CN116350762A - DeltatrpD mycobacterium smegmatis live vaccine for targeted inactivation of mycobacterium tuberculosis and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of tuberculosis immunotherapy, and in particular relates to a delta trpD mycobacterium smegmatis live vaccine for targeted inactivation of mycobacterium tuberculosis, and a preparation method and application thereof. The recombinant live vaccine taking the mycobacterium smegmatis with the trpD gene knocked out as a vector and simultaneously carrying three lysozyme gene coexpression vectors of tubercle bacillus phages is preserved with the number of CCTCC NO: C20221941. The preparation method comprises the following steps: preparing a trpD gene knockout mycobacterium smegmatis; constructing a recombinant eukaryotic coexpression system for phage lyase gene coexpression; the coexpression vector is introduced into the trpD gene knockout mycobacterium smegmatis to obtain the recombinant live vaccine. The vaccine can not survive in macrophages, and after the vaccine is cracked and dead, an Mtb phage lysozyme gene coexpression system is released into cells to translate three tubercle bacillus targeting lysozyme proteins. Achieving the purpose of targeted treatment of active tuberculosis and latent tuberculosis caused by proliferation tuberculosis bacteria, dormant tuberculosis bacteria and drug-resistant tuberculosis bacteria.

Description

DeltatrpD mycobacterium smegmatis live vaccine for targeted inactivation of mycobacterium tuberculosis and preparation method and application thereof

Technical Field

The invention belongs to the field of tuberculosis immunotherapy, and in particular relates to a delta trpD mycobacterium smegmatis live vaccine for targeted inactivation of mycobacterium tuberculosis, and a preparation method and application thereof.

Background

Current drugs for killing mycobacterium tuberculosis are mainly antibiotics, but with the occurrence of drug resistance problems of pathogenic bacteria to most antibiotics, humans are gradually entering the "post-antibiotic" age where no drugs are available. At present, along with the spread of multi-drug-resistant and widely-drug-resistant tuberculosis strains, the conventional effect of killing the mycobacterium tuberculosis is very little, and the conventional antitubercular drugs such as isoniazid, rifampicin, fluoroquinolones and the like all act in a mode of inhibiting DNA replication or cell wall synthesis of active mycobacterium tuberculosis, so that the traditional antitubercular drugs have obvious effect of killing drug-sensitive tubercle bacillus in a proliferation state, but have very little or no effect on bacteria in a non-proliferation state and drug-resistant tubercle bacillus which are hidden in cells. The search for new anti-mycobacterium tuberculosis drugs and protocols is urgent.

The phage has high specificity to bacterial invasion, natural performance of natural schizomycete and tracking sterilization, and no obvious toxicity to animals and plants. Phages have attracted considerable attention from scientists as biological agents with their unique antimicrobial advantage. Infectious disease authority-nobel prize master Lederberg doctor pointed out: because of the emergence of drug-resistant bacteria, antibiotic therapy is no longer as effective as before, and the study of phages as antibacterial therapy should be highly appreciated.

Mtb-specific phage D29 is a virulent phage isolated from soil and has a genome of double-stranded DNA (GenBank accession No. AF022214) containing 49,136 bp. In vitro and in vivo studies demonstrated: d29 has strong affinity and cleavage to Mtb (including standard strain H37Rv and clinically isolated Mtb), and the resulting lyase has important potential value for the treatment of tuberculosis. For the common G+ bacteria, only the lyase LysnA can efficiently lyse peptidoglycan to damage cell walls and kill bacteria. For Mtb with a special cell wall structure, phage D29 needs to completely dissolve the cell wall of Mtb, and not only lysin a is needed to cleave peptidoglycan, but also another lyase lysin b is needed to break the connection of mycolic acid and arabinogalactan, so as to achieve the effect of completely breaking the cell wall of Mtb. Ordinary bacterial cell walls generally die after disruption, but Mtb is an intracellular parasitic bacterium, and some of the cell wall defect Mtb (cell wall deficient forms, CWD-form or L-form) remaining in the body of a latently infected person may still survive for a long period of time. Holin is another lytic factor encoded by phage, and the target site is in the bacterial cell membrane, and can be specifically inserted into the cytoplasmic membrane to form a hole to kill bacteria.

The phage can be used for killing intracellular mycobacterium tuberculosis, and is effective even under low pH and anoxic conditions, and research shows that the active phage has obvious bactericidal effect on bacteria in the propagation period and is effective on bacteria in the stationary period and drug-resistant bacteria. Current phage therapy has just emerged and most focus on the in vitro treatment of skin and mucosal surface infections. Therefore, the current whole phage therapy is difficult to achieve in vivo targeted drug delivery and does not easily reach the interior of macrophages; the whole bacterium has stronger immunogenicity, and can generate neutralizing antibodies after multiple in vivo applications, so that the phage loses the therapeutic effect.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a multi-target specific lyase recombinant therapeutic live vaccine which has gene therapy and immunotherapy effects, is used for targeted delivery and expression of a multi-target specific lyase recombinant therapeutic live vaccine aiming at Mtb: the problems existing in the prior art are cooperatively overcome from the two aspects of targeted killing bacteria and enhancing host immunity.

