficiency. However, it can be seen from the cleavage curve (FIG. 3) that at high OD induction, the cleavage protein is temperature controlled +.>Is less effective than temperature-controlled ID52-E and arabinose-induced ID52-E, and has OD ₆₀₀ The degree of drop has a significant difference; after bacterial ghosts are formed, the contents such as genome and protein flow out from the cells, and OD ₆₀₀ Thus, the bacterial ghost formation rate of the temperature control ID52-E and the arabinose induction ID52-E is obviously higher than that of the temperature control cracking protein +.>And the formation rate of the bacterial ghost of the arabinose-induced ID52-E is highest, so that the yield of the bacterial ghost is obviously improved.

TABLE 8 inactivation efficiency of different lytic plasmids in E.coli BL21 (DE 3)

Example 3: characterization of leakage of cell contents such as genome and intracellular proteins

E.coli BL21 containing different lytic plasmids were cultured to OD ₆₀₀ The value is about 1.6, 2mL of bacterial liquid is respectively collected and temporarily stored in a refrigerator at 4 ℃; respectively inducing the expression of the lysate E, sampling and measuring OD every 30min ₆₀₀ When OD ₆₀₀ When the value reaches the lowest point, respectively collecting 2mL of bacterial liquid; centrifuging the collected pre-induction bacterial liquid and the minimal lysis bacterial liquid at 5000rpm for 15min, and collecting bacterial precipitate; genome extraction of the collected precipitate using a bacterial genome extraction kit of Tiangen company; the extracted genome concentration (ng/. Mu.L) was detected by an ultra-micro spectrophotometer, and the genome leakage degree was calculated as% = (pre-induction genome concentration-post-induction genome concentration)/pre-induction genome concentration×100%.

Bacteria containing different lytic plasmids were separately cultured to OD ₆₀₀ The value is about 1.6, 5mL of bacterial liquid is respectively collected and temporarily stored in a refrigerator at 4 ℃; respectively inducing the expression of the lysate E, sampling and measuring OD every 30min ₆₀₀ When OD ₆₀₀ When the value reaches the lowest point, respectively collecting 5mL of bacterial liquid; centrifuging the collected bacterial liquid at 5000rpm for 15min, and collecting bacterial precipitate and culture medium supernatant respectively; respectively resuspending the thalli with 500 mu L of PBS, placing under an ultrasonic breaker (ice bath) to break the thalli, wherein the ultrasonic power is 100W,3s is on, 3s is off, and the ultrasonic is carried out for 5min; taking 40 mu L of crushed sample whole liquid and 40 mu L of centrifuged culture medium supernatant, respectively adding 10 mu L of 5 XSDS-beta-mercaptoethanol Loading buffer, uniformly mixing, centrifuging for 2min in a boiling water bath at 12000rpm, and then carrying out SDS-PAGE detection.

Consistent with the trend of the lysis curve (FIG. 3), characterization of bacterial genome leakage (FIG. 5A, B) showed that E.coli BL21 (DE 3) containing the araC-ParaBAD-ID52-E plasmid had the least residual genome (FIG. 5A) and the highest degree of genome leakage (FIG. 5B). And containsPlasmid E.coli BL21 (DE 3) had the most residual genome (FIG. 5A) and the least leakage of genome (FIG. 5B). SDS-PAGE results (FIG. 5C) showed that E.coli BL21 (DE 3) containing the araC-ParaBAD-ID52-E plasmid had the least amount of intracellular protein and the most protein content in the culture supernatant. Characterization of genomic and intracellular protein leakage showed that the araC-ParaBAD-ID52-E plasmid had the best effect on E.coli BL21 (DE 3) lysis, resulting in more cell content being exported to the external medium.

Example 4: efficient preparation of E.coli nissle1917 ghost

Construction of 4.1E.coliNissle 1917 delta araBAD (FRT engineering bacteria)

Since E.coli nissle1917 can use L-arabinose as a carbon source and its use rate is fast, when E.coli nissle 1917. DELTA. AraBAD:: FRT consumes L-arabinose, E.coli nissle 1917. DELTA. AraBAD: FRT easily resumes growth. Therefore, the Arabic sugar consumption related genes araB, araA and araD of E.coli nissle1917 are knocked out by adopting a lambda Red recombination technology, so that all L-arabinose can be used for inducing bacterial lysis, and the utilization efficiency of L-arabinose and the formation efficiency of bacterial ghost are improved.

