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Gene deletion type klebsiella pneumoniae attenuated live vaccine, preparation method and application

A Klebsiella pneumoniae and attenuated live vaccine technology, applied in the field of bioengineering, can solve the problems of limited protection range, complicated production process, and low protection power

Pending Publication Date: 2021-04-13
HUBEI UNIVERSITY OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) Injection of large doses of capsular polysaccharides will lead to immune tolerance and complicated production process, which limits the application of capsular polysaccharide vaccines
[0006] (2) The current Klebsiella pneumoniae vaccine components and subunit vaccines have limited protection range and low protection
However, Klebsiella pneumoniae has not been able to find a strain that can reduce toxicity and maintain immunity. The deletion of this gene meets the requirements of attenuated live vaccines, and low-dose injection can produce immune protection.

Method used

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  • Gene deletion type klebsiella pneumoniae attenuated live vaccine, preparation method and application
  • Gene deletion type klebsiella pneumoniae attenuated live vaccine, preparation method and application
  • Gene deletion type klebsiella pneumoniae attenuated live vaccine, preparation method and application

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preparation example Construction

[0039] Such as figure 1 As shown, the preparation method of the gene-deleted Klebsiella pneumoniae live attenuated vaccine provided by the embodiments of the present invention comprises the following steps:

[0040]S101, preparing a virulent Klebsiella pneumoniae NTUH-K2044 strain with a KP1_RS12260 gene deletion strain, and analyzing the pathogenicity of Klebsiella pneumoniae KP1_RS12260 gene deletion strains infecting mice through different routes;

[0041] S102, select the virulent Klebsiella pneumoniae NTUH-K2044 strain KP1_RS12260 gene deletion strain, and use LB medium to culture and proliferate overnight on a bacterial shaker at 37°C and 220r / min;

[0042] S103, on the next day, dilute it 1:100 into fresh LB culture medium and cultivate to OD600=1.4, centrifuge for 10 minutes to collect the centrifuged sediment, wash the bacteria twice with normal saline, and then resuspend with normal saline until the bacterial concentration reaches 10 5 CFU / ml;

[0043] S104, Effic...

Embodiment 1

[0046] Example 1: Preparation and evaluation of Klebsiella pneumoniae KP1_RS12260 gene deletion strain of the present invention.

[0047] 1. Construction of Klebsiella pneumoniae KP1_RS12260 gene deletion strain

[0048] A 1669bp gene fragment of the upper and lower flanking sequences of the Klebsiella pneumoniae KP1_RS12260 gene was obtained by fusion PCR. Cloned to the temperature-sensitive suicide carrier pKO3-Km to obtain the recombinant mutant cassette plasmid, and electroporated into the Klebsiella pneumoniae wild strain (WT strain) NTUH-K2044 to obtain the KP1_RS12260 gene deletion strain (ΔKP1_RS12260). Then the 1876bp fragment comprising the KP1_RS12260 gene coding region, the promoter binding region and the transcription termination region was cloned onto the pGEM-T-easy vector and transformed into the KP1_RS12260 gene deletion strain to obtain the complementing strain (C-KP1_RS12260) (see figure 2 ).

[0049] The gene sequence corresponding to the attenuated vacc...

Embodiment 2

[0060] Embodiment 2: preparation and efficacy test of Klebsiella pneumoniae KP1_RS12260 gene deletion live attenuated vaccine of the present invention.

[0061] Select the KP1_RS12260 gene deletion strain of the virulent Klebsiella pneumoniae NTUH-K2044 strain, use LB medium to culture and proliferate on a bacterial shaker at 37°C 220r / min overnight, and dilute it into fresh LB culture medium at 1:100 the next day Cultivate to about OD600=1.4, centrifuge for 10 minutes to collect the centrifuged sediment, wash the bacteria twice with normal saline, and then resuspend with normal saline until the bacterial concentration reaches about 10 5 CFU / ml, take 100ul resuspended bacteria solution and inject subcutaneously to immunize BALB / C mice, and the control group is injected with the same amount of PBS. Fourteen days after the initial infection, the same amount of bacteria was used to boost the immunization once again. Blood was collected from the tail vein on day 28 of the initial...

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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 application. The preparation method comprises the following steps: preparing a KP1-RS12260 gene deletion strain of a klebsiella pneumoniae virulent strain NTUH-K2044; culturing the KP1-RS12260 gene deletion strain of the klebsiella pneumoniae virulent strain NTUH-K2044 for multiplication with a lysogeny broth (LB) medium overnight on a bacteria shaking table; and diluting multiplicative strains into a fresh LB culture solution according to a ratio of 1:100 the next day for culture until OD600 is equal to 1.4, performing centrifuging for 10min and collecting centrifugal precipitates, then washing bacteria twice with normal saline, and performing re-suspending with the normal saline until the concentration of the bacteria reaches 10<5>CFU / ml. The invention can effectively solve the problems of limited protection range, low protection effect and the like of klebsiella pneumoniae vaccine components and subunit vaccines at present.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a live attenuated gene-deleted Klebsiella pneumoniae vaccine, a preparation method and an application. Background technique [0002] At present, Klebsiella pneumoniae (Klebsiella pneumoniae) is one of the important pathogens of community-acquired infections, and it can also cause outbreaks of nosocomial infections. After infection, it can cause pneumonia, bacteremia, and urinary system infections. Klebsiella pneumoniae high-viscosity strains (mainly K1, K2 serotypes, LD 50 <100CFU) can cause liver (kidney) abscess and brain infection through intestinal invasion infection, and the mortality rate is as high as 10%-30%. Due to the widespread use of broad-spectrum antibiotics, multidrug-resistant and pan-drug-resistant Klebsiella pneumoniae have gradually increased. Enzyme), the representative species of Klebsiella pneumoniae, the fatality rate of blood infection caused b...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/63C12N15/31C07K14/26A61K39/02A61P31/04C12R1/22
CPCC12N1/20C12N15/63C07K14/26A61K39/0266A61P31/04A61K2039/522
Inventor 李蓓徐丽
Owner HUBEI UNIVERSITY OF MEDICINE
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