In order to achieve the above purpose, the present invention adopts the following technical scheme: the DeltpD mycobacterium smegmatis live vaccine for target inactivation of mycobacterium tuberculosis takes the trpD mycobacterium smegmatis with knocked out genes as a vector, and simultaneously carries recombinant live vaccines of three lysozyme gene coexpression vectors of tubercle bacillus phages, wherein the preservation number is CCTCC NO: C20221941, and the recombinant live vaccines are classified and named: mycobacterium smegmatis 2022YFG,Mycobacterium smegmatis 2022YFG. Preservation unit: china center for type culture Collection, address: the preservation date of the university of Wuhan in China is 2022, 12 months and 12 days.

Further, the three lysozyme genes are LysinA, lysinB and Holin gene fragments.

A method for preparing a delta trpD mycobacterium smegmatis live vaccine of targeted inactivated mycobacterium tuberculosis, the method comprising:

step 1: preparing a trpD gene knockout mycobacterium smegmatis;

step 2: constructing a recombinant eukaryotic coexpression system for phage lyase gene coexpression;

step 3: the phage lyase gene coexpression vector is introduced into trpD gene knockout mycobacterium smegmatis to obtain the recombinant live vaccine.

Further, the step 1 specifically includes:

(1) Constructing a delta trpD deletion gene homologous exchange site, and integrating the site into the genome of a tool phage phiE 159 for knocking out mycobacteria genes to obtain a phagemid;

(2) Introducing phagemid into engineering bacteria, and carrying out in vitro amplification to obtain recombinant phage with high titer, and carrying out transfection on mycobacterium smegmatis;

(3) Coating transfected mycobacterium smegmatis on hygromycin-resistant solid culture medium, culturing for 2-3 days at 37 ℃, picking up monoclonal, and verifying by a PCR mode;

(4) The hygromycin resistance gene is eliminated by electrotransformation of the pJH532 plasmid into the verified correct gene knockout strain, and the gene knockout strain without resistance is obtained.

Further, the step 2 specifically includes:

(1) When the primer sequence is designed, proper enzyme cutting sites are introduced, and an OriM replicon gene fragment, a LysnA gene fragment, a LysnB gene fragment and a Holin gene fragment are obtained by using a PCR amplification technology;

(2) Inserting the properly sequenced OriM replicon gene fragment into NheI cleavage site of pBudCE4.1 plasmid to obtain vector pZM0Y;

(3) Splicing the Lysin B and the Holin gene segment by using a hydrophobic glycine linker coding sequence to obtain a Lysin B-link-Holin fusion gene segment;

(4) The obtained LysinA, lysinB-link-Holin fusion gene fragment is respectively connected into eukaryotic expression plasmids pcDNA3.1 to generate eukaryotic expression vectors pZFY-1 and pZFY-2, and the eukaryotic expression vectors are introduced into Top10 to select ampicillin resistance colonies for double enzyme digestion and sequencing identification;

(5) And (3) carrying out double digestion on plasmids pZFY-1 and pZFY-2, recovering and sequencing to identify correct LysnA genes and LysnB-link-Holin gene fragments, and sequentially subcloning the LysnA genes and the LysnB-link-Holin gene fragments into the obtained pZM0Y vector, wherein the LysnA is inserted between XhoI and KpnI, and the LysnB-link-Holin is inserted between HindIII and BamHI, so as to obtain a phage lyase gene coexpression vector pZM0A.

Further, the step 3 specifically includes:

(1) Washing and counting the cultured trpD gene knocked-out mycobacterium smegmatis, adding the washed and counted mycobacterium smegmatis into Mphi, and observing the Mphi targeting of the bacterium;

(2) The plasmid pZM0A is electrically transformed into trpD gene knocked out mycobacterium smegmatis, recombinant bacteria with Zeocin resistance are obtained through screening, and a large amount of recombinant mycobacterium therapeutic live vaccines are cultured and amplified.

Further, the primer of the LysnA gene segment, the LysnB gene segment and the Holin gene segment are respectively:

LysnA upstream primer: 5'-GGGATCCGATATCATGGCCGAGGACCA-3', which is SEQ ID NO. 1 sequence;

LysnA downstream primer: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 2 sequence;

lysin B upstream primer: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is SEQ ID NO. 3 sequence;

lysin B downstream primer: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 4 sequence;

holin upstream primer: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is SEQ ID NO. 5 sequence;

holin downstream primer: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 6 sequence.

Use of a Δtrpd mycobacterium smegmatis live vaccine targeted to inactivate mycobacterium tuberculosis for non-therapeutic in vivo or in vitro inactivation of mycobacterium tuberculosis.

Further, the step of inactivating the mycobacterium tuberculosis in vitro comprises the steps of:

introducing the DeltatrpD mycobacterium smegmatis live vaccine into macrophages, observing transfection efficiency by a fluorescence microscope, and respectively detecting the expression of LysinA, lysinB and Holin by an RT-PCR method; acid-fast staining to observe the sterilization condition of pZM0A to Mtb within M phi, and cell lysis and bacterial culture counting are carried out; hoechst staining observes the influence of pZM0A on macrophages, and at the same time, electron microscopy observes whether ultrastructural changes exist, including the change states of mitochondria, cell nuclei, endoplasmic reticulum and golgi apparatus.

