CN109468256B - Probiotic clone strain integrating four-copy F18 pilus operon gene and double-copy F4 pilus operon gene and construction method - Google Patents
Probiotic clone strain integrating four-copy F18 pilus operon gene and double-copy F4 pilus operon gene and construction method Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a probiotic clone strain integrating a four-copy F18 pilus operon gene and a two-copy F4 pilus operon gene and a construction method thereof, which comprises the following steps: pTargetT-X::PtetF18 and pTargetT-X::PtetConstructing a F4 recombinant plasmid; by the construction of a probiotic clone strain integrating a four-copy functional F18 pilus operon gene and a double-copy functional F4 pilus operon gene, a nonresistant four-copy F18 pilus gene and double-copy F4 pilus gene compound integrated clone strain is obtained. The invention has the beneficial effects that: the recombinant strain can effectively improve the adhesion performance to porcine intestinal passage epithelial cells; the orally immunized mice were able to produce immune sera, antibodies to IgG against the F18 fimbriae and against the F4 fimbriae; the serum of the mice can obviously reduce F18 after oral immunization+And F4+Adherence of the strain to the porcine intestinal continuous cell line IPEC-J2.
Description
Technical Field
The invention relates to the field of biotechnology application, in particular to the expression of exogenous functional F18 and F4 pilin through chromosome integration and stable genetic surface display of bacteria.
Background
Escherichia coli Nissle1917 is a strain which is nonpathogenic, has no known harmful effect on hosts so far, can provide health benefits for different hosts, and is used as a main active ingredient of probiotic products which are marketed in Europe and are named as Muteflon products, and is mainly used for human intestinal health regulation. In clinical application, the probiotic Nissle1917 strain is modified as a vector of genetic engineering, and the probiotic is used for development and development of products in the aspects of vaccines, tumor treatment, health products, diagnostic preparations and the like. The EcNc clone strain is a wild strain of an Escherichia coli Nissle1917 prototype, is subjected to gene modification to remove two intracellular cryptic plasmids pMUT1 and pMUT2, has the characteristic of carrying more (large) capacity of homologous or exogenous genes than a parent strain, and is prepared and obtained in the laboratory of the applicant.
In order to achieve the overexpression production of homologous or heterologous proteins or compounds in microorganisms, overexpression of plasmids is mostly used, which is easy to manipulate and regulate expression, but which is genetically unstable. The metabolic burden of plasmid replication, antibiotic resistance genes, overexpression and other heterologous genes can lead to host cell exhaustion, yield loss and even loss of original functions of host cells. In addition, antibiotics or other selective agents must be used in order to maintain the presence of plasmids in bacterial cells, thereby increasing the cost of the overall bioprocessing and also increasing the chances of transmission of drug resistant genes. In recent years, chromosomal expression of homologous or heterologous genes in bacteria has become increasingly favored in synthetic biology and biomedicine.
The conventional molecular biology methods have certain advantages on the integration of genes on the bacterial chromosome, but have self limitations, such as difficult integration of long-fragment genes, low efficiency, high off-target rate and the like. The method used by the patent is that the integration of exogenous large fragment DNA gene fragments (the length of F4 pilus operon coding gene is about 8Kb, and the length of F18 pilus operon coding gene is about 5.6Kb) is carried out on the chromosome of a probiotic Nissle1917 plasmid-free clone strain by a CRISPR/cas9 double-plasmid system.
Post-weaning diarrhea (PWD) and Edema Disease (ED) of piglets are common diseases in the pig industry in clinic, main pathogenic bacteria are F4 and F18 pilus positive enterotoxigenic escherichia coli ETEC, and the piglets are susceptible to high mortality, low weight, slow growth, high drug treatment cost and the like due to infection and morbidity after the piglets are planted through pilus adhesion. Clinically, antibiotics are mainly used for preventing and treating the disease, but with the generation and accumulation of drug-resistant bacteria in the production of the breeding industry, new prevention and control measures are urgently needed to be researched. The constructed probiotic Nissle1917 plasmid-free clonal bacterium EcNc stably carries genetic expression and surface display of functional F4 and F18 pili, and is expected to provide a new idea and strategy for prevention and control of diarrhea of weaned piglets.
Disclosure of Invention
In order to overcome the defects, the application displays multiple copies of F18 pilus and F4(K88) pilus by utilizing nonessential gene internal integration and stable expression in the chromosome of probiotic Nissle1917 plasmid-free clone bacterium EcNc strain, and is expected to be a candidate strain of a probiotic active vaccine for resisting piglet post-weaning diarrhea and edema disease.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: the probiotic clone strain integrating the four-copy functional F18 fimbriae operon gene and the double-copy F4 fimbriae operon gene is characterized in that the probiotic clone strain is stored in the China general microbiological culture Collection center (CGMCC), the storage address is the microbial research institute of China academy of sciences No. 3 of the West Lu No.1 Hospital in the North Chen of the sunward area in Beijing, the storage date is 2018.7.2, and the storage number is CGMCC No. 16047.
