CN110951666A - Double-protein expressed escherichia coli ghost and preparation method thereof - Google Patents

Double-protein expressed escherichia coli ghost and preparation method thereof Download PDF

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CN110951666A
CN110951666A CN201911380493.5A CN201911380493A CN110951666A CN 110951666 A CN110951666 A CN 110951666A CN 201911380493 A CN201911380493 A CN 201911380493A CN 110951666 A CN110951666 A CN 110951666A
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郭长明
袁橙
孟婷
张步彩
管远红
刘剑华
李东旭
高伟
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Jiangsu Agri Animal Husbandry Vocational College
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Abstract

The invention discloses a double-protein expressed escherichia coli ghost which is prepared from double-expression bacteriolysis plasmids, wherein the double-expression bacteriolysis plasmids are bacteriolysis plasmids which take pETDuet1 as a carrier and are inserted with E genes and staphylococcus aureus nuclease A at the same time so as to realize double-protein expression of the E genes and the staphylococcus aureus nuclease A. According to the invention, the gene fragments are respectively inserted into two independent multiple cloning sites of the vector, so that the problem of mutual influence of two protein structures in expression is effectively avoided, the degradation of bacterial DNA is realized, the obtained bacterial ghost product has high safety, the dual-protein expression is realized, and the dual-protein expression efficiency is high. The escherichia coli ghost prepared by using the pETDuet1-E-SNA bacteriolysis plasmid can degrade genetic materials (including drug-resistant genes and toxin genes) of escherichia coli, effectively improves the safety of ghost use, and solves the biological safety hidden trouble existing in ghost product use.

Description

Double-protein expressed escherichia coli ghost and preparation method thereof
Technical Field
The invention relates to the technical field of preparation of escherichia coli bacterial ghosts, and in particular relates to an escherichia coli bacterial ghost with double-protein expression and a preparation method thereof.
Background
In 1985, Lubitz et al found that the lytic gene E of the bacteriophage PhiX174 can form transmembrane pores after being expressed in gram-negative bacteria, and cytoplasm, organelles and the like can escape from the pores to form bacterial empty shells, which are called bacterial ghosts or bacterial shadows. Compared with formalin inactivated bacterial vaccine, the preparation process of the bacterial ghost does not damage the antigen structure of the bacterial thallus, and the immunogenicity of the bacterial ghost is well maintained, so that the bacterial ghost can be used as a novel inactivated vaccine, a vaccine carrier or an adjuvant. At present, E gene is mostly adopted to be singly expressed to prepare bacterial ghosts in related reports at home and abroad. However, this method cannot degrade bacterial DNA, and thus the bacterial ghost product obtained has biological safety hazards. In addition, the method can only realize independent expression of the E protein, and has the technical problem that simultaneous expression of other exogenous proteins is difficult to realize so as to further expand the functions of bacterial ghosts. Currently, researchers have also attempted to prepare ghosts by co-expressing two proteins in a host bacterium by using 15 flexible amino acid linkers to join the E gene and staphylococcus aureus nuclease a (SNA) gene in series. However, there is a problem that the expression efficiency and structure of two proteins affect each other.
Disclosure of Invention
The invention aims to provide a double-protein expressed escherichia coli ghost and a preparation method thereof aiming at the defects. According to the invention, the gene fragments are respectively inserted into two independent multiple cloning sites of the vector, so that the problem of mutual influence of protein structure and expression efficiency is effectively avoided, the degradation of bacterial DNA is realized, the obtained bacterial ghost product has high safety, the dual-protein expression is realized, and the dual-protein expression efficiency is high.
The technical scheme of the invention is as follows:
the invention provides a double-protein expressed escherichia coli ghost which is prepared from double-expression bacteriolysis plasmids, wherein the double-expression bacteriolysis plasmids are bacteriolysis plasmids which take pETDuet1 as a carrier and are inserted with E genes and staphylococcus aureus nuclease A (SNA) at the same time so as to realize double-protein expression of the E genes and the staphylococcus aureus nuclease A. The double expression bacteriolysis plasmid constructed by the invention is named pETDuet 1-E-SNA.
