CN112646763A - Gene deletion type Klebsiella pneumoniae attenuated live vaccine, preparation method and application - Google Patents

Gene deletion type Klebsiella pneumoniae attenuated live vaccine, preparation method and application Download PDF

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CN112646763A
CN112646763A CN202011201644.9A CN202011201644A CN112646763A CN 112646763 A CN112646763 A CN 112646763A CN 202011201644 A CN202011201644 A CN 202011201644A CN 112646763 A CN112646763 A CN 112646763A
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klebsiella pneumoniae
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李蓓
徐丽
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Hubei University of Medicine
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Abstract

The invention belongs to the technical field of bioengineering, and discloses a gene-deletion type Klebsiella pneumoniae attenuated live vaccine, a preparation method and an application thereof, wherein the preparation method comprises the following steps: preparing Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain; selecting Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and using LB culture mediumCulturing and proliferating on a bacterial shaker overnight; the following day, the ratio of 1: 100 diluted to fresh LB culture solution, culturing to OD600 ═ 1.4, centrifuging for 10min, collecting the centrifugal precipitate, washing the bacteria with normal saline for 2 times, and then resuspending the bacteria with normal saline to make the concentration of the bacteria reach 105CFU/ml. The invention can effectively solve the problems of limited protection range, low protection power and the like of the components and subunit vaccines of the Klebsiella pneumoniae vaccine at present.

Description

Gene deletion type Klebsiella pneumoniae attenuated live vaccine, preparation method and application
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a gene deletion type Klebsiella pneumoniae attenuated live vaccine, a preparation method and an application thereof.
Background
At present, Klebsiella pneumoniae (Klebsiella pneumoniae) is one of important pathogens of community-acquired infection, can also cause nosocomial infection outbreak, and can cause pneumonia, bacteremia, urinary system infection and the like after infection. Klebsiella pneumoniae high-viscosity strains (mainly K1 and K2 serotypes, LD) planted in intestinal tracts of patients50<100CFU) can cause liver (kidney) abscess and brain infection through intestinal invasion and infection, and the death rate is up to 10-30%. Due to the wide use of broad-spectrum antibiotics, the multidrug-resistant and pan-resistant klebsiella pneumoniae is increasing, and klebsiella pneumoniae has become a representative strain for producing extended-spectrum beta-lactamases (ESBLs) and carbapenemases (KPC enzymes), and the lethality rate of blood infection caused by klebsiella pneumoniae reaches up to 50%.
Therefore, vaccine research is an important direction for the research of preventing and treating klebsiella pneumoniae. Vaccines in general include inactivated vaccines, attenuated live vaccines, recombinant vaccines, subunit vaccines, and the like. For klebsiella pneumoniae infection, vaccines currently studied mainly include: 1. a capsular polysaccharide vaccine. Although multivalent capsular polysaccharide vaccines can provide immunity to multiple serotypes of klebsiella pneumoniae, injection of large doses of capsular polysaccharide can result in immune tolerance and complex manufacturing processes, limiting their application. 2. Protein subunit vaccine: mainly concentrated on its outer membrane protein and pilin. 3. A ribosomal vaccine. Attenuated live vaccines are a main direction of vaccine research, and the vaccines are weakened in pathogenicity after being injected but can be proliferated in a body to sufficiently activate the immune system of the body so as to generate immune protection. For example, Yersinia pestis EV76 live attenuated vaccine strain, Brucella live attenuated vaccine S2 strain, etc. But the protection range of the components and subunit vaccines of the Klebsiella pneumoniae vaccine is limited at present, and the protection capability is low. Therefore, a new klebsiella pneumoniae vaccine is urgently needed.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) injection of large doses of capsular polysaccharide results in immune tolerance and complex manufacturing processes, limiting the application of capsular polysaccharide vaccines.
(2) At present, the protective range of the components and subunit vaccines of the Klebsiella pneumoniae vaccine is limited, and the protective power is low.
The difficulty in solving the above problems and defects is: attenuated live vaccines are a common type of vaccine for preventing bacterial infectious diseases because they can be propagated in vivo after injection to effectively activate the immune system in vivo and generate immunological memory. But the Klebsiella pneumoniae can not find a strain which can reduce toxicity and maintain immunity, the deletion of the gene meets the requirements of attenuated live vaccines, and low-dose injection can generate immune protection.