The construction method of the probiotic clone strain integrating the four-copy functional F18 pilus operon gene and the double-copy F4 pilus operon gene is characterized by comprising the following steps:
1)pTargetT-X::Ptetf18 and pTargetT-X:: PtetConstructing a F4 recombinant plasmid;
2) the resistance-free four-copy F18 pilus gene and double-copy F4 pilus gene compound integrated clone is obtained by integrating four-copy functional F18 pilus operon gene and double-copy functional F4 pilus operon gene expression and displaying on the surface of the thallus of the probiotic clone.
The step 1) is specifically as follows: the primers used and the sequences of N20 containing PAM are shown in table 2 and table 3. By using
The PCR amplification reaction was performed with ultra-high fidelity DNA polymerase (Vazyme, P501) (as shown in Table 1). In the present application, X represents a target site of a specific gene on the EcNc chromosome, and the specific gene includes yjcS, cadAP, lacZ, yieN/trkD, maeB, nth/tppB.
First, a linearized pTargetF-X fragment containing the N20 sequence of target site X was reverse PCR amplified from the pTargetF plasmid DNA template using the pB032-X/pB033 primer, and the resulting linearized fragment was then used
II one-step cloning kit (Vazyme, C112) circularizing the PCR product to obtain a first circularized product, transforming the first circularized product into competent cell DH5 α to construct pTargetT-X recombinant plasmid (one for each site). XupsteamHA-F/XupsteamHA-R and XdownsteamHA-F/XdownsteamHA-R amplification primers were used to amplify upstream and downstream homology arms of target sites from Nissel 1917 genomic template.PtetFWD/ptetF 18REV-X (or ptetF4REV-X) was used to amplify DNA template with P from pBR322F4/F18DNA templatetetF4 (or F18) pilus operon gene of the promoter. IPCRpTargetF-F (HindIII)/IPCRpTargetF-R (PstI) was used to linearize pTargetT-X. Use of
The four PCR product fragments are respectively cyclized by a MultiS one-step cloning kit (Vazyme, C113) to obtain a second cyclization product, which comprises an upper homologous arm and a lower homologous arm of a target site, an F4/F18 fimbrial operon gene fragment containing a Ptet promoter and a linearized pTargetT-X fragment, and then the second cyclization product is transformed into a competent cell DH5 α to construct pTargetT-X, wherein the sequence of the cyclization connection of the four fragments is that the upper homologous arm fragment of the target site and the fragment containing P contain the P of the Ptet F18/F4 series recombinant plasmidstetF18 pilus operon gene fragment of promoter or P-containingtetAn F4 pilus operon gene fragment of the promoter, a homologous arm fragment downstream of the target site, and a linearized pTargetT-X fragment.
Step 2) in particular the procedure for the construction of the integrated recombinant strain is annotated in the attached FIG. 1. pCas plasmid is electrotransformed into EcNc to construct the EcNc/pCas recombinant strain.EcNc/pCas competent cells are prepared (EcNc/pCas competent cells are selected in the first round to be inserted into the yjcS site; in the second round, EcNc/pCas competent cells are prepared with the yjcS site inserted therein so that the latter round is reinserted into the cadAP site; in the third round, EcNc/pCas competent cells with the yjcS, cadAP site inserted therein are prepared; in the third round, 10mM of final concentration of L-arabinose is added thereto to facilitate induction of late lambda-Red homologous recombination. 50. mu. l of Nc/pCas competent cells are taken (competent cells used in the first round are non-inserted Nc/pCas competent cells; in the first round are competent cells are viable cells with no insertion into a Nc/pCas competent cell culture tube; in the first round, after incubation with the PCR-transgenic strain for about 10mM of the third round, the PCR, the third round, the PCR is inoculated into a PCR-10. the cell culture medium, the PCR is inoculated into a cell culture medium containing the PCR-10. the cell strain of the EcNc-10. the PCR-pTXtref-10. the PCR-10. the plasmid containing the plasmid DNA strain is inoculated into the cell strain-tref-10. the cell strain-tref-trex strain-tref-trex strain-trex-is inoculated into the cell line-trex-was inoculated into the cell line-trex-tretetF4down-R, the two pairs of primers, if they have a positive band, prove successful integration, plus one pair of identifying primers, YZXPtetF18up-F/YZXPtetF18down-R (or YZXPtetF4up-F/YZXPtetF4down-R), which if only a band of about 600bp appears, prove integration failure, if a band of more than 5600bp or 8000bp appears, prove integration success, then select the positive integration recombinant strain to be tested again, after obtaining the positive integration strain, further remove the resistant plasmid (i.e., pTargetT-X:: PtetF18/F4 series recombinant plasmid), put the Nc strain containing pCas and pTargetT-X:: PtetF18/F4 plasmid into about 5ml Ptkanamycin (50mg/liter) containing Pty (50mg/liter) and IPTG (0.5mM) in a single cell containing pTargetF 3526 kD, insert into a shake flask, after verifying the susceptibility of the single cell strain containing pTarvyt, insert it into a strain containing pTargetF 3 mg/26 mg of pTarT-X in a shake flask, after inserting it into a test, after inserting it into a strain containing pTarvyt-X for verification in a streak test, inserting it into a single cell containing 50 mg/26 mg of a test, inserting it into a streak test, and inserting it into a streak test, after inserting it into a streak test, inserting it under a test, inserting it under 30 mg of about 26 mg of a streak test, inserting it under a streaktetF18 pilus operon gene fragment of promoter, and P-containing inserted in maeB, nth/tppB sitetetF4 pilus operon gene fragment of promoter.