The double-expression bacteriolysis plasmid is constructed by inserting an E gene fragment and a yellow staphylococcal nuclease A gene fragment in sequence according to two Multiple Cloning Sites (MCS) sequences of a pETDuet1 vector.
The construction method of the dual-expression bacteriolytic plasmid comprises the following steps:
(1) the E gene was synthesized with the E sequence of the lytic gene of bacteriophage PhiX174 (published according to NCBI) and ligated into the pUC57 vector; designing E gene fragment forward/reverse PCR primers according to a first Multiple Cloning Site (MCS) sequence of a vector pETDuet1, and amplifying by taking the pUC57 vector connected with the E gene as a template, so that the 5 'and 3' tail ends of a PCR product respectively have a sequence and a restriction enzyme site which are consistent with the two tail ends of a linearized vector, and completing the construction of a recombinant plasmid inserted with the E gene; after the Escherichia coli expresses the lytic protein E of the phage, the bacteria form a transmembrane channel structure on a cell membrane under the action of the lytic protein and osmotic pressure, and cytoplasmic contents in the bacteria are discharged through the channel.
(2) The sequence of the non-signal peptide extracellular region is synthesized by using the sequence of the staphylococcus aureus nuclease A, the sequence is 453bp and is connected to a pUC57 vector, the second MCS sequence of the vector pETDuet1 in the recombinant plasmid with the E gene inserted is inserted into a staphylococcus aureus nuclease A gene fragment, and the construction of the recombinant plasmid with the E gene and the staphylococcus aureus nuclease A gene inserted is completed.
The preparation method of the double-protein expressed escherichia coli ghost comprises the following steps:
(1) preparing a dual-expression bacteriolytic plasmid: firstly, synthesizing an E gene by using a lytic gene E sequence of a bacteriophage PhiX174 and connecting the E gene to a pUC57 vector; designing E gene fragment forward/reverse PCR primers according to a first MCS sequence of a vector pETDuet1, and amplifying by taking the pUC57 vector connected with the E gene as a template, so that the 5 'and 3' tail ends of a PCR product respectively have a sequence and a restriction enzyme cutting site which are consistent with the two tail ends of a linearized vector, and completing the construction of a recombinant plasmid inserted with the E gene; then, a 453bp signal peptide-free extracellular region sequence is synthesized by a staphylococcus aureus nuclease A sequence and is connected to a pUC57 vector, a staphylococcus aureus nuclease A gene fragment is inserted into a second MCS sequence of the vector pETDuet1 in the obtained recombinant plasmid with the inserted E gene, and the construction of the recombinant plasmid with the inserted E gene and the inserted staphylococcus aureus nuclease A gene is completed.
(2) Preparing the Escherichia coli ghost with double protein expression: firstly, transforming the dual-expression bacteriolytic plasmid into host bacteria; and carrying out shake culture on the bacterial liquid of the host bacteria, adding an IPTG inducer into the bacterial liquid for induction expression when the OD600 is 0.4-0.6, sampling every 30min to measure the OD600 value, stopping shake culture when the OD600 value is not changed, and stopping induction to obtain the escherichia coli ghost with double-protein expression.
The host bacterium is BL21(DE3)。
The culture temperature of the shaking culture is 37 ℃.
The final concentration of the IPTG inducer in the bacterial liquid is 1.2 mmol/L.
And (3) adopting β -propiolactone to inactivate the escherichia coli ghost expressed by the double proteins obtained by shaking culture.
The inactivation treatment comprises the steps of adding β -propiolactone into the solution of the Escherichia coli ghost with double protein expression until the final concentration is 0.025%, placing the solution at 37 ℃ for acting for 1h, centrifuging the bacterial ghost liquid, collecting the precipitate, washing the precipitate with PBS solution, then re-suspending the precipitate, adding β -propiolactone again until the final concentration is 0.05%, and placing the precipitate at 37 ℃ for acting for 2h to complete the inactivation treatment.
The PBS solution was a sterilized PBS solution with a pH of 7.4.
