CN112111438A - Preparation method of Salmonella indiana ghost - Google Patents
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Abstract
The invention discloses a preparation method of Salmonella indiana ghost, which comprises the steps of respectively connecting Phi X174 phage lysis gene E with a gene sequence of SEQ ID NO.1 and Staphylococcus aureus nuclease A with a gene sequence of SEQ ID NO.2 into a pTCV vector, constructing a temperature-controlled double-gene expression bacteriolysis plasmid pTCK03, converting Salmonella indiana, and heating to induce expression to obtain the Salmonella indiana ghost. The method can effectively improve the cracking efficiency of the Salmonella indiana ghost and thoroughly degrade genetic materials, and is an ideal preparation method of the Salmonella indiana ghost.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to a preparation method of salmonella indiana ghost.
Background
The Salmonella Indiana is a serotype which is widely popular in China in recent years, and the pathogen has a high prevalence rate in food-borne animals, raw meat food and breeding environments at present, becomes a popular serotype which is second to Salmonella enteritidis, and has attracted high attention in the fields of livestock breeding, food safety, public health and the like.
A great deal of research shows that clinical antibacterial drugs cannot effectively control the spread of pathogens due to the fact that epidemic strains of the Salmonella indiana generally have high drug resistance, and therefore other prevention and control technologies need to be developed. The vaccine is an important tool for preventing and controlling infectious diseases, but among various reported salmonella vaccines, inactivated whole-bacterium vaccine and subunit vaccine have good safety and can also stimulate strong antibody reaction, but the cellular immune reaction is weak, and only partial protection can be provided for the alimentary tract permanent planting, systemic transmission and the like of pathogeny; although live vaccines can replicate, colonize, invade the digestive tract and internal organs of a host and induce a strong immune response, the potential risk of back mutation and spread cannot be avoided. Therefore, it is very important to find a new safe and effective vaccine preparation method for the prevention and control of Salmonella indiana.
Bacterial ghosts are a novel inactive vaccine which is emerging in recent years, and the principle of the vaccine is that gram-negative bacteria generate bacterial empty shells through the controlled expression of a PhiX174 bacteriophage lytic gene E. The expression of the lysis gene E of the PhiX174 phage can fuse the inner and outer membranes of bacteria to form a transmembrane channel with the diameter of about 40-200nm, so that nucleic acid, ribosome or other cytoplasmic contents are lost under the action of osmotic pressure, and all cell surface structures and components which are the same as those of live bacteria are completely reserved, thereby having good immunogenicity and being capable of inducing an organism to generate stronger immune response. In addition, the bacterial ghosts are easy to produce and store, and can be used as excellent delivery vectors of nucleic acids, antigens and medicaments.
At present, some reports about preparation and application of salmonella ghosts are available, and the advantages of salmonella ghosts compared with traditional inactivated vaccines are proved again in experiments, but no research report about preparation of salmonella indiana ghost is available so far, and compared with the development trend of ghost vaccines, the preparation of salmonella ghosts at present still needs to be improved: the vectors are single-expression temperature control systems based on pR starting Phi X174 phage lytic gene E, and have the advantages of large volume, unfavorable transformation, low action efficiency and overlong induction time; ② lack of residual genome detection, has potential gene transfer risk. According to the research, staphylococcus aureus nuclease A is introduced into the research on preparation of the salmonella indiana ghost, a temperature control double expression vector for respectively starting a Phi X174 phage lytic gene E and a staphylococcus aureus nuclease A gene based on pR/pL is constructed, codon optimization is carried out on the Phi X174 phage lytic gene E and the staphylococcus aureus nuclease A, the bacteriolysis efficiency of the staphylococcus indiana salmonella is improved, and the preparation of a high-efficiency safe ghost vaccine is further realized.
Disclosure of Invention
The invention aims to provide a preparation method of the Salmonella indiana ghost, which can effectively improve the cracking efficiency of the Salmonella indiana ghost and thoroughly degrade genetic materials.