Further, the step of inactivating the mycobacterium tuberculosis in vivo comprises:

(1) Animal modeling: a model of latent infection and active tuberculosis infection in mice;

(2) The DeltatrpD mycobacterium smegmatis live vaccine was nasally administered to mice, 5X 106CFU,0.1 ml/time, once a week for four weeks;

(3) Each experimental group is killed in 1 st month, 3 rd month and 5 th month after the first administration, left lung and partial spleen tissues are taken under the aseptic condition to be accurately weighed, ground, diluted by a multiple ratio and inoculated on an improved Rogowski culture medium plate, and colony count is carried out after culture for 6 weeks at 37 ℃; 4% paraformaldehyde fixation of lung tissues, paraffin embedding and slicing, and HE staining and observation; PPD-specific spleen lymphocyte proliferation assay; after the PPD stimulation, the mouse spleen lymphocytes are secreted into the culture supernatant and the cytokine content in the mouse blood is quantitatively detected by ELISA.

The Deltpd mycobacterium smegmatis live vaccine for targeted inactivation of mycobacterium tuberculosis, and the preparation method and application thereof have the advantages that:

the recombinant live vaccine takes mycobacterium smegmatis with trpD gene knockout (delta trpD) as a vector and simultaneously carries three lysozyme gene coexpression vectors of tubercle bacillus (Mtb) phage. In the vaccine, live carrier mycobacterium smegmatis has good macrophage targeting (macrophages are main infection and parasitic cells of tubercle bacillus), after entering the macrophages, delta trpD mycobacterium smegmatis cannot survive in the macrophages due to the deletion of trpD genes, so that the delta trpD mycobacterium smegmatis is cracked and dead, and an Mtb phage lysozyme gene co-expression system carried by the delta trpD mycobacterium smegmatis cannot be released into cells, 3 lysozyme proteins LysinA/LysinB-link-Holin with good tubercle bacillus targeting can be translated in the macrophages, and can act on different structural sites of tubercle bacillus respectively; meanwhile, after bacterial lysis and death, the host immunity can be enhanced. Thereby achieving the aim of targeted treatment of active tuberculosis and latent tuberculosis caused by the proliferation tuberculosis bacteria, dormant tuberculosis bacteria and drug-resistant tuberculosis bacteria.

Drawings

FIG. 1A shows the PCR amplification results of Lysin A, lysin B and Holin according to the present invention (1:LysinA;2:DNA Marker;3:LysinB;4:Holin);

FIG. 1B is a diagram showing the electrophoresis of the recombinant plasmids pZFY1, pZFYB and pZFYH (B) for double digestion (5: double digestion of pZFY1; double digestion of pZFYB; double digestion of 7: pZFYH; 8: DNA Marker);

FIG. 2 is a sequence diagram of Lysin A (A), lysin B (B) and Holin C genes according to the embodiment of the invention;

FIG. 3A is a PCR identification chart of ZFY-2 according to an embodiment of the present invention (1: lysin B-link-Holin;2: DNA Marker);

FIG. 3B is a diagram of the dual cleavage assay according to an embodiment of the present invention (3: dual cleavage of pZFY-2; 4: DNA Marker);

FIG. 4 is a schematic diagram showing the construction of a Lysin A/Lysin B-linker-Holin eukaryotic coexpression plasmid pZM0A according to the embodiment of the invention;

FIG. 5A is a PCR identification chart of pZM0A according to an embodiment of the present invention (1, lysiA; 2:OriM;3, lysiB+Holin);

FIG. 5B is a graph showing OriM sequencing results according to an embodiment of the present invention;

FIG. 6 is a graph showing the results of pZM0A and double digestion (1: empty plasmid; 2: lysnA double digestion; 3: lysnB+Holin double digestion; 4: oriM double digestion; 5: DNA Marker) according to the embodiment of the present invention;

FIG. 7A shows transfection efficiency according to an embodiment of the present invention;

FIG. 7B shows the expression detection of Lysin A, lysin B and Holin in Mphi according to the embodiment of the present invention (1:Lysin A;2:Lysin B;3:Holin;4:DNA Marker);

FIG. 8A is a graph showing the bactericidal effect of acid fast staining for detection of pZM0A on intracellular Mtb according to an embodiment of the invention (a: infection control group; b: pZM0A treatment group);

FIG. 8B is a graph showing the bactericidal effect of cultured colony count assay pZM0A on intracellular Mtb (a: infection control group; B: pZM0A treatment group) according to the example of the present invention;

FIG. 9A is a Hoechst staining pattern of the effect of pZM0A on normal macrophages according to an embodiment of the invention;

FIG. 9B is an ultrastructural view of the effect of pZM0A on normal macrophages according to an embodiment of the present invention;

FIG. 10 is a graph showing the mode of action of the three lyase systems of the present invention for cleavage of Mtb;

FIG. 11 shows M.phi.targeting for detection of Mycobacteria by acid fast staining in accordance with an embodiment of the present invention;

FIG. 12A is a graph showing the therapeutic effect of a live vaccine (a: infection control group; b: live vaccine treatment group) on tuberculosis by pulmonary HE staining test according to an embodiment of the present invention;

FIG. 12B is a graph showing the therapeutic effect of live vaccine on tuberculosis (a: infection control group; B: live vaccine treatment group) according to the embodiment of the present invention;

FIG. 13A is a graph showing the proliferation activity of spleen lymphocytes according to an embodiment of the present invention;

FIG. 13B is a graph showing cytokine detection according to an embodiment of the present invention;

FIG. 14A is a diagram of lung tissue of a healthy mouse in physiological saline group according to an embodiment of the present invention;

FIG. 14B is a lung tissue map of a healthy mouse of a live vaccine group according to an embodiment of the present invention.