The significance of solving the problems and the defects is as follows: k1 serotype Klebsiella pneumoniae is a common strain of Klebsiella pneumoniae which can cause severe infection such as invasive infection and the like in normal people, and the death rate after infection can reach 10%. The development of the vaccine can overcome the defects that subunit vaccines and protein vaccines need large-dose inoculation or the duration time of immune protection is short, and the like, effectively stimulate the immune system of an organism in an all-around way, and can reduce the morbidity and mortality after inoculation.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a gene-deletion type Klebsiella pneumoniae attenuated live vaccine, a preparation method and application, aiming at solving the problems of limited protection range, low protection capability and the like of the existing Klebsiella pneumoniae vaccine components and subunit vaccines.
The invention is realized in such a way, the preparation method of the gene deletion type Klebsiella pneumoniae attenuated live vaccine comprises the following steps:
preparing a Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and analyzing pathogenicity of mice infected by the Klebsiella pneumoniae KP1_ RS12260 gene deletion strain in different ways;
selecting Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and culturing and proliferating the Klebsiella pneumoniae virulent NTUH-K2044 strain on a bacterial shaker at 37 ℃ and 220r/min for overnight by using an LB culture medium;
step three, in the following day, according to the proportion of 1: 100 diluted to fresh LB culture solution, culturing to OD600 ═ 1.4, centrifuging for 10min, collecting the centrifugal precipitate, washing the bacteria with normal saline for 2 times, and then resuspending the bacteria with normal saline to make the concentration of the bacteria reach 105CFU/ml；
And step four, performing a Klebsiella pneumoniae KP1_ RS12260 gene deletion attenuated live vaccine efficacy test.
Further, in the first step, the preparation method of the klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain comprises the following steps:
(1) obtaining a 1669bp gene fragment of the upper and lower flanking sequences of the Klebsiella pneumoniae KP1_ RS12260 gene by using a fusion PCR method;
(2) cloning to temperature sensitive suicide plasmid pKO3-Km to obtain recombinant mutation box plasmid, and electrically transferring to Klebsiella pneumoniae wild strain (WT strain) NTUH-K2044 to obtain KP1_ RS12260 gene deletion strain (delta KP1_ RS 12260);
(3) a1876 bp fragment containing the coding region, the promoter binding region and the transcription termination region of the KP1_ RS12260 gene is cloned to a pGEM-T-easy vector and transformed into a KP1_ RS12260 gene deletion strain to obtain a anaplerotic strain (C-KP1_ RS 12260).
Further, in the first step, the method for analyzing pathogenicity of mice infected by the Klebsiella pneumoniae KP1_ RS12260 gene deletion strain in different pathways comprises the following steps:
(1) infecting the abdominal cavity with Klebsiella pneumoniae wild strain, KP1_ RS12260 gene deletion strain and anaplerosis strain, and calculating the median lethal dose of different strains;
(2) by abdominal infection 104CFU calculated mouse survival.
Further, in step four, the efficacy test of the Klebsiella pneumoniae KP1_ RS12260 gene deletion attenuated live vaccine comprises the following steps:
(1) injecting 100ul of the heavy suspension liquid subcutaneously to immunize BALB/C mice, and injecting a control group with the same amount of PBS;
(2) booster again with the same bacterial load 14 days after the primary infection;
(3) taking blood from tail veins at 28 days of primary immunization, mixing the blood of every 3 mice, separating serum after coagulation, detecting the antibody of Klebsiella pneumoniae in the serum by using a whole bacteria ELISA method, and adding different mouse sera into a giant phagocytic cell culture solution to analyze whether the serum of the Klebsiella pneumoniae KP1_ RS12260 gene deletion attenuated live vaccine group mouse can perform opsonization phagocytosis experiments to enhance phagocytosis of bacteria by phagocytes;
(4) 10 of Klebsiella pneumoniae virulent NTUH-K2044 strain 35 days after primary immunization4CFU was injected subcutaneously to infect mice and mice survival was observed.
The invention also aims to provide the gene deletion type Klebsiella pneumoniae attenuated live vaccine prepared by the preparation method of the gene deletion type Klebsiella pneumoniae attenuated live vaccine.
The invention also aims to provide the application of the gene deletion type Klebsiella pneumoniae attenuated live vaccine in the preparation of the medicine for preventing and treating Klebsiella pneumoniae.