4.2E.coliNissle 1917 DeltaaraBAD:: efficient preparation of FRT bacterial shadows

Electrically transferring the recombinant plasmid araC-ParaBAD-ID52-E into E.coliNissle1917 delta araBAD of FRT electric transfer competence, wherein the electric transfer voltage is 2.0kV, and screening by taking chloramphenicol as resistance to obtain recombinant E.coliNissle1917 delta araBAD of FRT monoclonal;

the obtained recombinant E.coliNissle1917 delta araBAD is obtained by that FRT monoclonal is inoculated into LB liquid culture medium containing 25 mug/mL chloramphenicol, cultured overnight at 37 ℃ and 220rpm, transferred into fresh LB liquid culture medium according to the volume ratio of 1:50 in the next day, when the culture is carried out at 37 ℃ and 220rpm until OD is about 2.5, 0.25, 0.5 and 1mg/mL inducer L-arabinose are respectively added to induce the expression of the lysate ID52E, and the OD is measured by sampling every 30min ₆₀₀ ，OD ₆₀₀ The minimum can be reduced to about 0.5 (fig. 8).

Example 5: scanning electron microscope observation of E.coli BL21 bacterial ghost and E.coli Nissle1917 bacterial ghost

10mL of the bacterial solutions before induction and after induction lysis in examples 2 and 4 are respectively collected, centrifuged at 4 ℃ and 5000rpm for 15min, and the supernatants are discarded; washing with PBS for 3 times, centrifuging at 4deg.C and 5000rpm for 15min each time, and washing off the culture medium; then 5mL of 2.5% glutaraldehyde special fixing solution for electron microscope is added for fixing overnight (more than 8 h) at 4 ℃; centrifuging at 4deg.C and 5000rpm for 15min, discarding supernatant, and removing fixative; soaking the bacterial liquid with 5mL of deionized water for 5min, centrifuging at 4 ℃ and 5000rpm for 15min, discarding the supernatant, and repeating for 3 times; re-suspending thallus with 1mL deionized water, dripping appropriate amount of bacterial liquid onto dried cell scraping sheet, embedding in filter paper sheet, sequentially soaking in 70%, 85%, 95% ethanol for 15min, and finally soaking in 100% ethanol for 15min (the process is repeated for 3 times), dewatering step by step, and ensuring that water in sample is removed completely; and (3) putting the sample and the filter paper sheet into a critical point drying instrument for drying, taking out the cell scraping sheet in the filter paper after the drying is finished, adhering the cell scraping sheet containing the sample onto conductive adhesive, then putting the conductive adhesive into a metal spraying instrument for metal spraying treatment, and finally observing the bacterial ghost form under a field emission scanning electron microscope.

As shown in fig. 6, 6A is untreated morphological intact wild-type e.colli BL21;6B is a lytic plasmidThe bacterial ghost formed, which retains the cell membrane morphology, the cleavage tunnel is located in the center of the equator, and the cell membrane collapses to the empty cell lumen due to the drainage of the cell contents; 6C is a bacterial ghost formed by the cleavage plasmid pBV220-ID52-E, which retains the morphological structure of the cell membrane, the cleavage pore canal is positioned at two poles, shrinkage occurs at the cleavage pore canal, and presumably the cleavage pore canal is in the initial stage of cleavage, and the cell content is not completely discharged, so that the cell membrane is only slightly shrunken at the cleavage pore canal; 6D is a bacterial ghost formed by the lysis plasmid araC-ParaBAD-ID52-E, which retains the cell membrane morphology and structure, the lysis channel is centered at the equator, and the cell membrane collapses to an empty cell lumen due to the drainage of the cell contents. The arrow indicates the cleavage tunnel.

As shown in fig. 9, the e.collnissle 1917 ghost maintained the intact cell morphology and membrane structure with its transmembrane channels located at the equator or the dipoles of the bacterial cells (fig. 9B). Arrows represent cleavage tunnels.