Detailed Description

The following description of the embodiments is provided in conjunction with the accompanying drawings.

The reagents and apparatus in the examples below were conventional experimental reagents and apparatus.

Example 1:

a delta trpD mycobacterium smegmatis live vaccine of targeted inactivated mycobacterium tuberculosis takes the mycobacterium smegmatis with the trpD gene knocked out as a carrier, and simultaneously carries recombinant live vaccines of three lysozyme gene coexpression carriers of tubercle bacillus phages, and the preservation number is CCTCC NO: C20221941. The three lysozyme genes are LysinA, lysinB and Holin gene fragments.

A method for preparing a delta trpD mycobacterium smegmatis live vaccine of targeted inactivated mycobacterium tuberculosis, the method comprising:

preparation of Mycobacterium smegmatis with trpD gene knockout (DeltatrpD):

the delta trpD mycobacterium smegmatis gene knockout technical process comprises the following steps: firstly, constructing delta trpD deletion gene homologous exchange site (AES), and then integrating the delta trpD deletion gene homologous exchange site into the genome of a tool phage phiE 159 for knocking out mycobacteria genes to obtain phagemid (phasmid); introducing phagemid into engineering bacteria, obtaining recombinant phage with high titer through in vitro amplification, and transfecting mycobacterium smegmatis ATCC 607; the transfected mycobacterium smegmatis is coated on a hygromycin-resistant solid culture medium, cultured for 2-3 days at 37 ℃, and monoclonal is selected and verified by means of PCR and the like. Finally, the hygromycin resistance gene is eliminated by electrotransformation of pJH532 plasmid to verify the correct gene knockout strain, and the gene knockout strain without resistance is obtained.

When the live vaccine is used for treating tuberculosis, after the live vaccine targets to enter macrophages of a host, mycobacterium smegmatis cannot survive in host cells due to trpD gene knockout, so that a recombinant eukaryotic coexpression system for coexpression of the carried phage lyase genes is released; however, when tryptophan can be externally supplied during in vitro culture, the DeltatrpD mycobacterium smegmatis can be rapidly propagated in a large quantity in a liquid culture medium.

2. Obtaining of recombinant eukaryotic Co-expression System pBud-LysnA/LysnB-link-Holin (designated pZM 0A) for phage lyase Gene Co-expression:

(1) Construction of eukaryotic expression vectors pZFY-1 and pZFY-2 for expressing Mtb phage LysnA and LysnB-link-Holin respectively

D29 phage genomic DNA was extracted according to the instructions of the viral genomic DNA extraction kit (Dalian TaKaRa). The upstream primer and the downstream primer are designed according to the D29 phage LysinA, lysinB registered by GenBank and the Holin coding sequence, and are synthesized by Beijing three-blog radix Polygalae biological technology Co.

LysnA upstream: 5'-GGGATCCGATATCATGGCCGAGGACCA-3', which is SEQ ID NO. 1 sequence;

downstream of LysinA: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 2 sequence;

lysin B upstream: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is SEQ ID NO. 3 sequence;

downstream of LysinB: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 4 sequence;

holin upstream: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is SEQ ID NO. 5 sequence;

downstream of Holin: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 6 sequence;

and performing PCR reaction by taking the genomic DNA of the D29 phage as a template to obtain LysinA, lysinB and Holin genes. After three gene fragments were recovered from the PCR product, lysinA was digested with the endonucleases EcoRV/HindIII and LysinB was digested with EcoRV/HindIII, holin was digested with EcoRV/HindIII (purchased from Daida TaKaRa, also purchased from Shenzhen Crystal Mei, UK NEB, promega, U.S.A.), and cloned into the vectors pcDNA3.1 digested with the corresponding endonucleases (purchased from Novagen) to generate eukaryotic expression vectors pZFY1 (pcDNA3.1-LysinA), pZFYB (pcDNA3.1-LysinB) and pZFYH (pcDNA3.1-Holin), respectively. pZFY1, pZFYB and pZFYH are respectively introduced into Top10, and ampicillin resistant colonies are picked for double digestion and sequencing identification. And (3) connecting and sequencing correct LysnB and Holin by a link sequence by using a recombinant PCR method, connecting the obtained LysnB-link-Holin gene into eukaryotic expression plasmid pcDNA3.1 to generate pZFY-2, and identifying whether the pZFY-2 is successfully constructed by using a PCR technology, double digestion and sequencing.