The E gene fragment forward/reverse PCR primer sequences are shown in EpET-F and
EpET-R; the sequence of the SNA gene fragment forward/reverse PCR primer is shown in Table 1
SNA-F and SNA-R.
TABLE 1E Gene and SNA Gene fragment Forward/reverse PCR primer sequences
Figure BDA0002342096600000041
The E gene was synthesized based on the E sequence of the lytic gene of the NCBI published phage PhiX 174. The E sequence of the lytic gene of the phage PhiX174 (namely the E gene sequence of the invention) is as follows:
Figure BDA0002342096600000051
Figure BDA0002342096600000052
s. aureus nuclease A is a sequence of the extracellular domain without a signal peptide synthesized from the SNA sequence published by NCBI. The sequence of the extracellular region of the staphylococcus aureus nuclease A without the signal peptide (namely the sequence of the staphylococcus aureus nuclease A) is as follows:
Figure BDA0002342096600000053
Figure BDA0002342096600000054
pETDuet1 vector was purchased from Beijing Liuhua Dagen science and technology Co., Ltd; the pUC57 vector was purchased from Heihua Dagenetechnology, Inc. of Beijing.
The invention has the beneficial effects that:
(1) according to the invention, a bacteriolytic plasmid for expressing a phage E gene is constructed on the basis of a pETDuet1 vector, and a double-expression bacteriolytic plasmid for simultaneously expressing the E gene and staphylococcus aureus nuclease A is constructed by utilizing two multiple cloning sites of pETDuet1, so that the bacterial DNA degradation is realized while the bacterial ghost is prepared, the safety of bacterial ghost products is improved, the bacterial ghost can be combined with a double-expression vector pACYCDuet-1, the simultaneous expression of 4 genes is realized, and the application range of the bacterial ghost products is expanded. The escherichia coli ghost prepared by using the pETDuet1-E-SNA bacteriolysis plasmid can realize the degradation of genetic materials (including drug-resistant genes and toxin genes) in escherichia coli, effectively improve the safety of the use of the ghost and solve the problem of biological potential safety hazard in the use of ghost products.
(2) Compared with the single-protein escherichia coli ghost, the escherichia coli ghost with double-protein expression has high cracking efficiency, particularly has high cracking efficiency reaching 97.91% under the condition of an inducer with the concentration of 1.2mmol/L, and because IPTG induces the Lac promoter, T7 RNA polymerase controlled by the Lac promoter is indirectly induced to express, the T7 promoter of a vector is activated, and the high-efficiency expression of a target gene is realized.
(3) According to the invention, the gene fragments are respectively inserted into two independent multiple cloning sites of the vector, so that the problem that two exogenous protein structures are mutually influenced is effectively avoided, the degradation of bacterial DNA is realized, the obtained bacterial ghost product has high safety, the dual-protein expression is realized, and the dual-protein expression efficiency is high.
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FIG. 1 shows a transmission electron micrograph of pETDuet1-E-SNA/BL21(DE3) ghost in example 1 of the present invention.
FIG. 2 shows an electrophoretic analysis chart in test example 2 of the present invention.
Detailed Description
Example 1
Preparation of double-protein expressed Escherichia coli ghost comprises the following steps:
(1) preparing a double-expression bacteriolysis plasmid pETDuet 1-E-SNA: the E gene (276bp) was synthesized first based on the E sequence of the lytic gene of the NCBI published phage PhiX174 and ligated into the pUC57 vector, designated pUC 57-E.E gene fragment forward/reverse PCR primers were designed based on the first MCS sequence of vector pETDuet1, and amplified using pUC57-E as a template, so that the 5 'and 3' termini of the PCR product had sequences and restriction sites identical to those of the linearized vector. According to
Figure BDA0002342096600000071
IIOne Step cloning kit instructions, complete the construction of recombinant plasmid pETDuet 1-E. Then, the sequence of the non-signal peptide extracellular region of 453bp was synthesized based on the SNA sequence published by NCBI and ligated to pUC57 vector, named pUC57-SNA, and the fragment of the inserted SNA gene was designed based on the second MCS sequence of pETDuet1 in the recombinant plasmid of the inserted E gene obtained as described above, and the fragment was inserted according to the sequence of SNA published by NCBI
Figure BDA0002342096600000072
II One Step cloning kit instructions, complete the construction of recombinant plasmid pETDuet 1-E-SNA.