In order to realize the purpose, the invention provides a preparation method of the salmonella indiana ghost, which is characterized in that Phi X174 phage lytic gene E with a gene sequence of SEQ ID NO.1 and staphylococcus aureus nuclease A with a gene sequence of SEQ ID NO.2 are respectively connected into a pTCV vector to construct a temperature-controlled double-gene expression bacteriolytic plasmid pTCK03, and after the salmonella indiana is transformed, the salmonella indiana ghost is obtained through temperature rise induction expression.
In a preferred embodiment of the invention, the temperature-controlled bacteriolytic plasmid pTCK03 is transformed into Salmonella indiana, then inoculated into LB liquid medium containing chloramphenicol, shake-cultured until OD600nm reaches 0.3-0.4, heated to 40-44 ℃ to induce expression, and CaCl is added at the same time2And MgCl2And continuously culturing for 3-4 h to prepare the Salmonella indiana ghost.
In a preferred embodiment of the invention, the Salmonella indiana ghost has a lysis rate of more than 99.9%.
The invention also relates to the Indiana salmonella ghost prepared by the preparation method and application of the Indiana salmonella ghost in preparation of vaccines or medicines for preventing or treating Indiana salmonella infection
The invention also relates to a gene optimization sequence of the Phi X174 phage lytic gene E based on the codon preference of Salmonella indiana, which is characterized in that the nucleotide sequence is shown as SEQ ID NO.1 in the sequence table.
On the other hand, the invention relates to a gene optimization sequence of staphylococcus aureus nuclease A based on the codon preference of salmonella indiana, which is characterized in that the nucleotide sequence is shown as SEQ ID NO.2 in a sequence table.
The invention also relates to a temperature-controlled lysis plasmid pTCK03 of Salmonella indiana, which is characterized in that pFLX107 original plasmid is transformed into a temperature-controlled double-gene expression vector from a temperature-controlled single-gene expression vector, and then the optimized sequence of the Phi X174 phage lysis gene E and the optimized sequence of the staphylococcus aureus nuclease A are respectively connected to construct the pTCK03 temperature-controlled lysis plasmid.
The invention discloses a gene optimization sequence of Phi X174 phage lytic gene E and staphylococcus aureus nuclease A based on Salmonella indiana codon preference and a high-efficiency temperature-control bacteriolytic plasmid of Salmonella indiana for the first time, lays a foundation for preparing high-efficiency safe bacterial ghosts of Salmonella indiana, and provides an idea for developing other pathogenic bacterial ghosts.
Drawings
FIG. 1 is a physical map of cleavage plasmids pTCK 01-pTCK 03.
FIG. 2 is a bacteriolytic curve of recombinant Salmonella indiana S1105.
FIG. 3A is a pTCV/S1105 total genomic electrophoretic analysis.
FIG. 3B shows the total genomic electrophoretic analysis of pTCK 01/S1105.
FIG. 3C is a total genomic electrophoretic analysis of pTCK 02/S1105.
FIG. 3D is a total genomic electrophoretic analysis of pTCK 03/S1105.
FIG. 4A is a scanning electron microscope observation of Salmonella indiana prior to induction of the lysoplasmid.
FIG. 4B is a scanning electron microscope observation of Salmonella indiana after plasmid induction.
FIG. 4C is a transmission electron microscope observation of Salmonella indiana before plasmid induction.
FIG. 4D is a transmission electron microscope observation of Salmonella indiana before plasmid induction.
Detailed Description
The following examples are further detailed descriptions of the present invention.
Example 1 codon optimization of PhiX174 phage lytic Gene E and Staphylococcus aureus nuclease A
According to the known complete genome sequence of the Salmonella indiana in a GenBank database, the codon preference of the Salmonella indiana is counted and analyzed, and the gene sequence of the Phi X174 phage lytic gene E and the Staphylococcus aureus nuclease A is subjected to codon optimization through gene design, so that the codon use frequency of the gene and the host is matched to improve the protein expression level. The Phi X174 phage lytic gene E and the staphylococcus aureus nuclease A gene sequences after codon optimization are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Example 2 construction of lysis plasmid containing PhiX174 phage lytic Gene E and Staphylococcus aureus nuclease A
Compared with the fusion expression effect of staphylococcus aureus nuclease A and different exogenous DNA fragments, the biological engineering report 2016,32(12): 1654-1663) is used as a template, a combined terminator rrnBT1T2 in the pFLX107 plasmid is replaced by a single strong terminator rrnBT2 by a conventional molecular biological means, and then another single strong terminator rrnBT2 is inserted between enzyme cutting sites PstI and SacI in the plasmid to construct a plasmid pTCV containing two single strong terminators rrnBT1 and rrnBT 2.