Results: the LysinA, lysinB and Holin gene fragments were successfully amplified using PCR techniques (FIG. 1A); the constructed vectors pZFY1, pZFYB and pZFYH are completely correct through double enzyme digestion identification (figure 1B); the bidirectional sequencing result shows that the amplified LysinA gene (figure 2A), lysinB gene (figure 2B) and Holin gene (figure 2C) have completely correct sequences; the PCR results (FIG. 3A) and the double digestion assay results (FIG. 3B) of pZFY-2 indicate successful construction.

Conclusion: successful construction of eukaryotic expression vectors pZFY-1 (pcDNA3.1-LysnA) and pZFY-2

(pcDNA3.1-LysinB-link-Holin)。

(2) Construction of phage LysnA/LysnB-link-Holin eukaryotic Co-expression plasmid pZM0A, main framework construction schematic (FIG. 4)

The Mtb genome is used as a template, and the PCR amplification technology is used to obtain the OriM replicon gene fragment.

OriM upstream: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is SEQ ID NO. 7 sequence;

downstream of OriM: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 8 sequence;

the properly sequenced OriM replicon gene fragment was inserted into the NheI cleavage site of the pBudCE4.1 plasmid to obtain the vector pZM0Y, and double cleavage and sequencing were performed to determine whether the construction of pZM0Y was successful. The pZFY-1 was digested with the endonuclease EcoRV/HindIII and the pZFY-2 was digested with EcoRV/HindIII, and the LysnA gene and the LysnB-link-Holin gene fragment were recovered, respectively, and subcloned into pZM0Y in sequence (LysnA was inserted between HindIII and BamHI; lysnB-link-Holin gene fragment was inserted between XhoI and KpnI) to yield pZM0A. And (5) carrying out double digestion and PCR to determine whether the pZM0A is successfully constructed.

Results: FIG. 5A is a PCR identification map of pZM0A (1, lysin A;2: oriM;3, lysin B+Holin); FIG. 5B is a diagram of OriM sequencing results; the PCR technology is applied to successfully amplify the OriM replicon gene fragment; the double enzyme digestion identification result shows that pZM0Y is successfully constructed; sequencing results show that the amplified OriM replicon gene sequence is completely correct.

FIG. 6 is a graph showing the results of pZM0A and double digestion (1: empty plasmid; 2: lysin A double digestion; 3: lysin B+Holin double digestion; 4: oriM double digestion; 5: DNA Marker); the PCR identification and the double enzyme digestion identification are successful in constructing pZM0A.

Conclusion: the eukaryotic co-expression vector pZM0A carrying the mycobacterial replicon OriM gene fragment was successfully constructed.

3. The phage lyase gene coexpression vector is introduced into trpD gene knockout mycobacterium smegmatis to obtain the recombinant live vaccine.

The pZM0A is electrically transformed into the trpD gene knockout mycobacterium smegmatis, and the trpD gene knockout (delta trpD) mycobacterium smegmatis is propagated in an in vitro liquid culture medium (supplemented with tryptophan), so that a large amount of low-cost amplification of the recombinant therapeutic live vaccine in bacteria is realized.

Example 2:

use of a Δtrpd mycobacterium smegmatis live vaccine targeted to inactivate mycobacterium tuberculosis for non-therapeutic in vitro inactivation of mycobacterium tuberculosis.

In vitro cell layer experiments detect the delivery and expression of therapeutic genes, the killing effect on Mtb in macrophages and the safety of host cells by the gene recombinant live vaccine.

Respectively introducing pZM0A into cultured macrophages by using liposome, observing transfection efficiency by using a fluorescence microscope, and respectively detecting LysinA, lysinB and Holin expression by using methods such as RT-PCR; acid-fast staining to observe the sterilization condition of pZM0A to Mtb within M phi, and cell lysis and bacterial culture counting are carried out; hoechst staining observes the influence of pZM0A on macrophages, and at the same time, electron microscopy observes whether ultrastructural changes exist, including the changing states of mitochondria, cell nuclei, endoplasmic reticulum, golgi apparatus and the like.

Results: the transfection efficiency was detected by introducing pZM0A into the cultured mouse macrophage cell line RAW264.7 with liposomes and reached more than 75% (FIG. 7A); the expression of LysinA, lysinB and Holin genes in cells was detected (fig. 7B); pZM0A has a pronounced bactericidal effect on intracellular infection Mtb (FIGS. 8A, 8B); the Hoechst staining (FIG. 9A) and electron microscope observation (FIG. 9B) results show that pZM0A has no obvious toxic or side effect on normal macrophages (FIG. 9), and the sterilization mechanism is schematically shown in FIG. 10.

The invention utilizes the synergy of gene therapy and immunotherapy, namely the gene therapy of Mtb obligate multiple lyase and the immunotherapy of a targeting vector

Gene therapy of Mtb ampholytic multiple lyase: the nature of phage specific lysis to kill host bacteria is that specific phage lyase (LysinA, lysinB, holin) is relied on, and the invention combines the 3 genes to carry out intracellular expression so as to directly kill Mtb of intracellular infection; second, immunotherapy with live vector delivering the above gene therapy drug: mycobacterium smegmatis with trpD gene knockout (delta trpD) carries mycobacteria common antigen as a cellular immune adjuvant, and can effectively perform secondary immune excitation protection on organisms infected with Mtb, play an immune treatment function and cooperatively kill the intracellular Mtb by cooperating with therapeutic genes.