After the six sites are integrated and recombined, the pTargetT-X of the last round is removed, the PtetF18/F4 plasmid is removed, another plasmid pCas is finally removed (the plasmid is removed only after the last round is completed), the recombinant clone is placed in a non-resistant L B culture medium for passage at 42 ℃ until the EcNc recombinant clone integrating F4 and F18 in a non-resistant multicopy is finally obtained, namely, one site is integrated in each round, the integration sequence is yjcS, cadAp, lacZ, yieN/trkD, maeB and nth/tppB, the corresponding pTargetT-X is removed after each round of integration, PtetF18/F4 recombinant plasmid is removed, and the pTargetT-X of the last round is removed after the last round of integration, the PtetF18/F4 plasmid is removed, and the recombinant pCas is removed.
The invention also provides a probiotic clone strain which is obtained by the construction method and compositely integrated with the four-copy functional F18 pilus operon gene and the double-copy F4 pilus gene. Use of a probiotic clonal strain integrating four copies of a functional F18 fimbrial operon gene and two copies of a F4 fimbrial operon gene as a candidate strain for a live probiotic vaccine. The probiotic clone strain integrating four copies of functional F18 pilus operon gene and two copies of F4 pilus operon gene is used as a medicine for treating post-weaning diarrhea and edema disease of piglets.
A biological technique for inserting the exogenous gene segment in the locus of Nissle1917 plasmid-free clone EcNc bacterial chromosome genome is based on the gene editing technique using the CRISPR/cas9 dual-plasmid system, and the biological technique for constructing the recombinant plasmid pTargetT-X:: PtetF18/K88 based on the gene editing technique using the CRISPR/cas9 dual-plasmid system, which is based on the recombination plasmid pTargetT-X-plasmid in the CRISPR/cas9 dual-plasmid system, the recombinant plasmid pTargetT-38 system pTargetT-plasmid pTartPtetF-18/Kgsp, linearization and multi-segment splicing cloning DNA recombination technique, and the recombination of pTargetT-38 system pTargetT-plasmid pTargetPTT-X-18/Kncr 88, the recombinant plasmid DNA for integrating the gene sequence of pTargetDNA-DNA-ORF 6326, the recombinant plasmid DNA-ORF-26, the recombinant plasmid-DNA-sequence, and the recombinant plasmid-DNA-sequence of the recombinant plasmid, and the recombinant plasmid-DNA-sequence of the recombinant plasmid, and the recombinant plasmid, including the gene sequence of the recombinant plasmid, and the gene sequence of the recombinant+Resistance, followed by the next round (next site) of integration insertion assay. When the six sites are integrated and recombined, the pTargetT-X of the last round is removed, and after PtetF18/K88 plasmid is subcultured at 42 ℃ finally to eliminate pCas substancePlasmid (temperature sensitive plasmid) until the recombinant strain loses kanamycin Kan+Resistant, becoming a recombinant strain without resistant multiple copies of the integrated F4/F18 fimbrial operon genes.
The invention utilizes the characteristic that the CRISPR/cas9 double-plasmid system has the high-efficiency editing of bacterial genome (including integration insertion, knockout and point mutation), and the CRISPR/cas9 double-plasmid system has a mechanism that self-plasmid is easy to remove and no resistance gene is remained. The construction of the escherichia coli probiotics Nissle1917 plasmid-free clone bacterium EcNc stably carrying genetic expression F4 and F18 pili does not influence the self biological properties of the host bacterium EcNc, and the preparation of the recombinant integration strain is expected to become the construction of anti-F4+And F18+The preparation method of the probiotic live vaccine candidate strain for post-weaning diarrhea and edema disease of piglets caused by the strain realizes the preparation of the novel probiotic live vaccine candidate strain.
Compared with the prior art, the invention has the beneficial effects that: 1. the original plasmid of the Nissle1917 probiotics is removed, no new exogenous plasmid is introduced, and no resistance gene exists. 2. The recombinant strain does not need to use antibiotics to maintain the expression of the exogenous gene for a long time. 3. After multiple passages, the exogenous pilin (F4 and F18 pilus) is still stably and persistently expressed. 4. The successful construction of the candidate strain of the live vaccine does not affect the known biological characteristics of the probiotic host bacteria, such as growth performance and the like. 5. Compared with parent strains, the recombinant strain can effectively improve the adhesion performance to porcine intestinal passage epithelial cells (IPEC-J2 and IPEC-1). 6. Orally immunized mice produce immune sera, antibodies to IgG of F18 and F4 fimbriae. 7. The serum of the mice can obviously reduce F18 after oral immunization+And F4+Adherence of strains (e.g., pig farm field isolate 8516 and virulent strain 3030-2) to the porcine intestinal passaged epithelial cell line IPEC-J2.
