Recombinant helicobacter pylori protein vaccine and preparation method thereof

A technology of Helicobacter pylori and protein is applied in the field of recombinant Helicobacter pylori protein vaccine and its preparation, and achieves the effects of important social value, economic value and high immune effect.

Inactive Publication Date: 2017-10-27
成都亿妙生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The strongest mucosal immune adjuvants currently recognized are cholera enterotoxin (CT) and Escherichia coli heat-labile enterotoxin (LT), but CT and LT have strong toxicity to humans, which limits their application.

Method used

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  • Recombinant helicobacter pylori protein vaccine and preparation method thereof
  • Recombinant helicobacter pylori protein vaccine and preparation method thereof
  • Recombinant helicobacter pylori protein vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Synthetic gene:

[0033] Using the nucleic acid sequence of Escherichia coli heat-labile enterotoxin gene (GenBank: AB011677.1) published by GenBank as a template for transformation, the specific design is as follows:

[0034] The gene encoding LT is 1148bp in length, including: LTA signal peptide (18 amino acids) gene, LTA (240 amino acids) gene, LTB signal peptide (21 amino acids) gene and LTB (103 amino acids) gene . There is a frameshift reading frame between the end of the gene encoding LTA and the beginning of the gene encoding LTB signal peptide, so the gene encoding the entire LT is a continuous DNA sequence. Its complete gene nucleotide sequence is shown in Seq ID No.4.

[0035] After bioinformatics analysis and structural analysis, the 100-585bp coding nucleotide sequence of the LTA subunit gene was removed, and the Helicobacter pylori urease A subunit gene fragment ureA 1 Insert it to replace the removed LT A subunit gene coding nucleotide sequence 100-585...

Embodiment 2

[0038] Recombinant plasmid pET-11c / lt-ureA 1 construction, such as figure 1 Shown:

[0039] The expression vector was pET-11c, which was purchased from Novagen. The expression vector carries the ampicillin resistance gene, is a mode of chemical substance IPTG induction control, and has a high expression level. Before induction, lacIq encodes a repressor protein, which can be combined with the operator gene in the operon to control the transcription of adjacent structural genes; when the inducer exists, the repressor protein is affected by the inducer, and its configuration occurs Changes, so that the loss of affinity with the operator is released, and the polymerase can transcribe the structural gene and then express it. Insert the recombinant target gene containing LTA signal peptide and the ltB gene containing LTB signal peptide behind the NdeI site of the plasmid, and pET-11c can co-express the recombinant target protein LTA in the host bacteria 1 -UreA 1 and LTB prote...

Embodiment 3

[0046] Expression, purification and identification of target protein:

[0047] The host bacteria used was E.coli / BL21(DE3), which was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Genotype: F-ompT gal dcm lon hsdSB(rB-mB-)λ(DE3[lacI lacUV5-T7 gene 1 ind1 sam7 nin5]). The bacterial strain is used as a host for protein expression of high-efficiency exogenous genes using T7 RNA polymerase as an expression system. The expression of the T7 phage RNA polymerase gene is controlled by the lacUV5 promoter in the DE3 region of the lambda phage, which is integrated on the chromosome of BL21. The strain is suitable for the expression of non-toxic protein.

[0048] (1) Conversion

[0049] Take the recombinant plasmid pET-11c / lt-ureA 1 E.coli / BL21(DE3) competent cells (TIANGEN, Code: CB105) were transformed according to the operating instructions, and positive recombinants were screened on ampicillin-resistant LB plates.

[0050] (2) Identification of recombinant en...

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Abstract

The invention discloses a recombinant helicobacter pylori protein vaccine and a preparation method thereof. The active ingredient recombinant fusion protein of the vaccine consists of recombinant LTAl-Ureal protein and LTB protein, the amino acid sequence of the recombinant LTAl-Ureal protein is shown as Seq ID No.1, the amino acid sequence of the LTB protein is shown as Seq ID No.2. Epitope-containing gene segments of Hp urease A subunit is inserted in an LTA subunit-encoded gene, a toxic part-containing segment is replaced to prepare a recombinant plasmid so as to express and obtain recombinant fusion protein polymer as a vaccine antigen, the fusion antigen and LTB protein pentamer are combined to form a hexamer structure, so that not only can the structure basis and activity of an LT mucosa adjuvant be remained, but also the toxicity can be removed, the immune response of organism muscosa can be effectively induced through immunity of mucosa path, to generate specific IgA antibody. The recombinant helicobacter pylori protein vaccine provides a vaccine manner for preventing and treating infection of helicobacter pylori.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a recombinant Helicobacter pylori protein vaccine and a preparation method thereof. Background technique [0002] Helicobacter pylori (Hp) is a helical, microaerophilic gram-negative bacillus that mainly colonizes the human gastric mucosa and has caused infection of more than 50% of the world's population (Lee A. Prevention of Helicobacter pylori infection. Scand-J - Gastroenterol. 1996. Suppl 215:11-15.). Hp infection is an important pathogenic factor of chronic gastritis, peptic ulcer and gastric mucosa-associated lymphoid tissue lymphoma (MALT), and is closely related to the occurrence of gastric cancer. It has been listed as a class I carcinogen by WHO. At present, the triple therapy of proton pump inhibitor plus two antibiotics is the first-line treatment for Hp eradication recommended globally. However, with the widespread use of antibiotics in the treatment of Hp infecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/02A61P31/04
CPCC07K14/245A61K39/0208A61K2039/541C12N9/80C12N15/70C12Y305/01005
Inventor 刘开云
Owner 成都亿妙生物科技有限公司
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