The sterilization mechanism is irrelevant to whether the target Mtb is a multi-drug resistant strain or not, and whether the target Mtb is proliferation and dormant, so that the problems of intracellular latency, multi-drug resistance, reburning, recurrence and the like in tuberculosis prevention and treatment are hopeful to be solved.

Multiple targeting sterilization strategy-targeting of drug delivery carrier and targeting of direct sterilization drug are combined ingeniously

First is targeting of the delivery vehicle: the mycobacterium smegmatis with trpD gene knockout (delta trpD) is used as a vector to realize targeting. The requirement for a new generation of gene therapy vectors is that they should have good safety, targeting and cheapness. Mycobacterium smegmatis itself is a normal flora of the human body and has good macrophage targeting. The mycobacterium smegmatis delivery system is able to target bactericidal plasmids well into macrophages (the primary infected cells of Mtb).

Second, targeting of the multi-linked bactericidal lyase: the 3 lyases target different structural parts of the bacteria to be destroyed respectively and kill Mtb in a combined way. The main obstacle of the direct treatment of intracellular bacterial infection by conventional phages is poor targeting, and meanwhile, a human body can generate phage neutralizing antibodies, so that the treatment is ineffective; the invention targets the expression of Mtb phage lysis functional protein in M phi cells, greatly reduces the molecular weight and the contact of the molecular weight with an immune system, and bypasses the barriers, thereby opening up an idea for treating a plurality of refractory bacterial infections by utilizing natural phage, and having important research value and scientific significance.

In a word, the recombinant live vaccine constructed by the invention can safely target and can efficiently and specifically kill the intracellular Mtb, and has great significance in the research on how to remove the latent multi-drug resistant Mtb. The method can radically cure the tuberculosis patients and LTBI patients, not only can reduce the chance of developing into active tuberculosis, but also can eliminate potential infectious sources, and has very important scientific significance and application prospect for treating and controlling the tuberculosis; meanwhile, a brand new idea is created for exploring and solving the troublesome problems of intracellular latent infection, multi-drug resistance and the like of other pathogenic bacteria; the subject is also aimed at providing a brand new idea and beneficial exploration for applying the novel phage biological preparation to the treatment of intracellular infectious diseases.

Example 3:

use of a Δtrpd mycobacterium smegmatis live vaccine targeted to inactivate mycobacterium tuberculosis for non-therapeutic in vivo inactivation of mycobacterium tuberculosis.

Animal modeling: a model of latent infection and active tuberculosis infection in mice;

animal experiments evaluate the therapeutic effect of the recombinant therapeutic live vaccine: recombinant mycobacterium smegmatis: nasal drip (5X 106CFU,0.1 ml/time), once a week for four weeks of treatment;

treatment and overall treatment effect detection:

each experimental group was sacrificed at 1 month, 3 months and 5 months after the first treatment, and the following treatments were performed: accurately weighing left lung and part of spleen tissues under a sterile condition, grinding, diluting by a multiple ratio, inoculating to a modified Rogowski-Qin culture medium plate, and culturing at 37 ℃ for 6 weeks to count colonies; the pathological effect is as follows: 4% paraformaldehyde fixation of lung tissues, paraffin embedding and slicing, and HE staining and observation; PPD-specific spleen lymphocyte proliferation assay (MTT method); after the PPD stimulation, the mouse spleen lymphocytes are secreted into the culture supernatant and the cytokine content in the mouse blood is quantitatively detected by ELISA.

Results: mycobacterium smegmatis has a pronounced Mphi targeting (FIG. 11); the lung HE staining (fig. 12A) and colony count (fig. 12B) showed a significant decrease in lung load for the recombinant mycobacterium smegmatis live vaccine treatment group compared to the infection control group; MTT method results show that the treatment group has higher spleen lymphocyte proliferation activity than the infection group (figure 13A), and the cell culture supernatant and the cytokines INF-gamma and TNF-alpha in serum have obviously increased content (figure 13B); the recombinant live vaccine had no obvious toxic side effects on mice (fig. 14).

Conclusion: the recombinant mycobacterium smegmatis live vaccine has good tuberculosis treatment effect, and has no obvious toxic or side effect on organisms.

The therapeutic vaccine of the invention has sterilization high efficiency: lysinA, lysinB and Holin are jointly applied to realize multi-target efficient cracking and killing of Mtb. After the live vaccine is subjected to nasal administration, the mycobacterium smegmatis is rapidly phagocytized and cracked by M phi in a body, and the Mtb phage LysnA/LysnB-link-Holin triple lyase double independent promoters eukaryotic coexpression plasmid is released, three specific lyases which are respectively and independently targeted to Mtb are transcribed and synthesized in macrophage cytoplasm by utilizing the vigorous protein synthesis capability of macrophages, so that not only can the latent infected cell wall Mtb and cell wall defect L-type Mtb be killed, but also the proliferation type Mtb and drug-resistant Mtb can be killed efficiently; meanwhile, the mycobacterium smegmatis can be used as a cellular immune adjuvant to play an immune treatment role, activate immune memory of an infected Mtb organism, and jointly kill the infected Mtb in vivo by cooperating with gene therapy, so that a comprehensive treatment effect on the Mtb infection is obtained.