By combining all the technical schemes, the invention has the advantages and positive effects that: the gene-deletion Klebsiella pneumoniae attenuated live vaccine provided by the invention can effectively solve the problems of limited protection range, low protection capability and the like of the existing Klebsiella pneumoniae vaccine components and subunit vaccines.
Experiments show that compared with a PBS control group mouse, the serum of the Klebsiella pneumoniae KP1_ RS12260 gene-deleted attenuated live vaccine group mouse has the advantages that the titer of the anti-Klebsiella pneumoniae serum antibody is obviously increased, and the serum can obviously enhance the phagocytosis of macrophages on Klebsiella pneumoniae wild strains in vitro, so that the serum antibody can play a role in opsonophagocytosis, and is favorable for enhancing the phagocytosis and sterilization of Klebsiella pneumoniae during infection. The survival rate experiment result of the mice shows that the survival rate of the Klebsiella pneumoniae KP1_ RS12260 gene-deleted attenuated live vaccine group mice is obviously improved compared with that of a PBS control group, and shows that the resistance to Klebsiella pneumoniae infection after immunization is enhanced.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a preparation method of a gene-deleted Klebsiella pneumoniae attenuated live vaccine provided by the embodiment of the invention.
FIG. 2 is a schematic diagram of the construction of a KP1_ RS12260 gene deletion strain provided in the examples of the present invention.
FIG. 3 is a graph showing the survival rate of mice infected with KP1_ RS12260 gene-deleted strain in different ways.
FIG. 4 is a schematic diagram showing the phagocytic capacity of macrophages for Klebsiella pneumoniae wild-type and KP1_ RS12260 gene-deleted strains under different conditions according to the embodiment of the present invention.
FIG. 5 is a schematic diagram of the survival rate of mice immunized by the attenuated live vaccine of Klebsiella pneumoniae with KP1_ RS12260 gene deletion provided in the embodiments of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a gene-deletion type Klebsiella pneumoniae attenuated live vaccine, a preparation method and application thereof, and the invention is described in detail with reference to the accompanying drawings.
As shown in fig. 1, the preparation method of the gene-deleted klebsiella pneumoniae attenuated live vaccine provided by the embodiment of the invention comprises the following steps:
s101, preparing a Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and analyzing pathogenicity of mice infected by the Klebsiella pneumoniae KP1_ RS12260 gene deletion strain in different ways;
s102, selecting Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and culturing and proliferating the Klebsiella pneumoniae virulent NTUH-K2044 strain overnight by using an LB (LB) culture medium on a bacterial shaker at 37 ℃ and 220 r/min;
s103, on the next day, according to the ratio of 1: 100 diluted to fresh LB culture solution, culturing to OD600 ═ 1.4, centrifuging for 10min, collecting the centrifugal precipitate, washing the bacteria with normal saline for 2 times, and then resuspending the bacteria with normal saline to make the concentration of the bacteria reach 105CFU/ml；
S104, performing a Klebsiella pneumoniae KP1_ RS12260 gene deletion attenuated live vaccine efficacy test.
The preparation method of KP1_ RS12260 gene-deleted klebsiella pneumoniae attenuated live vaccine provided by the invention can also be implemented by other steps by a person of ordinary skill in the art, and the preparation method of KP1_ RS12260 gene-deleted klebsiella pneumoniae attenuated live vaccine provided by the invention in fig. 1 is only a specific example.
The technical solution of the present invention is further described with reference to the following examples.
Example 1: the invention relates to preparation and evaluation of Klebsiella pneumoniae KP1_ RS12260 gene deletion strain.
1. Construction of Klebsiella pneumoniae KP1_ RS12260 gene deletion strain
A1669 bp gene fragment of the upper and lower flanking sequences of the Klebsiella pneumoniae KP1_ RS12260 gene is obtained by a fusion PCR method. Cloning to temperature sensitive suicide vector pKO3-Km to obtain recombinant mutation box plasmid, and electrically transferring to Klebsiella pneumoniae wild strain (WT strain) NTUH-K2044 to obtain KP1_ RS12260 gene deletion strain (delta KP1_ RS 12260). A1876 bp fragment comprising the coding region, promoter-binding region and transcription termination region of the KP1_ RS12260 gene was then cloned into a pGEM-T-easy vector and transformed into a KP1_ RS12260 gene-deleted strain to obtain a complementation strain (C-KP1_ RS12260) (see FIG. 2).