The above recombinant plasmid pETDuet1-E-SNA was transformed into DH5αScreening positive clones on a resistant plate, extracting positive clone plasmids, and further identifying the positive clones by double enzyme digestion and sequencing.
(2) Preparing the Escherichia coli ghost with double protein expression: firstly, the pETDuet1-E-SNA bacteriolysis plasmid is transformed into BL21(DE3) host bacteria; performing shake culture on the bacterial liquid at 37 ℃, taking 2mL of bacterial liquid sample as a control sample before induction when OD600 is 0.5, then adding IPTG inducer with the concentration of 1.2mmol/L into the bacterial liquid to perform induction expression at 37 ℃, sampling every 30min to measure an OD600 value, stopping induction when the OD600 value is unchanged, and obtaining the escherichia coli ghost with double protein expression, which is named as pETDuet1-E-SNA/BL and is named as pETDuet1-E-SNA/BL21(DE3), and 2mL of ghost liquid was taken as a post-induction sample.
(3) Inactivating, adding β -propiolactone into the solution of Escherichia coli ghost with double protein expression to final concentration of 0.025%, placing at 37 deg.C, slowly oscillating for 1h, centrifuging ghost bacteria liquid, collecting precipitate, cleaning the precipitate with sterilized PBS solution with pH of 7.4, resuspending, adding β -propiolactone again to final concentration of 0.05%,putting the mixture at 37 ℃ for 2h to complete inactivation treatment, respectively coating the inactivated bacterial ghost solution on plates for sterile inspection, and finding out pETDuet1-E-SNA/BL after the inactivation treatment of β -propiolactone through plate coating inspection21(DE3) the ghost solution contains no viable bacteria. pETDuet1-E-SNA/BL21(DE3) A transmission electron micrograph of the bacterial ghosts is shown in FIG. 1.
The control sample before induction was diluted to 10 with physiological saline-4～10-6After induction, the sample was diluted to 10 with physiological saline-1～10-3The diluted control sample before induction and the sample after induction were each coated with 3 pieces of non-resistant LB plates, and incubated at 37 ℃ for 12 hours. And (3) selecting a plate with proper dilution for counting, taking the average value of CFU to calculate the cracking rate, and calculating to obtain the cracking rate of 97.91%. The calculation formula is as follows: the lysis rate was (1-CFU after induction/CFU before induction) × 100%. Test example 1 study on influence of IPTG inducers with different concentrations on different lysis rates of Escherichia coli ghosts
Comparative sample 1: pETDuet1-E/BL21(DE 3);
comparative sample 2: pETDuet1/BL21(DE 3);
the sample of the invention: pETDuet1-E-SNA/BL21(DE 3);
wherein, the reference 1 bacterial ghost pETDuet1-E/BL21The preparation method of (DE3) is:
(1) preparation of pETDuet 1-E: the E gene (276bp) was synthesized based on the E sequence of the lytic gene of the NCBI published phage PhiX174 and ligated into the pUC57 vector, designated pUC 57-E. E gene fragment forward/reverse PCR primers were designed based on the first MCS sequence of vector pETDuet1, and amplified using pUC57-E as a template, so that the 5 'and 3' termini of the PCR product had sequences and cleavage sites identical to those of the linearized vector. According to
Figure BDA0002342096600000091
II, completing the construction of a recombinant plasmid pETDuet1-E according to the instruction of a One Step cloning kit, transforming the recombinant plasmid into DH5 α, screening positive clones on a resistance plate, extracting positive cloning plasmids, and further identifying the positive clones by double enzyme digestion and sequencing.