Drawings
FIG. 1 is a schematic diagram of the construction of a probiotic EcNc integrated recombinant strain integrating multiple copies of the F4/F18 pilus-encoding operon genes;
FIG. 2 is a immunoblot of multiple copies of the integrated recombinant bacteria anti-FaeG and anti-F18;
m protein Standard marker, EcNc-E.coli Nissle1917 with two recessive plasmids deleted, MIS-multiple integration Strain, EcNc. DELTA. yjcS:: PtetF18ΔcadAp::PtetF18ΔlacZ::PtetF18ΔyieN/trkD::PtetF18ΔmaeB::PtetF4Δnth/tppB::PtetF4, wherein the primary antibody used in the left figure is FaeG resistant murine monoclonal antibody, and the primary antibody used in the right figure is F18 anti-pilus murine polyclonal antiserum;
FIG. 3 is a graph showing the results of an in vitro adhesion experiment of a multi-copy integrated recombinant bacterium;
EcNc E.coli Nissle1917 with two recessive plasmids deleted, MIS multiple integration Strain, EcNc. DELTA. yjcS:: PtetF18ΔcadAp::PtetF18ΔlacZ::PtetF18ΔyieN/trkD::PtetF18ΔmaeB::PtetF4Δnth/tppB::PtetF4; the graph shows the percentage change in the ability of the integrants to adhere to porcine-derived, intestinal passage epithelial cells relative to the EcNc strain, with the cells used in the left panel being IPEC-J2 cells; the cells used in the right panel are IPEC-J2 cells;
FIG. 4 is a graph showing the results of indirect E L ISA antibody titer detection;
EcNc-E.coli Nissle1917 with two recessive plasmids deleted, EcNc. DELTA. yjcS:: PtetF18ΔcadAp::PtetF18ΔlacZ::PtetF18ΔyieN/trkD::PtetF18ΔmaeB::PtetF4Δnth/tppB::PtetF4；
Orally immunizing BA L B/c mice with age of 8 weeks twice by EcNc and MIS recombinant strains respectively at two weeks, collecting and separating obtained immune serum to perform indirect E L ISA test, and testing and verifying the change of antibody titer of the mice in the test against F4 (main subunit FaeG protein) and F18 pilus;
FIG. 5 shows the results of in vitro cell adhesion inhibition experiments;
EcNc-E.coli Nissle1917 with two recessive plasmids deleted, EcNc. DELTA. yjcS:: PtetF18ΔcadAp::PtetF18ΔlacZ::PtetF18ΔyieN/trkD::PtetF18ΔmaeB::PtetF4Δnth/tppB::PtetF4, orally immunizing BA L B/c mice with age of 8 weeks twice by EcNc and MIS recombinant strains respectively at two-week intervals,the obtained immune serum tests verify that the immune serum is F4/F18+The adhesion inhibiting activity of ETEC on porcine intestinal cells;
the graph shows the percentage change of inhibition of serum of an immune mouse orally integrated with recombinant bacteria on ETEC-adhered porcine intestinal epithelial cells relative to EcNc strains, the cells used in the left graph are IPEC-J2 cells, and the ETEC bacteria used in the left graph are pathogenic F4 positive escherichia coli strains 3030-2; the cells used in the right panel are IPEC-J2 cells, and the ETEC bacteria used are pathogenic F18 positive E.coli strain 8516;
the probiotic clone strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is the microbiological research institute of China academy of sciences No. 3 of North West Lu No.1 of the Korean district in Beijing, the preservation date is 2018.7.2, and the preservation number is CGMCC No. 16047; classified and named as Escherichia coli.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The famous probiotic escherichia coli Nissle1917 (EcN) is a nonpathogenic escherichia coli probiotic that has been shown to confer health benefits on different hosts, and escherichia coli Nissle1917 is a nonpathogenic strain that confers health benefits on different hosts without finding known adverse effects on the host, and is used primarily for human gut health regulation as the major active ingredient of the products marketed in europe under the name Mutaflor. The famous probiotic escherichia coli Nissle1917 (EcNc) is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is microorganism institute of China academy of sciences, No. 3, West Lu No.1 Hospital, North American district, Beijing, the preservation date is 2018.7.2, and the preservation number is CGMCC No. 16045.
In the experiment, the probiotic Nissle1917 plasmid-free clone EcNc is used as a vaccine vector to construct recombinant probiotics for stably expressing F4/F18 pili, and the recombinant probiotics are used as a daily feed additive to immunize piglets to control postweaning diarrhea (PWD) of the piglets. The F4/F18 pilus integrated recombinant probiotic can be specifically bound to an F4/F18 pilus receptor of porcine intestinal cellsPreferentially occupy the receptor site and induce mucosal immunity against F4 and F18, thereby blocking the pathogenic bacteria F4/F18+The adhesion of ETEC to the intestinal tract of the piglets effectively and directly prevents the infection of diarrhea and edema disease of the weaned piglets.