The gene sequence corresponding to the attenuated vaccine is SEQ ID NO: 1:
atgactaattttttgttcaatataaaaaatcactatttgcgagtcgctattgctgaactggttgatgaagcgatgaaggc cgccgggcggccacactatcagttttctcaacaatgggatgcaggatcgatggcgcaagctgatgtgatattcacggaaat ggtcgcgggggagtggtacttgtgtcaggatctttttcagcatgccccggaacaatatacgctttttattttcccggacaatga acacgctaccgtagatgagggtttaccgaattgtctgcagcatgcggtatttatgccgcctcacgctcgcgttcagcggctg aaagatgagatagctaacgccatcgagcgtccgctgctaccgcggcaagacccgccgttcaatcgtctgcggcgttgcat taattgcgcctgcaggtcggtcagcgacgcgcaaaccaaagtcatttacgcgttcagtattggcctgagcccgcatgaagt cgccgctgcgctaaatattagtcccaaaacgattcattcacataaaaagaatattatgagtaaatttaatttgaatagccgtca gcagtttaacaaccttgtgcaactgctcgccaaacgctga。
the protein sequence corresponding to the attenuated vaccine is SEQ ID NO: 2:
mtnflfniknhylrvaiaelvdeamkaagrphyqfsqqwdagsmaqadvif
temvagewylcqdlfqhapeqytlfifpdnehatvdeglpnclqhavfmpp
harvqrlkdeianaierpllprqdppfnrlrrcincacrsvsdaqtkviyafsi
glsphevaaalnispktihshkknimskfnlnsrqqfnnlvqllakr
2. pathogenicity analysis of mice infected by Klebsiella pneumoniae KP1_ RS12260 gene deletion strain in different ways
The abdominal cavity is infected with Klebsiella pneumoniae wild strain, KP1_ RS12260 gene deletion strain and anaplerosis strain, and half of lethal dose of different strains is calculated, the result shows that WTLD50 is approximately equal to 500CFU, and delta KP1_ RS12260LD50>5×105CFU, C-KP1_ RS12260LD50 ≈ 1000 CFU. The pathogenicity of the bacteria is obviously reduced after the gene is deleted. By abdominal infection 104CFU calculation of mouse survival it was also found that all mice infected with the wild strain died and all mice infected with the KP1_ RS12260 gene-deleted strain survived (see FIG. 3). The result of subcutaneous infection with the same bacterial quantity is the same as that of abdominal infection, which shows that the pathogenicity of mice infected by different ways with KP1_ RS12260 gene deletion strain is reduced. Infection in mice 104CFU-deleted strains did not develop disease.
3. Phagocytosis and sterilization capacity of macrophages on Klebsiella pneumoniae KP1_ RS12260 gene deletion strain is enhanced
In vitro macrophage phagocytosis experiments show that the phagocytic bactericidal ability of the macrophages to the Klebsiella pneumoniae KP1_ RS12260 gene deletion strain is obviously enhanced (see figure 4).
Example 2: the invention discloses preparation and efficacy test of a Klebsiella pneumoniae KP1_ RS12260 gene deletion attenuated live vaccine.
Selecting Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, culturing and proliferating overnight on an LB culture medium on a bacterial shaker at 37 ℃ and 220r/min, and culturing according to the ratio of 1: 100 diluted to fresh LB culture solution, culturing to OD600 ═ 1.4, centrifuging for 10min, collecting the centrifugal precipitate, washing the bacteria with normal saline for 2 times, and then resuspending the bacteria with normal saline until the bacteria concentration reaches about 105CFU/ml, 100ul of the resuspended solution was injected subcutaneously into immunized BALB/C mice, and the control group was given an equal amount of PBS for injection. The same bacterial load was boosted once more 14 days after the primary infection. Taking blood from tail vein at 28 days of initial immunization, mixing blood of every 3 mice, separating serum after coagulation, detecting anti-Klebsiella pneumoniae antibody in the serum by using a whole bacteria ELISA method, and adding different mouse sera into macrophage culture solution to analyze whether the serum of the Klebsiella pneumoniae KP1_ RS12260 gene-deleted attenuated live vaccine group mice can perform opsonization phagocytosis experiment to enhance phagocytosis of bacteria by phagocytes. 10 of Klebsiella pneumoniae virulent NTUH-K2044 strain 35 days after primary immunization4CFU was injected subcutaneously to infect mice and mice were observed for survival.