(2) Preparing E eggWhite-expressing E.coli ghost: firstly, the pETDuet1-E bacteriolysis plasmid is transformed into BL21(DE3) host bacteria; then, the bacterial liquid is subjected to shake culture at 37 ℃, and when the OD600 is 0.5, 2mL of bacterial liquid sample is taken as a control sample before induction; equally dividing the bacterial liquid into 5 parts, respectively adding IPTG inducers with the concentrations of 0.5mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and 1.5mmol/L into the 5 parts of bacterial liquid to respectively perform induction expression at 37 ℃, sampling every 30min to measure an OD600 value, stopping the induction when the OD600 value is not changed, obtaining the Escherichia coli ghost expressed by E protein induced by the 5 concentrations of inducers, and naming the Escherichia coli ghost as pETDuet1-E/BL21(DE3), and 2mL of ghost liquid after each induced expression was taken as a sample after induction.
(3) Inactivating, namely adding β -propiolactone into E protein expressed escherichia coli ghost solution to the final concentration of 0.025%, placing the solution at 37 ℃ for slow oscillation for 1h, centrifuging ghost bacteria liquid, collecting precipitate, cleaning the precipitate with sterilized PBS solution with the pH of 7.4, then re-suspending, adding β -propiolactone again to the final concentration of 0.05%, placing the solution at 37 ℃ for 2h, completing inactivation, respectively taking inactivated ghost solution, coating plates for aseptic inspection, and discovering through coating plate inspection that pETDuet1-E/BL is inactivated after β -propiolactone21(DE3) the ghost solution contains no viable bacteria.
The control sample before induction was diluted to 10 with physiological saline-4～10-6After induction, the sample was diluted to 10 with physiological saline-1～10-3The diluted control sample before induction and the sample after induction were each coated with 3 pieces of non-resistant LB plates, and incubated at 37 ℃ for 12 hours. And selecting a plate with proper dilution for counting, taking the average value of CFU to calculate the cracking rate, and calculating the cracking rate of the IPTG inducer at the concentrations of 0.5mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and 1.5 mmol/L.
pETDuet1/BL21(DE3) of comparative sample 2 was prepared as follows:
(1) transformation of pETDuet1 plasmid into BL21(DE3) host bacteria; then, the bacterial liquid is subjected to shake culture at 37 ℃, and when the OD600 is 0.5, 2mL of bacterial liquid sample is taken as a control sample before induction; the bacterial liquid is divided into 5 parts, and the 5 parts areIPTG inducers with the concentrations of 0.5mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and 1.5mmol/L are respectively added into the bacterial liquid to carry out induction expression at the temperature of 37 ℃, the induction is stopped after 3 hours, and 2mL of bacterial ghost liquid is taken as a sample after the induction.
(3) And (2) inactivation treatment, namely adding β -propiolactone into the bacterial liquid of the ghost slough to the final concentration of 0.025%, placing the bacterial liquid of the ghost slough at 37 ℃ for slow oscillation for 1 hour, centrifuging the bacterial liquid of the ghost slough and collecting precipitates, cleaning the precipitates by using a PBS (phosphate buffer solution) with the pH of 7.4 after sterilization, then re-suspending the precipitates, adding β -propiolactone again to the final concentration of 0.05%, placing the precipitates at 37 ℃ for 2 hours to complete inactivation treatment, respectively coating the inactivated bacterial ghost solution on plates for aseptic inspection, and finding that viable bacteria still exist in the solution after β -propiolactone inactivation treatment through plate coating inspection.
The control sample before induction was diluted to 10 with physiological saline-4～10-6After induction, the sample was diluted to 10 with physiological saline-1～10-3The diluted control sample before induction and the sample after induction were each coated with 3 pieces of non-resistant LB plates, and incubated at 37 ℃ for 12 hours. And selecting a plate with proper dilution for counting, and taking the average value of CFU to calculate the cracking rate of the IPTG inducer at the concentration of 0.5mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and 1.5mmol/L of the IPTG inducer.