Using pBV220-E plasmid (Fu, L.and C.Lu.A Novel double Vector coexpression PhiX174 Lysis E Gene and staphylococcus Nuclease A Gene on the Basis of Lambda Promoter pR and pL, reproduction quality.molecular Biotechnology,2013,54(2): 436. 444.), the PhiX174 phage Lysis Gene E sequence was PCR-amplified, ligated into pTCV plasmid after double digestion identification, E.coli DH 5. alpha. was transformed by heat shock and spread on LB plate containing chloramphenicol, and pTCK01 (FIG. 1) expressing PhiX174 phage Lysis Gene E was constructed after PCR identification and sequencing verification.
Further using pKF396M-4 plasmid (Fu, L.and C.Lu.A Novel double Vector coexpression PhiX174 lysine E Gene and staphylococcus Nuclease A Gene on the Basis of Lambda Promoter pR and pL, representational. molecular Biotechnology,2013,54(2): 436:. 444.) as a template, the left Promoter and staphylococcus aureus Nuclease A Gene sequences were PCR-amplified, ligated into pTCK01 plasmid after double digestion identification, and pTCK02 plasmid expressing PhiX174 phage lytic Gene E and staphylococcus aureus Nuclease A was constructed after PCR identification and sequencing verification (FIG. 1).
The codon-optimized Phi X174 phage lytic gene E and the gene sequence of staphylococcus aureus nuclease A are synthesized by the committee Saimer Feishhl science and technology (China) Co., Ltd.), and after PCR amplification and enzyme digestion identification, pTCK02 plasmid is sequentially connected to replace the original Phi X174 phage lytic gene E and staphylococcus aureus nuclease A gene to construct pTCK03 plasmid (figure 1).
Example 3 preparation of Salmonella Indiana ghost
The pTCK03 bacteriolytic plasmid was transformed into a Salmonella indiana strain, which was then inoculated into LB liquid medium containing chloramphenicol (35. mu.g/mL), followed by shaking culture at 28 ℃. When the OD600nm of the culture solution reaches 0.3-0.4, the temperature is increased to 42 ℃ to induce the expression of the Phi X174 phage lytic gene E and the staphylococcus aureus nuclease A gene. Simultaneously adding CaCl into the culture solution2And MgCl2The final concentrations were 10mM and 1mM, respectively, to maximize the activity of the Staphylococcus aureus nuclease A gene. And culturing for 3-4 h to prepare the Salmonella indiana ghost.
Example 4 recombinant bacteriolysis kinetics test
Plasmids pTCV, pTCK01, pTCK02 and pTCK03 were each separately electroporated into the strain of Salmonella Indiana S1105 (GONGJ, ZENG X, ZHANG P, et al. Characterisation of the engineering multidrug-resistant Salmonella enterica serovar Indiana strains in China. emery Microbes infection, 2019,8(1):29-39.) and inoculated into 100mL of LB liquid medium containing chloramphenicol and cultured with shaking at 28 ℃. When the OD600nm of the culture solution reaches 0.3-0.4, the temperature is increased to 42 ℃ to induce the expression of the Phi X174 phage lytic gene E and the staphylococcus aureus nuclease A gene. Adding CaCl into the culture solution2And MgCl2The final concentrations were 10mM and 1mM, respectively. The cultures were sampled at regular intervals for OD600nm and monitored for bacterial growth. And (5) properly diluting the bacterial liquid after 0, 2h and 4h of induction, counting the viable bacteria on a flat plate, and calculating the bacteriolysis efficiency. The calculation formula is as follows: the bacteriolysis rate is (1-cfu after induction/cfu before induction) x 100%.