The result shows that the serum antibody titer of the Klebsiella pneumoniae KP1_ RS12260 gene-deleted live attenuated vaccine group mouse serum against the Klebsiella pneumoniae is obviously increased compared with the PBS control group mouse, and the serum can obviously enhance the phagocytosis of macrophages on the Klebsiella pneumoniae wild strain in vitro, which indicates that the serum antibody can play the role of opsonophagocytosis and is beneficial to enhancing the phagocytosis and sterilization of the Klebsiella pneumoniae during infection (see FIG. 4). The survival rate experiment result of the mice shows that the survival rate of the Klebsiella pneumoniae KP1_ RS12260 gene-deleted attenuated live vaccine group mice is obviously improved compared with that of a PBS control group (see figure 5), and the resistance to Klebsiella pneumoniae infection after immunization is enhanced.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.
Sequence listing
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<120> gene deletion type Klebsiella pneumoniae attenuated live vaccine, preparation method and application
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atgactaatt ttttgttcaa tataaaaaat cactatttgc gagtcgctat tgctgaactg 60
gttgatgaag cgatgaaggc cgccgggcgg ccacactatc agttttctca acaatgggat 120
gcaggatcga tggcgcaagc tgatgtgata ttcacggaaa tggtcgcggg ggagtggtac 180
ttgtgtcagg atctttttca gcatgccccg gaacaatata cgctttttat tttcccggac 240
aatgaacacg ctaccgtaga tgagggttta ccgaattgtc tgcagcatgc ggtatttatg 300
ccgcctcacg ctcgcgttca gcggctgaaa gatgagatag ctaacgccat cgagcgtccg 360
ctgctaccgc ggcaagaccc gccgttcaat cgtctgcggc gttgcattaa ttgcgcctgc 420
aggtcggtca gcgacgcgca aaccaaagtc atttacgcgt tcagtattgg cctgagcccg 480
catgaagtcg ccgctgcgct aaatattagt cccaaaacga ttcattcaca taaaaagaat 540
attatgagta aatttaattt gaatagccgt cagcagttta acaaccttgt gcaactgctc 600
gccaaacgct ga 612
<210> 2
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<213> Artificial Sequence (Artificial Sequence)
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Met Thr Asn Phe Leu Phe Asn Ile Lys Asn His Tyr Leu Arg Val Ala
1 5 10 15
Ile Ala Glu Leu Val Asp Glu Ala Met Lys Ala Ala Gly Arg Pro His
20 25 30
Tyr Gln Phe Ser Gln Gln Trp Asp Ala Gly Ser Met Ala Gln Ala Asp
35 40 45
Val Ile Phe Thr Glu Met Val Ala Gly Glu Trp Tyr Leu Cys Gln Asp
50 55 60
Leu Phe Gln His Ala Pro Glu Gln Tyr Thr Leu Phe Ile Phe Pro Asp
65 70 75 80
Asn Glu His Ala Thr Val Asp Glu Gly Leu Pro Asn Cys Leu Gln His
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Ala Val Phe Met Pro Pro His Ala Arg Val Gln Arg Leu Lys Asp Glu
100 105 110
Ile Ala Asn Ala Ile Glu Arg Pro Leu Leu Pro Arg Gln Asp Pro Pro
115 120 125
Phe Asn Arg Leu Arg Arg Cys Ile Asn Cys Ala Cys Arg Ser Val Ser
130 135 140
Asp Ala Gln Thr Lys Val Ile Tyr Ala Phe Ser Ile Gly Leu Ser Pro
145 150 155 160
His Glu Val Ala Ala Ala Leu Asn Ile Ser Pro Lys Thr Ile His Ser
165 170 175
His Lys Lys Asn Ile Met Ser Lys Phe Asn Leu Asn Ser Arg Gln Gln
180 185 190
Phe Asn Asn Leu Val Gln Leu Leu Ala Lys Arg
195 200

Claims (10)

1. An attenuated live vaccine evaluation method, which is characterized in that the attenuated live vaccine evaluation method uses a Klebsiella pneumoniae KP1_ RS12260 gene deletion strain.
2. The live attenuated vaccine evaluation method of claim 1, wherein the live attenuated vaccine has the gene sequence of SEQ ID NO: 1.