1、pTargetT-X::PtetConstruction of F18/F4 recombinant plasmid
The primers used and the sequences of N20 containing PAM are shown in table 2 and table 3. By using
The ultrahigh fidelity DNA polymerase (Vazyme, P501) was used for PCR amplification (the reaction system and procedure are shown in Table 1).
TABLE 1 PCR amplification reaction System during plasmid construction
The PCR cycle parameters were: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10sec, annealing at 45-72 ℃ (depending on the primer) for 30sec, extension at 72 ℃ for 25sec/kb (depending on the PCR product length), 35 cycles, and final extension at 72 ℃ for 10 min. After the PCR reaction is finished, the product is subjected to agarose gel electrophoresis at the concentration of 1.0-1.5% (depending on the length of the amplified product fragment), and then gel cutting is carried out to recover and purify the corresponding PCR product for further experiments.
In the present application, X represents a target site of a specific gene on the EcNc chromosome, and the specific gene includes yjcS, cadAP, lacZ, yieN/trkD, maeB, nth/tppB.
First, a linearized pTargetF-X fragment containing the N20 sequence of target site X was reverse PCR-amplified from the pTargetF plasmid DNA template using the pB032-X/pB033 primer (the PCR reaction system and PCR cycle parameters were as described in Table 1 above), and the resulting linearized fragment was subsequently used
II one-step cloning kit (Vazyme, C112) circularization of the PCR product, transformation into competent cell DH5 α to construct pTargetT-X recombinant plasmids XuppesamHA-F/XuppesamHA-R and XdowncaseamHA-F/XThe downsteadha-R amplification primers were used to amplify the upstream and downstream homology arms of the target site from the Nissle1917 genomic template (PCR reaction system and PCR cycling parameters were performed as described in table 1 above). Use of ptetFWD/ptetF18REV-X (or ptetF4REV-X) for amplification of P-bearing DNA from pBR322F4/F18DNA templatetetThe F4 (or F18) pilus operon gene of the promoter (PCR reaction system and PCR cycle parameters were performed as shown in Table 1 above). IPCRpTargetF-F (HindIII)/IPCRpTargetF-R (PstI) was used to linearize pTargetT-X (PCR reaction system and PCR cycle parameters were as described in Table 1 above). Use of
The four PCR product fragments were circularized separately by the MultiS one-step cloning kit (Vazyme, C113) and included homology arms upstream and downstream of the target site, F4/F18 fimbrial operon gene fragment containing Ptet promoter, linearized pTargetT-X fragment, and transformed into competent cell DH5 α to construct pTargetT-X:: PtetF18/F4 series recombinant plasmid.
2. Construction of multicopy F4/F18 Probiotics EcNc integration Strain
Procedures for integration of recombinant Strain construction see FIG. 1 for comments, pCas plasmid electrotransformation into EcNc to construct EcNc/pCas recombinant strains preparation of EcNc/pCas competence, a final concentration of 10mM of L-arabinose was added to it to aid induction of late lambda-Red homologous recombination.50. mu.l of competent cells were taken and mixed with about 100ng of pTargetT-X:PtetF 18/F4 series DNA, the mixture was placed in a 0.1-cm electrode cup (Bio-Rad) for 1.8kV electrotransformation, the click product was placed in a 1.5ml finger and incubated with 1ml of frozen L B medium for about 5min, followed by shake recovery at 30 ℃ for 1 hour, followed by plating on L B plates containing kanamycin (50mg/liter) and spectinomycin (50mg/liter) and overnight incubation at 30 ℃ of coloniestetF18up-F/YZXptetF18up-R and YZxptetF18down-F/YZXptetF18down-R (or YZXP)tetF4up-F/YZXPtetF4up-R and YZXPtetF4down-F/YZXPtetF4down-R), if the PCR result of the two pairs of primers has a positive band, the PCR result proves thatThe integration is successful; an additional pair of identifying primers, YZXPtetF18up-F/YZXPtetF18down-R (or YZXP)tetF4up-F/YZXPtetF4down-R), if the PCR product of the primer has a band of only about 600bp, the integration is proved to be failed, and if the PCR product has a band of more than 5600bp or 8000bp, the integration is proved to be successful), and then a positive integration recombinant strain is selected to be sequenced and identified again.
After obtaining positive integration strains, the resistant plasmids were further removed EcNc strain containing pCas and pTargetT-X:: PtetF18/F4 plasmid was grown in about 5ml of L B medium containing kanamycin (50mg/liter) and IPTG (0.5mM) at 30 ℃ for longer than 14 hours on a shaker, after which a few colonies were picked up and streaked on L B plate containing kanamycin (50mg/liter), and the grown single colonies were streaked on spectinomycin (50mg/liter) L B plate to verify its sensitivity to spectinomycin, which confirmed removal of pTargetT-X:: PtetF18/F4 plasmid, followed by the next round (next site) of integration insertion test.