The preparation method of the sample pETDuet1-E-SNA/BL21(DE3) comprises the following steps:
(1) preparing a double-expression bacteriolysis plasmid pETDuet 1-E-SNA: the E gene (276bp) was synthesized first based on the E sequence of the lytic gene of the NCBI published phage PhiX174 and ligated into the pUC57 vector, designated pUC 57-E. The E gene fragment forward/reverse PCR primers were designed based on the first MCS sequence of vector pETDuet 1. The PCR product was amplified using pUC57-E as a template so that the 5 'and 3' termini of the PCR product had sequences and cleavage sites corresponding to the two termini of the linearized vector, respectively. According to
Figure BDA0002342096600000112
IIOne Step cloning kit instructions, complete the construction of recombinant plasmid pETDuet 1-E. Then, according to the SNA sequence published by NCBI, the 453bp signal peptide-free extracellular region sequence was synthesized and ligated to pUC57 vectorNamed pUC 57-SNA. The second MCS sequence of vector pETDuet1 in the recombinant plasmid inserted with the E gene obtained above was used to design a fragment inserted with the SNA gene, and the fragment was designed in accordance with the sequence
Figure BDA0002342096600000111
II One Step cloning kit instructions, complete the construction of recombinant plasmid pETDuet 1-E-SNA.
The above recombinant plasmid pETDuet1-E-SNA was transformed into DH5αScreening positive clones on a resistant plate, extracting positive clone plasmids, and further identifying the positive clones by double enzyme digestion and sequencing.
(2) Preparing the Escherichia coli ghost with double protein expression: firstly, the pETDuet1-E-SNA bacteriolysis plasmid is transformed into BL21(DE3) host bacteria; performing shake culture on the bacterial liquid at 37 ℃, taking 2mL of bacterial liquid sample as a control sample before induction when OD600 is 0.5, taking 5 parts of bacterial liquid, respectively adding IPTG inducers with the concentrations of 0.5mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and 1.5mmol/L into the 5 parts of bacterial liquid, respectively performing induction expression at 37 ℃, sampling every 30min to measure an OD600 value, stopping induction when the OD600 value is unchanged, obtaining the Escherichia coli ghost with double protein expression, and naming the Escherichia coli ghost as pETDuet1-E-SNA/BL21(DE3), and 2mL of ghost liquid was taken as a post-induction sample.
(3) Inactivating, namely adding β -propiolactone into the escherichia coli ghost solution with double protein expression to the final concentration of 0.025%, placing the solution at 37 ℃ for slow oscillation for 1h, centrifuging the bacterial ghost solution, collecting precipitate, cleaning the precipitate with sterilized PBS (PBS) solution with the pH of 7.4, then re-suspending, adding β -propiolactone again to the final concentration of 0.05%, placing the solution at 37 ℃ for 2h, completing the inactivation, respectively taking inactivated bacterial ghost solution, coating plates for sterile inspection, and discovering through coating plate inspection that pETDuet1-E-SNA/BL is inactivated after β -propiolactone21(DE3) the ghost solution contains no viable bacteria. pETDuet1-E-SNA/BL21(DE3) A transmission electron micrograph of the bacterial ghosts is shown in FIG. 1.
The control sample before induction was diluted to 10 with physiological saline-4～10-6After induction, the samples were diluted to 1 with physiological saline0-1～10-3The diluted control sample before induction and the sample after induction were each coated with 3 pieces of non-resistant LB plates, and incubated at 37 ℃ for 12 hours. And selecting a plate with proper dilution for counting, taking the average value of CFU to calculate the cracking rate, and calculating the cracking rate of the IPTG inducer at the concentration of 0.5mmol/L, 0.8mmol/L, 1mmol/L, 1.2mmol/L and 1.5mmol/L of the IPTG inducer. The calculation formula of the cracking rate of the comparison samples 1-2 and the sample of the invention under each IPTG inducer concentration condition is as follows: the lysis rate was (1-CFU after induction/CFU before induction) × 100%. The calculation results are shown in table 3:
TABLE 3 cleavage rates at different IPTG inducer concentrations
Figure BDA0002342096600000131
The result shows that the cracking rate of the escherichia coli ghost expressed by the double proteins is highest only when the IPTG final concentration is 1.2mmol/L, compared with the escherichia coli ghost expressed by the double proteins, the cracking efficiency of the escherichia coli ghost expressed by the single proteins is lower than that of the invention when the IPTG final concentration is 1.2mmol/L, but the bacterial ghost can not be cracked due to the induced expression of the TDpEuet 1 empty plasmid.
