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A kind of ehec O157: H7 recombinant Lactobacillus acidophilus vector vaccine and its preparation method and application

A technology of EHECO157, Lactobacillus acidophilus, applied in the field of genetic engineering, can solve the problems such as no Lactobacillus acidophilus, and achieve the effect of good immunogenicity and immune protection effect

Active Publication Date: 2019-08-30
广州南医智诚医疗产业投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, there is no research report on the expression of EHEC O157:H7 antigen by Lactobacillus acidophilus at home and abroad

Method used

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  • A kind of ehec O157: H7 recombinant Lactobacillus acidophilus vector vaccine and its preparation method and application
  • A kind of ehec O157: H7 recombinant Lactobacillus acidophilus vector vaccine and its preparation method and application
  • A kind of ehec O157: H7 recombinant Lactobacillus acidophilus vector vaccine and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1: the preparation of EHEC O157:H7 recombinant Lactobacillus acidophilus live vector vaccine strain

[0046] like figure 1 Shown, the concrete method of the present invention is:

[0047] 1. Design and synthesis of primers

[0048] Search the EspA gene sequence (KJ549678.1) and Tir M gene sequence (NC_002655.2) from GenBank, introduce the Pst I restriction site at the 5′ end of the EspA upstream primer, remove the stop codon TAA with the downstream primer, and introduce the linker (ggaggcggaagtggaggaggtagc), The primers were designed as follows: EspA-P1: 5′-AAAACTGCAGGATGGATACATCAAATGCA (SEQ ID No.2), EspA-P2: 5′-GCTACCTCCTCCACTTCCGCCTCCTTTACCAAGGGATATTGCTG (SEQ ID No.3); Tir M upstream primer was introduced into the linker, and the downstream primer was introduced into the restriction site Hind III, Tir M-P1:5′-GGAGGCGGAAGTGGAGGAGGTAGC AGCCCAACCACGACCGAC (SEQ ID No.4), Tir M-P2:5′-CCCAAGCTTTTAGGCTTGCTGTTT GGCCTCTT (SEQ ID No.5), primers were synthesized by...

Embodiment 2

[0081] sExample 2: Recombinant Lactobacillus acidophilus expressing EHEC O157:H7EspA-Tir M protein has immune protection to mice infected with O157:H7

[0082] 1. Inoculate the recombinant Lactobacillus acidophilus in MRS broth containing 0.1ug / ml erythromycin, culture anaerobically at 37°C until the logarithmic growth phase, collect the bacterial sediment, and adjust the bacterial density to 10 10 CFU / ml immunized mice.

[0083] 2. Study on the immunogenicity of recombinant Lactobacillus acidophilus: select 4-5 week-old SPF grade BALB / c mice, and inject 0.1ml 10 into each group with 15 mice 10 CFU / ml of recombinant Lactobacillus acidophilus, and set PBS negative control at the same time, the immunization program is: 0~3d, 7~10d, 21~24d.

[0084] 2.1 Before each immunization and 10 days after the last immunization, the blood was collected by docking the tail, and after standing at room temperature for 2 hours, the serum was collected by centrifugation (1:50). The specific IgG...

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Abstract

The invention relates to a recombinant Lactobacillus acidophilus vector vaccine for EHEC (enterohaemorrhagic E.coli) O157:H7 as well as a preparation method and an application of the recombinant Lactobacillus acidophilus vector vaccine. Recombinant Lactobacillus acidophilus is a live vector bacterium transformed into double-epitope protective antigen containing EHEC O157:H7 EspA-Tir M, the constructed recombinant Lactobacillus acidophilus strain can perform in-vitro passage stably with or without evolutionary pressure, immunization of a mouse through an oral vaccine and proceeding of a challenge assay prove that the Lactobacillus acidophilus live vector vaccine has good immunogenicity and immunoprotection effect, and the live vector vaccine is safe and non-toxic and has good immunoprophylaxis effect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to the development of a live vector vaccine, in particular to a method and application for preparing the EHEC O157:H7 bivalent recombinant Lactobacillus acidophilus vector vaccine. Background technique [0002] Enterohaemorrhagic E.coli (EHEC) O157:H7 is mainly transmitted through the digestive tract, and clinically can cause hemorrhagic enteritis, hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, etc., and even death. Since Riley et al. first identified severe hemorrhagic colitis caused by EHEC O157:H7 in 1983, sporadic cases and outbreaks caused by EHECO157:H7 have continued to occur worldwide, and the global incidence is on the rise. At present, there is still no effective prevention and control measures for EHEC O157:H7 infection. Although most antibiotics can kill it, the released Shiga toxin will increase the risk of hemolytic uremic syndrome. In view o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/62A61K39/108A61P31/04C12R1/23
Inventor 范宏英林如琴吴娴波龙北国张益多
Owner 广州南医智诚医疗产业投资有限公司