Example 6: e.coli BL21 ghost and E.coli nissle1917 ghost observed by transmission electron microscope

10mL of the bacterial solutions before induction and after induction lysis in examples 2 and 4 are respectively collected, centrifuged at 4 ℃ and 5000rpm for 15min, and the supernatants are discarded; washing with PBS for 3 times, centrifuging at 4deg.C and 5000rpm for 15min each time, and washing off the culture medium; then 5mL of 2.5% glutaraldehyde special fixing solution for electron microscope is added for fixing overnight (more than 8 h) at 4 ℃; centrifuging at 4deg.C and 5000rpm for 15min, discarding supernatant, removing the fixing solution, soaking the bacterial liquid in 5mL deionized water for 5min, centrifuging at 4deg.C and 5000rpm for 15min, discarding supernatant, and repeating for 3 times; the thalli are resuspended by 1mL of deionized water, a proper amount of fungus liquid is sucked by a liquid-transferring gun and is dripped on a carbon film copper net which is prepared in advance, the mixture is stood for 5 to 10 minutes at room temperature, and the residual water around the mixture is sucked by water-absorbing paper. A proper amount of 3% phosphotungstic acid dye liquor is absorbed by a liquid-transfering gun and covered on a carbon film copper net attached with thalli, and the carbon film copper net is kept stand for 5min, and the redundant dye liquor is absorbed by absorbent paper. And (5) absorbing a proper amount of double distilled water, placing the double distilled water on a carbon film copper net, washing away unbound dye liquor, and repeating the operation for 2 times. Finally, standing at room temperature, evaporating to dryness, and observing under a transmission electron microscope, wherein bacterial cells with different color depths are observed in the field of view due to different transmittance of bacterial shadows and complete bacteria.

As shown in fig. 7, 7A is an untreated wild-type e.coli BL21 with intact morphology, and the cells as a whole have a uniform black color due to the inclusion of cells; e.coli BL21 bacterial ghost is light in color and transparent in light color due to the outward discharge of cell contents, and 7B is a lytic plasmidThe E.coli BL21 bacterial ghost formed retains the cell membrane morphological structure, but the bacterial ghost has lower formation rate, and most bacteria in the visual field still do not discharge cell contents, which indicates that the lysis effect is poor. 7C is an E.coli BL21 ghost formed by cleavage of plasmid pBV220-ID52-E, which retains the cell membrane morphology and structure, most bacteria have excreted cell contentsAnd forming bacterial shadows. 7D is E.coli BL21 ghost formed by lysing plasmid araC-ParaBAD-ID52-E, the bacteria retain cell membrane morphological structure, and most of bacteria basically discharge cytoplasm to form ghost except a few bacteria, which shows that the lysis effect is best.

As shown in fig. 10, the content of e.colinissle1917 bacterial shadows has been completely discharged (fig. 10B) compared to untreated e.colinissle1917 (fig. 10A).

The invention provides a preparation method for preparing the bacterial ghost of the enterobacteriaceae, which remarkably improves the yield of the bacterial ghost and lays a technical foundation for large-scale preparation of the bacterial ghost of the enterobacteriaceae.

The above description of all the disclosed embodiments, however, is not intended to limit the scope of the invention in any way, as long as it is known to those skilled in the art to understand or practice the invention.

Sequence listing

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<400> 2

atggaacgct ggaccttaag cggcattctg gcgtttctgc ttctgctgag cctgcttctg 60

ccgagcctgc tgattatgtt tattccgagc acctttcgcc gcccggtgct gagctggaaa 120

gtgcagagcc tgccgaaaac cagcctgatg gtgagcaacg cgcttctgcg cccgccgaac 180

tgcagcccgc ttctgtttag ctttgtgccg gaaaccaaaa ccctgctggt gatgccgaaa 240

cagaccagcg tgaacaacta tgcgctgaaa gaactgtgca acgaaaaaat taacagcccg 300

ctgcgtcgc 309

<210> 3

<211> 3462

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ctgcagccaa gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta 60

aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg 120

tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 180

ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 240

aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca 300

aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga 360

cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt 420

tttgcgtttc tacaaactct tgatcggcac gtaagaggtt ccaactttca ccataatgaa 480

ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct 540

aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa 600

gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg 660

gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt 720

attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac 780

ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact 840

gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata 900

tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt 960

gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac 1020

gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa 1080

ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc 1140

catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg 1200

taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 1260

tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 1320

gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 1380

cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 1440

gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 1500

gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac 1560

tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 1620

ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 1680

cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 1740

gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 1800

gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 1860

gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 1920

cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 1980

tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 2040

cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 2100

cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgctt atctttccct 2160

ttatttttgc tgcggtaagt cgcataaaaa ccattcttca taattcaatc catttactat 2220

gttatgttct gaggggagtg aaaattcccc taattcgatg aagattcttg ctcaattgtt 2280

atcagctatg cgccgaccag aacaccttgc cgatcagcca aacgtctctt caggccactg 2340

actagcgata actttcccca caacggaaca actctcattg catgggatca ttgggtactg 2400

tgggtttagt ggttgtaaaa acacctgacc gctatccctg atcagtttct tgaaggtaaa 2460

ctcatcaccc ccaagtctgg ctatgcagaa atcacctggc tcaacagcct gctcagggtc 2520

aacgagaatt aacattccgt caggaaagct tggcttggag cctgttggtg cggtcatgga 2580

attaccttca acctcaagcc agaatgcaga atcactggct tttttggttg tgcttaccca 2640

tctctccgca tcacctttgg taaaggttct aagcttaggt gagaacatcc ctgcctgaac 2700

atgagaaaaa acagggtact catactcact tctaagtgac ggctgcatac taaccgcttc 2760

atacatctcg tagatttctc tggcgattga agggctaaat tcttcaacgc taactttgag 2820

aatttttgta agcaatgcgg cgttataagc atttaatgca ttgatgccat taaataaagc 2880

accaacgcct gactgcccca tccccatctt gtctgcgaca gattcctggg ataagccaag 2940

ttcatttttc tttttttcat aaattgcttt aaggcgacgt gcgtcctcaa gctgctcttg 3000

tgttaatggt ttcttttttg tgctcatacg ttaaatctat caccgcaagg gataaatatc 3060

taacaccgtg cgtgttgact attttacctc tggcggtgat aatggttgca tgtactaagg 3120

aggttgtatg gaacaacgca taaccctgaa agattatgca atgcgctttg ggcaaaccaa 3180

gacagctaaa gatctctcac ctaccaaaca atgcccccct gcaaaaaata aattcatata 3240

aaaaacatac agataaccat ctgcggtgat aaattatctc tggcggtgtt gacataaata 3300

ccactggcgg tgatactgag cacatcagca ggacgcactg accaccatga aggtgacgct 3360

cttaaaaatt aagccctgaa gaagggcagc attcaaagca gaaggctttg gggtgtgtga 3420

tacgaaacga agcattggtt aaaaattaag gaggaggaat tc 3462

<210> 4

<211> 1233

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac ggaactcgct 60

cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc 120

aacattgcga ccgacggtgg cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg 180

gctgatacgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct ggcggaaaag 240

atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga tatcaaaatt 300

gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat tatccatcgg 360

tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct caagcagatt 420

tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga tttgcccaaa 480

caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaccccg tattggcaaa 540

tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt aaacccactg 600

gtgataccat tcgcgagcct ccggatgacg accgtagtga tgaatctctc ctggcgggaa 660

cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc tgatttttca ccaccccctg 720

accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt cgataaaaaa 780

atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg cattaaacga 840

gtatcccggc agcaggggat cattttgcgc ttcagccata cttttcatac tcccgccatt 900

cagagaagaa accaattgtc catattgcat cagacattgc cgtcactgcg tcttttactg 960

gctcttctcg ctaaccaaac cggtaacccc gcttattaaa agcattctgt aacaaagcgg 1020

gaccaaagcc atgacaaaaa cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca 1080

cattgattat ttgcacggcg tcacactttg ctatgccata gcatttttat ccataagatt 1140

agcggatcct acctgacgct ttttatcgca actctctact gtttctccat acccgttttt 1200

ttgggaattc gagctctaag gaggttataa aaa 1233

<210> 5

<211> 45

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ttggttaaaa attaaggagg aattcatggt acgctggact ttgtg 45