The results of the inactivation of β -propiolactone at various IPTG inducer concentrations for the above-described controls 1-2 and the inventive samples are shown in Table 4:
TABLE 4 inactivation Effect of 4 β -propiolactone on samples of different induced concentrations
Figure BDA0002342096600000141
The results show that β -propiolactone treatment can completely inactivate the residual viable bacteria in pETDuet1-E-SNA/BL21(DE3) bacterial ghosts and pETDuet1-E/BL21(DE3) bacterial ghosts, but not in pETDuet1/BL21(DE3) solutions, when the final concentration of IPTG is 0.5mmol/L-1.5 mmol/L.
Test example 2 safety test
Taking pETDuet1-E-SNA/BL in example 121(DE3) 1mL each of the inoculum samples before (0h) and after (2h) inductionAnd separating and removing the supernatant for later use. Extracting DNA from the thallus precipitate by a boiling cracking method: the precipitate was dissolved in 100. mu.L of PBS (pH7.4), subjected to boiling water bath (10min) and ice bath (5min), centrifuged at 12000g for 10min to take the supernatant, and 5. mu.L of the sample was subjected to 1% agarose gel electrophoresis to analyze the degrading activity of SNA protein on bacterial DNA.
pETDuet1-E/BL was prepared in the same manner21(DE3) samples of the inoculum after induction (2h) were used as a control. Wherein, pETDuet1-E/BL21(DE3) is the one prepared in test example 1.
The results are shown in FIG. 2, where: 1 is pETDuet1-E-SNA/BL21(DE3) inducing 0h of bacterial ghost culture supernatant; 2 is pETDuet1-E-SNA/BL21(DE3) induction of 0h bacterial ghost DNA; 3 is pETDuet1-E-SNA/BL21(DE3) inducing 2h of ghost culture supernatant; 4 is pETDuet1-E-SNA/BL21(DE3) induction of 2h bacterial ghost DNA; 5 is pETDuet1-E/BL21(DE3) inducing 2h of ghost culture supernatant; 6 is pETDuet1-E/BL21(DE3) Induction of 2h bacterial ghost DNA.
The results showed 0h after IPTG induction, pETDuet1-E-SNA/BL21(DE3) No obvious genomic band was seen in the ghost culture supernatant lane (1 in FIG. 2); while a large fragment genomic band was visible in the ghost lysis sample at the same time point (2 in fig. 2). The bacterial DNA exudes under the action of E protein on the perforation of a bacterial membrane, and pETDuet1-E/BL 2h after induction21(DE3) No genomic band in the DNA sample (6 in FIG. 2), and pETDuet1-E/BL 2h after induction21(DE3) undegraded genomic bands (5 in FIG. 2) appeared in the ghost culture supernatant samples. After 2h of IPTG induction, pETDuet1-E-SNA/BL is under the action of SNA protein21(DE3) bacterial ghost DNA was degraded, and after induction, neither the bacterial culture supernatant nor the bacterial lysate lanes showed large genomic bands, but only about 100bp bands (3 in FIG. 2, 4 in FIG. 2). Therefore, the invention can realize the degradation of genetic materials (including drug-resistant genes and toxin genes) of the escherichia coli, and effectively improve the use safety of the bacterial ghost.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed.
Sequence listing
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<120> double-protein expressed escherichia coli ghost and preparation method thereof
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gcgctgaatt tacggaaaac attattaatg gcgtcgagcg tccggttaaa gccgctgaat 180
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Claims (10)

1. The bacterial ghost is characterized in that the bacterial ghost is prepared from double-expression bacteriolysis plasmids, and the double-expression bacteriolysis plasmids are bacteriolysis plasmids which take pETDuet1 as a vector and are inserted with E genes and staphylococcus aureus nuclease A at the same time to realize double-protein expression of the E genes and the staphylococcus aureus nuclease A.
2. The double-protein-expressed escherichia coli ghost according to claim 1, wherein the double-expression bacteriolysis plasmid is constructed by inserting an E gene fragment and a staphylococcus aureus nuclease A gene fragment into two polyclonal site sequences of a pETDuet1 vector in sequence.