3. the live attenuated vaccine assessment method according to claim 1, wherein said live attenuated vaccine corresponds to klebsiella pneumoniae KP1_ RS12260 protein sequence of SEQ ID NO: 2.
4. a preparation method of a gene-deleted Klebsiella pneumoniae attenuated live vaccine is characterized in that the preparation method of the gene-deleted Klebsiella pneumoniae attenuated live vaccine comprises the following steps:
preparing Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and analyzing pathogenicity of mice infected by the Klebsiella pneumoniae KP1_ RS12260 gene deletion strain in different ways;
selecting Klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene deletion strain, and culturing and proliferating on a bacterial shaker overnight by using LB culture medium;
diluting to a fresh LB culture solution for culture in the next day, centrifugally collecting centrifugal precipitates, washing the bacteria with physiological saline, and then resuspending the bacteria with the physiological saline;
and (4) carrying out a Klebsiella pneumoniae KP1_ RS12260 gene deletion attenuated live vaccine efficacy test.
5. The method for preparing the gene-deleted klebsiella pneumoniae attenuated live vaccine according to claim 4, wherein the method for preparing the klebsiella pneumoniae virulent NTUH-K2044 strain KP1_ RS12260 gene-deleted strain comprises the following steps:
(1) obtaining a 1669bp gene fragment of the upper and lower flanking sequences of the Klebsiella pneumoniae KP1_ RS12260 gene by using a fusion PCR method;
(2) cloning to temperature sensitive suicide vector pKO3-Km to obtain recombinant mutation box plasmid, and electrically transferring to Klebsiella pneumoniae wild strain NTUH-K2044 to obtain KP1_ RS12260 gene deletion strain delta KP1_ RS 12260.
6. The method for preparing the gene-deleted klebsiella pneumoniae attenuated live vaccine according to claim 4, wherein the method for analyzing the pathogenicity of mice infected by different ways with the klebsiella pneumoniae KP1_ RS12260 gene-deleted strain comprises the following steps:
(1) infecting the abdominal cavity with Klebsiella pneumoniae wild strain, KP1_ RS12260 gene deletion strain and anaplerosis strain, and calculating the median lethal dose of different strains;
(2) by abdominal infection 104CFU calculated mouse survival.
7. The method for preparing the gene-deleted Klebsiella pneumoniae attenuated live vaccine of claim 4, wherein the Klebsiella pneumoniae virulent NTUH-K2044 KP1_ RS12260 gene-deleted strain is selected, and cultured and propagated overnight in LB medium on a bacterial shaker at 37 ℃ and 220 r/min.
8. The method of claim 4, wherein the gene-deleted live attenuated Klebsiella pneumoniae vaccine is administered at a rate of 1: 100 were diluted into fresh LB medium to an OD600 of 1.4.
9. The method of claim 8, wherein the gene-deleted live attenuated Klebsiella pneumoniae vaccine is obtained by centrifuging for 10min, collecting the centrifuged precipitate, washing the bacteria with physiological saline for 2 times, and then resuspending the bacteria in physiological saline until the concentration of the bacteria reaches 105CFU/ml。
10. The method for preparing the gene-deleted live attenuated Klebsiella pneumoniae vaccine of claim 4, wherein the efficacy test of the live attenuated Klebsiella pneumoniae KP1_ RS12260 gene-deleted vaccine comprises the following steps:
(1) injecting 100ul of the heavy suspension liquid subcutaneously to immunize BALB/C mice, and injecting a control group with the same amount of PBS;
(2) booster again with the same bacterial load 14 days after the primary infection;
(3) taking blood from tail veins at 28 days of primary immunization, mixing the blood of every 3 mice, separating serum after coagulation, detecting an anti-Klebsiella pneumoniae antibody in the serum by using a whole bacteria ELISA method, and adding different mouse sera into a macrophage culture solution to analyze whether the serum of the Klebsiella pneumoniae KP1_ RS12260 gene-deleted attenuated live vaccine group mouse can perform an opsonization phagocytosis experiment to enhance phagocytosis of bacteria by phagocytes;
(4) after primary immunization, mice are infected by subcutaneous injection of Klebsiella pneumoniae virulent NTUH-K2044 strain, and the survival rate of the mice is observed;
10 of Klebsiella pneumoniae virulent NTUH-K2044 strain 35 days after primary immunization4CFU was injected subcutaneously to infect mice and mice were observed for survival.
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