After the integration and recombination of all six sites, pTargetT-X was removed, and after the PtetF18/F4 plasmid, another plasmid pCas was finally removed, the recombinant clone was passaged in a medium of L B without resistance at 42 ℃ until finally an EcNc recombinant clone integrating multiple copies of F4 and F18 without resistance was obtained.
The primers in Table 2 are SEQ ID Nos. 1 to 64 from top to bottom. The primers in Table 3 are SEQ ID Nos. 65 to 70 from top to bottom.
TABLE 2 primers used in this experiment
Remarking: x represents a target site on the EcNc chromosome, such as yjcS, cadAP, lacZ, yieN/trkD, maeB, nth/tppB.
TABLE 3 EcNc chromosomal target site sequences used in this assay plus PAM three bases
Remarking: the PAM three bases are the last three bases of the four sequences.
FIG. 2 is an immunoblot of multicopy complex integration of recombinant bacteria MIS anti-F18 and anti-F4. The F18 pilus and the F4 pilus can be displayed on the recombinant probiotic EcNc in a functional expression way through a glass plate agglutination test and a Western blot test. In a standard immunoblot assay, the F4 strain FaeG major subunit and F18 fimbriae subunit FedC expressed in the EcNc recombinant integrate strain (protein samples prepared by boiling whole bacteria) were detected and recognized by F4 monoclonal antibody hybridoma supernatant (36/41,1:300 dilution) and mouse anti-F18 multi-antiserum (1: 500 dilution) antibodies. Boiled whole-mycoprotein samples of EcNc strain, pathogenic ETEC3030-2 strain and 8516 strain were used as negative control, positive control of F4 and positive control of F18, respectively. M, size marker; EcNc Escherichia coli Nissle1917, nth, EcNc. DELTA. nth/tppB: PtetF 4; MIS, multiple integration strain, EcNc Δ yjcS: PtetF18 Δ pcadA: PtetF18 Δ lacZ: PtetF18 Δ yieN/trkD: PtetF18 Δ maeB: PtetF4 Δ nth/tpp B: PtetF 4.
FIG. 3 is a graph of the results of in vitro adhesion experiments of four-copy integrated recombinant bacteria MIS. The graph shows the percentage change in the ability of four copies of integrant MIS to adhere to porcine-derived, intestinal passaged epithelial cells relative to the EcNc strain, using IPEC-J2 and IPEC-1 cells. As can be seen from fig. 3, the adhesion ability of recombinant probiotics to the porcine intestinal passage epithelial cells IPEC-J2 and IPEC-1 was significantly improved (140.23% ± 12.12% (. x., P ═ 0.0067), 183.19% ± 27.92% (. P ═ 0.0063), respectively) relative to EcNc to porcine intestinal passage epithelial cells IPEC-J2 and IPEC-1 (100 ± 5.852%, 100 ± 9.821%), further demonstrating that the recombinant bacteria were able to express surface functionality and displayed F18 and F4 pili. In fig. 3, the data are presented as Standard Error (SEM) of the mean of three replicates.
The antibody titers of F18 and F4 expressed by the recombinant bacteria (which are excellent in immunogenicity) can be detected by performing animal experiments by using a BA L B/c mouse model, so that the antibody titers of the F18 and F4 pilus IgG can be obtained, the anti-F18 and F4 expressed by the recombinant bacteria can be detected, the anti-F4 (FaeG) antibody titer of the MIS immune group is 2.141 +/-0.130 (the anti-F4 (FaeG) antibody titer of the EcNc group is 0.925 +/-0.409 (the difference is significant, P0.0220)), and the anti-F18 antibody titer of the immune MIS immune group is 1.8525 antibody titer of the EcNc group is 0.972 +/-0.273 (the anti-F890.273 of the EcNc group is 0.890.273).
FIG. 5 shows the results of in vitro cell adhesion inhibition experiments, in which EcNc and four copies of integrated recombinant bacterium MIS were orally immunized twice, respectively, at two weeks intervals, to 8-week-old BA L B/c mice, and the obtained immune sera were tested to verify that they had pili F18+And F4+The adhesion inhibiting activity of ETEC on porcine intestinal cells. The graph shows that the percentage of inhibition of sera of mice immunized with orally integrated recombinant bacteria against porcine intestinal epithelial cells by ETEC3030-2 and 107/86 strains varied by 54.01. + -. 6.96% and 68.71. + -. 7.76% respectively, relative to EcNc strain (100. + -. 11.23% and 100. + -. 7.07%), the cells used in the left panel were IPEC-J2 cells, and the ETEC bacteria used were pathogenic F4 pilus positive E.coli strain 3030-2; the right picture uses IPEC-J2 cell as cell, uses ETEC bacteria as pathogenic F18 positive colibacillus strain 8516, and uses in vitro cytostatic test to prove that the immune mouse serum can obviously inhibit F18+And F4+Adherence of wild species (e.g., pig farm field isolates 8516 and 3030-2) to porcine-derived intestinal passaged epithelial cells IPEC-J2.
