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Fusobacterium nucleatum FomA protein vaccine, preparation method and applications thereof

A fusobacterium nucleatum and protein technology, applied in the field of biological vaccines, can solve the problems of the preparation of colon tumor vaccines that have not been seen, and achieve significant protective effects, good stability, and the effects of delaying the occurrence and development

Active Publication Date: 2019-02-01
SHANDONG UNIV QILU HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] At present, there is no relevant report on the preparation of colon tumor vaccine by Foma protein

Method used

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  • Fusobacterium nucleatum FomA protein vaccine, preparation method and applications thereof
  • Fusobacterium nucleatum FomA protein vaccine, preparation method and applications thereof
  • Fusobacterium nucleatum FomA protein vaccine, preparation method and applications thereof

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preparation example Construction

[0040] In a third typical embodiment of the present disclosure, there is provided a method for preparing an anti-colon tumor Fusobacterium nucleatum protein vaccine, the method comprising: connecting the Foma gene with the signal peptide sequence removed and connected with the MBP tag gene to In the expression vector, transform Escherichia coli, induce expression, and purify the FomA recombinant protein containing the MBP tag; then digest the prepared FomA recombinant protein containing the MBP tag to remove the MBP tag protein, and obtain the anti-colon tumor nucleated shuttle bacillus protein vaccine.

[0041] Further, the FomA gene sequence without the signal peptide is shown in SEQ ID NO: 3.

[0042] The FomA recombinant protein antigen prepared in the present disclosure contains MBP tag. It has been verified by experiments that linking the FomA protein sequence with the MBP tag can not only enhance the expression of FomA protein when exogenously expressed, but also improv...

Embodiment 1

[0060] A preparation method of Fusobacterium nucleatum FomA recombinant protein vaccine, comprising the steps of:

[0061] (1) Ligate the Foma gene that removes the signal peptide and connects with the MBP tag gene to the PetDuet-1 carrier to obtain the expression vector, and introduce the expression vector into Escherichia coli to obtain the expression strain; In LB medium, shake culture at 37°C;

[0062] Wherein, the FomA gene sequence is shown in SEQ ID NO: 1, 1-60 bp is the sequence corresponding to the signal peptide, and the sequence shown in SEQ ID NO: 3 is the FomA gene sequence without the signal peptide sequence.

[0063] (2) When the expression strain proliferates until the OD600 is 0.5-0.6, add IPTG with a mass concentration of 0.1% (that is, the IPTG in the culture system is 0.1%), induce expression overnight at 18°C, collect the cells, and lyse to obtain the protein;

[0064] The FomA protein sequence encoded by the FomA gene is shown in SEQ ID NO: 2, wherein 1-...

Embodiment 2

[0070] The Foma recombinant protein vaccine prepared in Example 1 was used to immunize C57BL / 6 mice by gavage, and the control group C57BL / 6 mice were gavaged with normal saline, 50ug protein / mouse, and 7 healthy mice in each group. There was no significant difference in body weight of the mice. The mice were immunized three times with an interval of 7 days. The concentration of Foma-specific IgA antibody in the supernatant of mouse feces was detected 7 days after the last gavage. The concentration of IgA antibody in the fecal supernatant after the immune response was as follows: figure 2 As shown, the average IgA antibody concentration in the feces supernatant of the mice in the FomaA group was 7.71ug / mL, and the average IgA antibody concentration in the feces supernatant of the mice in the control group was 1.30ug / mL.

[0071] The results showed that after three immunizations, the antibody concentration in the feces supernatant of C57BL / 6 mice gavaged with FomA recombinant ...

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Abstract

The present disclosure relates to a Fusobacterium nucleatum FomA protein vaccine, a preparation method and applications thereof. The preparation method comprises: linking a signal-peptide-sequence-deleted FomA gene linked to a MBP tag to an expression vector, transforming into Escherichia coli, carrying out induced expression, carrying out affinity purification on FomA recombinant protein by usingAmylose Resin High Flow, and carrying out enzyme digestion with a tev enzyme to remove the MBP tag to obtain the Fusobacterium nucleatum FomA protein vaccine. According to the present invention, theFusobacterium nucleatum FomA protein vaccine has good stability, and can effectively improve the immunity of organisms to Fusobacterium nucleatum, improve the survival rate and delay the occurrence and development of colons tumor; and in APCMin / + mice models, the FomA protein vaccine has significant protecting effect on the mice, can be used as a new colon tumor vaccine candidate, and has great application prospects.

Description

technical field [0001] The disclosure belongs to the technical field of biological vaccines, and more specifically relates to a Fusobacterium nucleatum FomA protein vaccine as well as its preparation method and application. Background technique [0002] The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art. [0003] Tumor is an important medical problem in today's world, which has caused serious harm to global economic development, social stability and people's health. At present, the rapid development of biotechnology plays a key role in improving and enhancing the quality of human life. Vaccine, as a kind of biotechnology drug, has continuously made brilliant achievements in the fight against infectious diseases, and at the same time it has brought new opportunities for the field of tumor treatment. [0004] Fusobacterium nucleatum (F. nucleatum) is an obligate anaerobic Gram-negative ba...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/114A61K39/39A61P35/00A61P31/04C12N15/70C12N15/31
CPCA61K39/114A61K39/39A61K2039/55516A61P31/04A61P35/00C07K14/195C12N15/70
Inventor 左秀丽顾湘李理想李延青
Owner SHANDONG UNIV QILU HOSPITAL
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