After the recombinant bacteria containing pTCK 01-pTCK 03 plasmids are heated to 42 ℃ and induced for 30min, OD600nm rises slowly and then shows a descending trend, wherein the OD600nm value of pTCK01/S1105 rises slowly after 90min reaches the minimum after induction, the OD600nm values of pTCK02/S1105 and pTCK03/S1105 reach the minimum after 120min after induction and keep stable, and the OD600nm value of pTCK03/S1105 is lower. The OD600nm value of pTCV/S1105 as a control continued to increase throughout the experiment (FIG. 2).
The counting result of the viable bacteria on the flat plate basically conforms to the trend of the bacteriolysis curve. The viable count of pTCK01/S1105 also began to increase slowly after undergoing a drop, the lysis rates of 2h and 4h were 99.84% and 98.52%, respectively, with significant differences (p <0.05), while pTCK02/S1105 and pTCK03/S1105 induced no significant difference in lysis rates of 2h and 4h, respectively, but pTCK03/S1105 had a significantly higher lysis rate than pTCK01/S1105 and pTCK02/S1105(p < 0.05). The lysis rate (99.52%) of pTCK02/S1105 induced for 2h was lower than that of pTCK01/S1105 at the same induction time, but there was no significant difference, while the lysis rate (99.29%) after 4h was significantly higher than that of pTCK01/S1105 at the same time (p < 0.05). The viable cell count of pTCV/S1105 as a control continued to increase, and the viable cell counts after 2h and 4h were 9.12 and 18.95 times, respectively, before induction (Table 1). The bacteriolysis rate of pTCK03/S1105 is shown to be significantly higher than that of pTCK01/S1105 and pTCK02/S1105(p <0.05) by bacteriolysis kinetic monitoring.
TABLE 1 lysis efficiency of recombinant Salmonella indiana S1105
Example 5 recombinant bacterium genome electrophoresis detection
The recombinant bacteria were subjected to total genomic extraction using a DNA Mini Kit (Beijing Jepitm Biotechnology Co., Ltd.) with 1mL of each of the bacterial culture solutions taken from the initiation of induction at 42 ℃ and every 1h thereafter, and then analyzed by 0.8% agarose electrophoresis to confirm the degrading activity of Staphylococcus aureus nuclease A on the Salmonella indiana genome. Agarose electrophoresis results show that the pTCV/S1105 and pTCK01/S1105 genomes can be detected in the whole experiment period, but the genome concentration of the pTCV/S1105 and the genome concentration of the pTCK01/S1105 can be continuously increased as seen from the light and dark intensity of the electrophoresis bands (FIG. 3A), while the genome concentration of the pTCK01/S1105 genes is reduced after 1-3h of temperature rise induction than before induction, and is increased again after 4-5h (FIG. 3B); in the presence of calcium and magnesium ions, the genome of pTCK02/S1105 and pTCK03/S1105 was degraded after 1h of induction, but the former still showed degradation of residual fragments, while the latter degraded nucleic acid fragments more thoroughly (FIG. 3C, FIG. 3D). The above results indicate that the nucleic acid fragment of pTCK03/S1105 genome was degraded most completely.
Example 6 Ex.Indiana ghost Electron microscopy
Washing the bacterial ghosts with PBS, then suspending the bacterial ghosts in 2.5% glutaraldehyde, fixing the bacterial ghosts at 4 ℃ overnight, washing the bacterial ghosts with PBS for 3 times daily, 10min each time, then fixing the bacterial ghosts in 1% osmium tetroxide for 1.5h, washing the bacterial ghosts with PBS for 3 times, dehydrating the bacterial ghosts with ethanol step by step (50%, 70%, 80%, 90%, 100%) and replacing the dehydrated bacterial ghosts with isoamyl acetate for 20min each time, then drying the bacterial ghosts at a CO2 critical point, adhering the bacterial ghosts to a platform and spraying gold, and finally observing the bacterial ghosts under a scanning electron. The results show that the external morphology of ghost cells is similar to that of non-lysed cells, but the surface morphology is significantly shrunken, and transmembrane lysis channels are visible at the center or both poles of the cell surface (fig. 4A, 4B).