The invention is based on the clear background of the bacterial genome sequence of a probiotic Nissle1917 plasmid-free clone EcNc (two cryptic plasmids pMUT1 and pMUT2 of the bacteria are deleted), and the genome sequence is selectedUsing the CRISPR/cas9 two-plasmid system to construct the CRISPR/cas9 recombinant plasmid pTargetT-X containing E.coli F18 or F4 pilus operon gene with insertion site homology arms at both sides, PtetF18/F4. Further, the recombinant plasmid pTargetT-X is expressed in the specification PtetThe recombinant Nissle1917/pCas host bacteria expressing cas9 protein is transformed into F18/F4, the recombinant plasmid pTargetT-X is combined with the recombinant plasmid pTargetT-X in view of expressing cas9 protein on pCas plasmid, homologous recombination is carried out by using sgRNA targeting to guide the specific cutting target site of cas9 cutting enzyme and homologous recombinase (Gam, Exo and Bet) activity on PtetF18/F4, the integration of F18 or F4 pilus operon gene into the corresponding site of EcNc genome is completed, IPTG induction on pCas plasmid is initiated by IPTG induction dependent promoter (Ptrc), thereby initiating the targeting on pTargetT-X:: PtaretF 18/573K 5 downstream of PtarGetF promoter, the sgRNA (sgRG 1) of pMB1 replicon is initiated on pTargetT promoter, the replication initiation site on pMB 18/88 is initiated by Western plasmid, the efficient cutting of pTarGetT-T plasmid, the recombinant plasmid pTarT-K is initiated by PCR, the PCR is carried out after the transfection, the bacterial strain is cultured, the anti-adhesion gene is tested, the anti-adhesion gene is eliminated, the anti-adhesion gene of the recombinant plasmid of mouse-adhesion gene after the strain is tested, the anti-adhesion gene is tested, the+And F4+Adherence of strains (such as porcine field isolate 8516 and virulent strain 3030-2) to porcine intestinal passage cells IPEC-J2, which is importantThe preparation of the group integration strain is expected to become a method for constructing a probiotic live vaccine candidate strain for resisting piglet post-weaning diarrhea and edema disease.
The foregoing has described the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is intended to be covered by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Yangzhou university
<120> probiotic clone strain integrating four-copy F18 pilus operon gene and double-copy F4 pilus operon gene, and construction method thereof
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<141>2018-11-26
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actagtatta tacctaggac tgagctagct gtcaag 36
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ttctctagag tcgacctgca gctcgttccg ctcacacttc 40
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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ccaacaattt tgttcgcagt ttagaattac tgtatctcga aaacaat 47
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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cagggtaata gatctaagct taaagctgcc gccgattagc cc 42
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<212>DNA
<213>Escherichia coli
<400>28
ttttagccac cagcaagaaa ttagaattac tgtatctcga aaacaat 47
<210>29
<211>42
<212>DNA
<213>Escherichia coli
<400>29
ttctctagag tcgacctgca gatggcccgt gcgccaatga tc 42
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<211>41
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<213>Escherichia coli
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ataatggttt cttagacgtc gcgcttcggc gctttgcgag c 41
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<212>DNA
<213>Escherichia coli
<400>31
gccactcagg aactggtaac g 21
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<211>42
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
<400>36
aacactgcat tcggctggcc g 21
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<213>Escherichia coli
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<213>Escherichia coli
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<212>DNA
<213>Escherichia coli
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<213>Escherichia coli
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<210>41
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<212>DNA
<213>Escherichia coli
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<211>20
<212>DNA
<213>Escherichia coli
<400>42
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<213>Escherichia coli
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tatttcatgt attaatttgc tg 22
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<211>21
<212>DNA
<213>Escherichia coli
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attgttttcg agatacagta a 21
<210>45
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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cgtttacctg cattgcctt 19
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<213>Escherichia coli
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<211>21
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<213>Escherichia coli
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<213>Escherichia coli
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<213>Escherichia coli
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ggatatgtgg ggtaacgacg cgg 23
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<213>Escherichia coli
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Claims (7)
1. The probiotic clone strain integrating the four-copy functional F18 fimbriae operon gene and the double-copy F4 fimbriae operon gene is characterized in that the probiotic clone strain is stored in the China general microbiological culture Collection center (CGMCC), the storage address is the microbial research institute of China academy of sciences No. 3 of the West Lu No.1 Hospital in the North Chen of the sunward area in Beijing, the storage date is 2018.7.2, and the storage number is CGMCC No. 16047.
2. The probiotic clonal strain integrating four copies of a functional F18 pilus operon gene and two copies of a F4 pilus operon gene according to claim 1, wherein the construction method of the clonal strain comprises the following steps:
1）pTargetT-X::Ptetf18 and pTargetT-X::PtetConstructing a F4 recombinant plasmid;
2) the resistance-free four-copy F18 pilus gene and double-copy F4 pilus gene compound integrated clone is obtained by integrating four-copy functional F18 pilus operon gene and double-copy functional F4 pilus operon gene expression and displaying on the surface of the thallus of the probiotic clone.