The therapeutic vaccine provided by the invention has good targeting property: first, mycobacterium smegmatis has macrophage targeting: after entering the body, mphi is used as the first defense line of the immune system, and actively chemotaxis and phagocytize the vector. In addition, mycobacteria possess a variety of proteins that invade Mphi cells, such as Mce1A protein, and have the ability to actively invade Mphi. Targeting of Mtb phage LysnA/LysnB-link-Holin triple lyase: unlike the mechanism of action of antibiotics, lytic enzymes are highly specific for the lysis of Mtb and do not act on other insensitive bacteria and eukaryotic cells. The binding targets of this approach are on the cell wall and membrane of Mtb, without impediments to penetration through the particularly tough cell wall of Mtb.

The therapeutic vaccine of the invention has good biological safety: mycobacterium smegmatis is inherently a normal flora of the human body and is also a cellular immune adjuvant. When it is used as biological carrier for gene therapy, it is quickly cracked by phagocyte after entering body, so releasing large amount of gene therapy plasmid carried in it. Mphi is a terminal cell, disintegrates in a mode of naturally occurring programmed death and the like after the terminal cell exists in a body for a few months, and the killed exogenous germ genome and the M phi genome break down in the course of natural sterilization. This is different from muscle cells, and therefore there is basically no need to consider the risks of gene therapy such as drug resistance induced by the incoming plasmid, cancer induced after gene integration, lifetime existence, difficult clearance, etc.

The therapeutic vaccine of the invention is not easy to generate drug resistance: the biggest bottleneck encountered in phage whole-bacteria therapy is that targeting is difficult to achieve, and the whole bacteria have strong immunogenicity, and neutralizing antibodies can be generated after multiple in-vivo applications, so that phage lose therapeutic effects. The therapeutic vaccine removes turnip, and the carrier targets and carries target genes to express Mtb phage lyase in M phi cells, so that the molecular weight and the contact between the molecular weight and an immune system are greatly reduced, and the defect of full-bacterial therapy can be overcome, thereby opening a path for treating a plurality of refractory bacterial infections by utilizing natural phage.

The live vaccine of the invention can be obtained at low cost: mtb phage carried in gene recombination live vaccine

The LysnA/LysnB-link-Holin therapeutic genes can be multiplied in advance in an in vitro special culture medium by a mycobacterium smegmatis live vaccine to obtain a large number of therapeutic gene copies.

The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the essence of the present invention are intended to be included within the scope of the present invention.

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Claims (10)

1. The Deltpd mycobacterium smegmatis live vaccine for target inactivation of mycobacterium tuberculosis is characterized by comprising the following components in percentage by weight: the live vaccine takes the mycobacterium smegmatis with the trpD gene knocked out as a vector, and simultaneously carries the recombinant live vaccine of three lysozyme protein gene coexpression vectors of tubercle bacillus phages, and the preservation number is CCTCC NO: C20221941.

2. The Δtrpd mycobacterium smegmatis live vaccine targeted against inactivated mycobacterium tuberculosis of claim 1, wherein the Δtrpd mycobacterium smegmatis live vaccine is characterized by: the three lysozyme genes are LysinA, lysinB and Holin gene fragments.

3. The method for preparing the delta trpD mycobacterium smegmatis live vaccine of the targeted inactivated mycobacterium tuberculosis according to any one of claims 1 to 2, wherein the method is characterized in that: the preparation method comprises the following steps:

step 1: preparing a trpD gene knockout mycobacterium smegmatis;

step 2: constructing a recombinant eukaryotic coexpression system for phage lyase gene coexpression;

step 3: the phage lyase gene coexpression vector is introduced into trpD gene knockout mycobacterium smegmatis to obtain the recombinant live vaccine.

4. The method for preparing a live vaccine against Δtrpd smegmatic mycobacterium of claim 3, wherein step 1 specifically comprises:

(1) Constructing a delta trpD deletion gene homologous exchange site, and integrating the site into the genome of a tool phage phiE 159 for knocking out mycobacteria genes to obtain a phagemid;

(2) Introducing phagemid into engineering bacteria, and carrying out in vitro amplification to obtain recombinant phage with high titer, and carrying out transfection on mycobacterium smegmatis;

(3) Coating transfected mycobacterium smegmatis on hygromycin-resistant solid culture medium, culturing for 2-3 days at 37 ℃, picking up monoclonal, and verifying by a PCR mode;

(4) The hygromycin resistance gene is eliminated by electrotransformation of the pJH532 plasmid into the verified correct gene knockout strain, and the gene knockout strain without resistance is obtained.