3. The dual-protein-expressed escherichia coli ghost according to claim 2, wherein the construction method of the dual-expression bacteriolytic plasmid comprises the following steps:
(1) synthesizing the E gene by using the E sequence of the lytic gene of the phage PhiX174 and connecting the E gene to a pUC57 vector; designing E gene fragment forward/reverse PCR primers according to a first multiple cloning site sequence of a vector pETDuet1, and amplifying by taking the pUC57 vector connected with the E gene as a template, so that the 5 'and 3' tail ends of a PCR product respectively have a sequence and a restriction enzyme site which are consistent with the two tail ends of a linearized vector, and completing the construction of a recombinant plasmid inserted with the E gene;
(2) the sequence of the non-signal peptide extracellular region is synthesized by the sequence of the staphylococcus aureus nuclease A, the sequence is 453bp and is connected to a pUC57 vector, the second polyclonal site sequence of the vector pETDuet1 in the recombinant plasmid inserted with the E gene is inserted into a staphylococcus aureus nuclease A gene fragment, and the construction of the recombinant plasmid simultaneously inserted with the E gene and the staphylococcus aureus nuclease A gene is completed.
4. The preparation method of the double-protein expressed escherichia coli ghost according to claim 1, which is characterized by comprising the following steps:
(1) preparing a dual-expression bacteriolytic plasmid: firstly, synthesizing an E gene by using a lytic gene E sequence of a bacteriophage PhiX174 and connecting the E gene to a pUC57 vector; designing E gene fragment forward/reverse PCR primers according to a first multiple cloning site sequence of a vector pETDuet1, and amplifying by taking the pUC57 vector connected with the E gene as a template, so that the 5 'and 3' tail ends of a PCR product respectively have a sequence and a restriction enzyme site which are consistent with the two tail ends of a linearized vector, and completing the construction of a recombinant plasmid inserted with the E gene; then, a sequence of an extracellular region without a signal peptide is synthesized by using a sequence of the staphylococcus aureus nuclease A and is connected to a pUC57 vector, a second polyclonal site sequence of the vector pETDuet1 in the obtained recombinant plasmid with the inserted E gene is inserted into a gene fragment of the staphylococcus aureus nuclease A, and the construction of the recombinant plasmid with the inserted E gene and the inserted staphylococcus aureus nuclease A gene is completed.
(2) Preparing the Escherichia coli ghost with double protein expression: firstly, transforming the dual-expression bacteriolytic plasmid into host bacteria; and carrying out shake culture on the bacterial liquid of the host bacteria, adding an IPTG inducer into the bacterial liquid for induction expression when the OD600 is 0.4-0.6, sampling every 30min to measure the OD600 value, stopping shake culture when the OD600 value is not changed, and stopping induction to obtain the escherichia coli ghost with double-protein expression.
5. The method according to claim 4, wherein the host bacterium is BL21(DE3)。
6. The method according to claim 4, wherein the culture temperature of the shaking culture is 37 ℃.
7. The method of claim 4, wherein the IPTG inducer has a final concentration of 1.2mmol/L in the bacterial liquid.
8. The method according to claim 4, wherein the inactivation of the E.coli ghost expressed by the double proteins obtained by the shake culture is performed by using β -propiolactone.
9. The preparation method of claim 8, wherein the inactivation treatment comprises the steps of adding β -propiolactone to the solution of E.coli ghost with double protein expression to a final concentration of 0.025%, subjecting the solution to 37 ℃ for 1 hour, centrifuging the bacterial ghost solution, collecting the precipitate, washing the precipitate with PBS solution, resuspending the precipitate, adding β -propiolactone again to a final concentration of 0.05%, and subjecting the precipitate to 37 ℃ for 2 hours to complete the inactivation treatment.
10. The method according to claim 9, wherein the PBS solution is a PBS solution having a pH of 7.4 after sterilization.
CN201911380493.5A 2019-12-27 2019-12-27 Double-protein expressed escherichia coli ghost and preparation method thereof Pending CN110951666A (en)

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