3. The probiotic clonal strain integrating four copies of a functional F18 pilus operon gene and two copies of a F4 pilus operon gene according to claim 2, wherein in the construction method of the clonal strain, the step 1) is specifically as follows: by using pB032-XReverse PCR amplification of primers for amplification of plasmid containing target sites from pTargetF DNA templateXpTargetF-of the N20 sequence of (9)XLinearizing the fragment, followed by circularization of the resulting linearized fragment to obtain a first circularized product, which is transformed into competent cells DH5 α to construct pTargetT-XRecombinant plasmids;XupsteamHA-F/Xupteamha-R andXdownsteamHA-F/Xthe downsteadHA-R amplification primers were used to amplify the upstream and downstream homology arms of the target site from the Nissle1917 genomic template; ptetFWD/ptetF18REV-XOr ptetF4REV-XThe amplification primers were used to amplify DNA from pBR322-F18 or pBR322-F4Amplification of the band P in the platetetF18 or F4 pilus operon genes of the promoter; IPCRpTargetF-F (HindIII)/IPCRpTargetF-R (PstI) for linearizing pTargetT-XA plasmid; using Clonexpress®The MultiS one-step cloning kit includes the upstream and downstream homology arms of the target site and PtetF18 or F4 pilus operon gene fragment of promoter, linearized pTargetT-XThe fragments are circularized together to give a second circularized product which is subsequently transformed into competent cell DH5 α to construct pTargetT-X::PtetF18 or pTargetT-X::PtetF4 series recombinant plasmids;Xrepresents a target site of a specific gene on the EcNc chromosomeyjcS，cadAp，lacZ，yieN/trkD，maeB，nth/tppB(ii) a In the second cyclization product, the cyclization and connection arrangement sequence of the four fragments is as follows: target site upstream homology arm fragment containing PtetF18 pilus operon gene fragment of promoter or P-containingtetF4 pilus operon gene fragment of promoter, homologous arm fragment of target site downstream, and linearized pTargetT-XAnd (3) fragment.
4. The probiotic clonal strain integrating four copies of a functional F18 pilus operon gene and two copies of a F4 pilus operon gene according to claim 2, wherein in the construction method of the clonal strain, the step 2) is specifically as follows: the pCas plasmid was electro-transformed into the probiotic EcNc to construct EcNc/pCas recombinant strains; preparing EcNc/pCas competent cells, and taking EcNc/pCas competent cells and pTargetT-X::PtetF18 or pTargetT-X::PtetF4 recombinant plasmid, for competent cells and pTargetT-X::PtetF18 or pTargetT-X::PtetElectrically converting the mixture of F4 recombinant plasmid, culturing the electric shock product in frozen L B culture medium, recovering in 30 deg.C shaking table, coating it on L B plate containing kanamycin and spectinomycin, culturing at 30 deg.C overnight, selecting the suspicious positive clone growing on resistant plate, colony PCR identification, if the band greater than 5600bp shows that F18 is successfully integrated, and if the band greater than 8000bp shows that F4 is successfully integrated, sequencing and mixingDetermining whether a positive single copy F18 or F4 integrate strain is obtained; and removing the resistant plasmid to finally obtain a non-resistant four-copy F18 fimbrial gene and double-copy F4 fimbrial gene composite integrated clone strain.
5. The probiotic clonal strain integrating four copies of a functional F18 pilus operon gene and two copies of a functional F4 pilus operon gene as claimed in claim 4, wherein said clonal strain is constructed by a method in which the resistance plasmid is removed as follows: will contain pCas and pTargetT-X::PtetF18 or pTargetT-X::PtetThe EcNc strain of F4 plasmid was cultured in L B medium containing kanamycin and 0.5mM IPTG for 14-18 hours at 30 ℃ on a shaker, after which a little bacterial liquid was picked up and streaked on L B plate containing kanamycin, and the grown single colony was streaked on L B plate containing spectinomycin to verify its sensitivity to spectinomycin, thereby verifying whether pTargetT-X::PtetF18 or pTargetT-X::PtetF4 resistance plasmid; removal of pTargetT-X::PtetF18 or pTargetT-X::PtetAfter the F4 plasmid, another plasmid pCas was removed and the EcNc strain was passaged at 42 ℃ in non-resistant L B medium until a non-resistant four-copy F18 fimbriae gene and a two-copy functional F4 fimbriae operon gene integrated clone were finally obtained.
6. Use of a probiotic clonal strain of claim 1 integrating four copies of a functional F18 pilus operon gene and two copies of a functional F4 pilus operon gene as candidate strains for a live probiotic vaccine.
7. Use of the probiotic clonal strain integrating four copies of a functional F18 pilus operon gene and two copies of a functional F4 pilus operon gene according to claim 1 for the preparation of a biological preparation for the prevention and treatment of post-weaning diarrhea and edema disease in piglets.
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