5. The method for preparing the live vaccine of Δtrpd smegmatic mycobacterium for targeted inactivation of mycobacterium tuberculosis according to claim 3, characterized in that: the step 2 specifically includes:

(1) When the primer sequence is designed, proper enzyme cutting sites are introduced, and an OriM replicon gene fragment, a LysnA gene fragment, a LysnB gene fragment and a Holin gene fragment are obtained by using a PCR amplification technology;

(2) Inserting the properly sequenced OriM replicon gene fragment into NheI cleavage site of pBudCE4.1 plasmid to obtain vector pZM0Y;

(3) Splicing the Lysin B and the Holin gene segment by using a hydrophobic glycine linker coding sequence to obtain a Lysin B-link-Holin fusion gene segment;

(4) The obtained LysinA, lysinB-link-Holin fusion gene fragment is respectively connected into eukaryotic expression plasmids pcDNA3.1 to generate eukaryotic expression vectors pZFY-1 and pZFY-2, and the eukaryotic expression vectors are introduced into Top10 to select ampicillin resistance colonies for double enzyme digestion and sequencing identification;

(5) And (3) carrying out double digestion on plasmids pZFY-1 and pZFY-2, recovering and sequencing to identify correct LysnA genes and LysnB-link-Holin gene fragments, and sequentially subcloning the LysnA genes and the LysnB-link-Holin gene fragments into the obtained pZM0Y vector, wherein the LysnA is inserted between XhoI and KpnI, and the LysnB-link-Holin is inserted between HindIII and BamHI, so as to obtain a phage lyase gene coexpression vector pZM0A.

6. The method for preparing the live vaccine of Δtrpd smegmatic mycobacterium for targeted inactivation of mycobacterium tuberculosis according to claim 3, characterized in that: the step 3 specifically includes:

(1) Washing and counting the cultured trpD gene knocked-out mycobacterium smegmatis, adding the washed and counted mycobacterium smegmatis into Mphi, and observing the Mphi targeting of the bacterium;

(2) The plasmid pZM0A is electrically transformed into trpD gene knocked out mycobacterium smegmatis, recombinant bacteria with Zeocin resistance are obtained through screening, and a large amount of recombinant mycobacterium therapeutic live vaccines are cultured and amplified.

7. The method for preparing the delta trpD mycobacterium smegmatis live vaccine targeting inactivated mycobacterium tuberculosis according to claim 5, wherein the method is characterized in that: the primers of the LysinA gene fragment, the LysinB gene fragment and the Holin gene fragment in the (1) are respectively as follows:

LysnA upstream primer: 5'-GGGATCCGATATCATGGCCGAGGACCA-3', which is SEQ ID NO. 1 sequence;

LysnA downstream primer: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 2 sequence;

lysin B upstream primer: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is SEQ ID NO. 3 sequence;

lysin B downstream primer: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 4 sequence;

holin upstream primer: 5'-GGGATCCGATATCATGGCCGAGGACCAGC-3', which is the sequence of SEQ ID NO. 5;

holin downstream primer: 5'-GCGGCCGC AAGCTTCTACGCGGCCGAGGT-3', which is SEQ ID NO. 6 sequence.

8. Use of a Δtrpd mycobacterium smegmatis live vaccine according to any one of claims 1-2 for targeted inactivation of mycobacterium tuberculosis or prepared according to the method of any one of claims 3-7, characterized in that: the Δtrpd mycobacterium smegmatis live vaccine is used for non-therapeutic in vivo or in vitro inactivation of mycobacterium tuberculosis.

9. The use of a Δtrpd mycobacterium smegmatis live vaccine targeting inactivated mycobacterium tuberculosis according to claim 8, characterized in that:

the step of inactivating the mycobacterium tuberculosis in vitro comprises the following steps:

introducing the DeltatrpD mycobacterium smegmatis live vaccine into macrophages, observing transfection efficiency by a fluorescence microscope, and respectively detecting the expression of LysinA, lysinB and Holin by an RT-PCR method; acid-fast staining to observe the sterilization condition of pZM0A to Mtb within M phi, and cell lysis and bacterial culture counting are carried out; hoechst staining observes the influence of pZM0A on macrophages, and at the same time, electron microscopy observes whether ultrastructural changes exist, including the change states of mitochondria, cell nuclei, endoplasmic reticulum and golgi apparatus.

10. The use of a Δtrpd mycobacterium smegmatis live vaccine targeting inactivated mycobacterium tuberculosis according to claim 8, characterized in that:

the step of inactivating the mycobacterium tuberculosis in vivo comprises the steps of:

(1) Animal modeling: a model of latent infection and active tuberculosis infection in mice;

(2) Nasal administration of DeltatrpD mycobacterium smegmatis live vaccine to mice, 5X 10 ⁶ CFU,0.1 ml/time, once a week for four weeks;

(3) Each experimental group is killed in 1 st month, 3 rd month and 5 th month after the first administration, left lung and partial spleen tissues are taken under the aseptic condition to be accurately weighed, ground, diluted by a multiple ratio and inoculated on an improved Rogowski culture medium plate, and colony count is carried out after culture for 6 weeks at 37 ℃; 4% paraformaldehyde fixation of lung tissues, paraffin embedding and slicing, and HE staining and observation; PPD-specific spleen lymphocyte proliferation assay; after the PPD stimulation, the mouse spleen lymphocytes are secreted into the culture supernatant and the cytokine content in the mouse blood is quantitatively detected by ELISA.

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