<210> 6

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

acagccaagc ttggctgcag ttatttttca aactgcggat g 41

<210> 7

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

taaaaattaa ggaggaattc atggaacgct ggaccttaag 40

<210> 8

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

acagccaagc ttggctgcag ttatttttca aactgcggat g 41

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atggaacgct ggaccttaag 20

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcggcaaggt gttctggt 18

<210> 11

<211> 47

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tgcgccgacc agaacacctt gccgattatg acaacttgac ggctaca 47

<210> 12

<211> 49

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tgccgcttaa ggtccagcgt tccatttttt ataacctcct tagagctcg 49

Claims (12)

1. The efficient preparation method of the bacterial ghost of the enterobacteriaceae is characterized by comprising the following steps of:

(1) Connecting phage ID52 cleavage gene E with a temperature control vector by adopting an RF cloning method to construct a temperature control recombinant plasmid; the gene sequence of the phage ID52 cleavage gene E is shown in SEQ ID No. 2;

(2) Transferring the recombinant plasmid in the step (1) into competent cells of escherichia coli, and screening by taking chloramphenicol as resistance to obtain recombinant escherichia coli monoclonal;

(3) Inoculating the recombinant escherichia coli monoclonal obtained in the step (2) into an LB liquid medium containing chloramphenicol for shake culture; when the bacterial liquid OD ₆₀₀ When the value reaches more than 1.2, inducing the expression of the cracking gene E, and finally collecting bacterial ghosts after the induction expression.

2. The efficient preparation method of the bacterial ghost of the enterobacteriaceae is characterized by comprising the following steps of:

(1) Connecting phage ID52 cleavage gene E with a temperature control vector by adopting an RF cloning method to construct a temperature control recombinant plasmid; the gene sequence of the phage ID52 cleavage gene E is shown in SEQ ID No. 2;

(2) An arabinose induction type recombinant plasmid is constructed by adopting a seamless cloning homologous recombination method, and the process comprises the following steps: performing PCR amplification by taking the temperature-controlled recombinant plasmid constructed in the step (1) and the plasmid pKD46 as templates to obtain a linearization fragment lacking the temperature-sensitive repressor lambda cI857 gene and an arabinose-inducible promoter fragment, and performing recombination reaction on the two obtained fragments by adopting a seamless cloning homologous recombination method to construct the arabinose-inducible recombinant plasmid;

(3) Transferring the recombinant plasmid in the step (2) into competent cells of escherichia coli, and screening by taking chloramphenicol as resistance to obtain recombinant escherichia coli monoclonal;

(4) Inoculating the recombinant escherichia coli monoclonal obtained in the step (3) into an LB liquid medium containing chloramphenicol for shake culture; when the bacterial liquid OD ₆₀₀ When the value reaches more than 0.8, the expression of the cracking gene E is inducedAnd finally, collecting bacterial shadows after induced expression.

3. The method according to claim 1 or 2, wherein the vector in step (1) is pBV220 having a gene sequence shown in SEQ ID No. 3.

4. The method according to claim 2, wherein the gene sequence of the arabinose inducible promoter fragment in step (2) is shown in SEQ ID No. 4.

5. The method of claim 1 or 2, wherein the competent cells of escherichia coli areE. coli BL21、E. coliNissle1917 or DH5 alpha competent cells,E. colithe conversion method of BL21 and DH5 alpha is a heat shock method,E. coliNissle1917 is electrical conversion.

6. The method of claim 5, wherein the steps ofE. coliNissle1917A lambda Red recombination technique is adopted to combine the gene with the gene related to the consumption of arabinosearaB、araAA kind of electronic device with high-pressure air-conditioning systemaraDKnocking out.

7. The method according to claim 5, wherein the voltage of the electric conversion is 2.0-0%

3.0 kV。

8. The method according to claim 1, wherein the induction condition in the step (3) is to raise the temperature to 37-42 ℃.

9. The method according to claim 8, wherein the shaking culture conditions in the step (3) are 28 to 30℃and 200 to 220 rpm.

10. The method according to claim 2, wherein the induction condition in the step (4) is addition of an inducer L-arabinose.

11. The method according to claim 10, wherein the shaking culture conditions in the step (4) are 28 to 37℃and 200 to 220 rpm.

12. The method of claim 1 or 2, wherein the OD ₆₀₀ The value reaches more than 2.0.