And (3) sequentially soaking the bacterial ghosts dehydrated step by step in acetone/resin with the ratio of 1:1 to 1:3 for 1-2 h each time, then embedding the bacterial ghosts in pure resin, polymerizing the bacterial ghosts at 60 ℃ for 48h, preparing ultrathin sections, dyeing the ultrathin sections with uranium acetate and lead citrate, and finally observing the ultrathin sections under a transmission electron microscope. It was shown that the cytoplasmic contents of ghosts were released leaving only the bacterial ghost, and that the unlysed cells were filled and filled to a full extent, with a clear cytoplasmic content being visible (FIGS. 4C, 4D).
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
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<120> preparation method of salmonella indiana ghost
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gctctgaatc tgcgtaaaac cctgctgatg gcttcttctg ttcgtctgaa acctctgaat 180
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gcttttacca aaaaaatggt tgaaaacgct aaaaaaattg aagttgaatt tgataaaggt 240
cagcgtaccg ataaatatgg tcgtggtctg gcttatattt atgctgatgg taaaatggtt 300
aacgaagctc tggttcgtca gggtctggct aaagttgctt atgtttataa accgaacaac 360
acccatgaac agctgctgcg taaatctgaa gctcaggcta aaaaagaaaa actgaacatt 420
tggtctgaag ataacgctga ttctggtcag taa 453
Claims (8)
1. A preparation method of Salmonella indiana ghost is characterized in that Phi X174 phage lysis gene E with a gene sequence of SEQ ID No.1 and Staphylococcus aureus nuclease A with a gene sequence of SEQ ID No.2 are respectively connected into a pTCV vector to construct a temperature-controlled double-gene expression bacteriolysis plasmid pTCK03, and after Salmonella indiana is transformed, the Salmonella indiana ghost is obtained through heating induction expression.
2. The preparation method of claim 1, wherein the temperature-controlled lysis plasmid pTCK03 is transformed into Salmonella indiana, inoculated into LB liquid medium containing chloramphenicol, shake-cultured until OD600nm reaches 0.3-0.4, heated to 40-44 ℃ for induction expression, and CaCl is added2And MgCl2And continuously culturing for 3-4 h to prepare the Salmonella indiana ghost.
3. The method of claim 1, wherein the Salmonella indiana ghost has a lysis rate of greater than 99.9%.
4. Salmonella indiana ghost prepared by the preparation method according to any one of claims 1 to 3.
5. Use of the Salmonella indiana ghost of claim 4 in the manufacture of a vaccine or medicament for the prevention or treatment of Salmonella indiana infection.
6. A gene optimization sequence of Phi X174 phage lytic gene E based on Salmonella indiana codon preference is characterized in that the nucleotide sequence is shown as SEQ ID NO.1 in a sequence table.
7. A gene optimization sequence of staphylococcus aureus nuclease A based on the codon preference of Salmonella indiana is characterized in that the nucleotide sequence is shown as SEQ ID NO.2 in a sequence table.
8. A temperature-controlled lysis plasmid pTCK03 of Salmonella indiana is characterized in that a pFLX107 original plasmid is transformed into a temperature-controlled double-gene expression vector from a temperature-controlled single-gene expression vector, and then the optimized sequence of the Phi X174 phage lysis gene E of claim 6 and the optimized sequence of the staphylococcus aureus nuclease A of claim 7 are respectively connected to construct the pTCK03 temperature-controlled lysis plasmid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115044598A (en) * | 2022-06-22 | 2022-09-13 | 华南理工大学 | Efficient preparation method of bacterial ghost of enterobacteriaceae |
| CN117106626A (en) * | 2023-07-03 | 2023-11-24 | 江苏省家禽科学研究所 | Indiana salmonella Sincyz and application thereof |
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