Food grade lactic acid bacteria active carrier Group A rotavirus vaccine and preparation method thereof

A group A rotavirus, lactic acid bacteria technology, applied in the direction of antiviral agents, viral antigen components, drug combinations, etc., to achieve the effects of low cost, stable properties, and simple production process

Inactive Publication Date: 2014-03-26
刘占良 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0027] However, up to now, there is no overall solution for the p

Method used

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  • Food grade lactic acid bacteria active carrier Group A rotavirus vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Codon optimization of antigen-encoding genes

[0053] Because rotavirus genes are expressed in eukaryotic cells, their codon usage preference is different from that of lactic acid bacteria. In order to successfully express the rotavirus gene in lactic acid bacteria cells, it is necessary to optimize its nucleotide sequence and remove rare codons. Its optimization principle is:

[0054] TTC→TTT; GTA→GTT; TCC→TCT; GAG→GAA; TGC→TGT; AGA→CGT; AGG→CGT

[0055] 1) Taking the gene encoding the VP4 protein of human rotavirus G2 serotype as an example, its optimized nucleotide sequence is as follows, and the two underlined parts are the sequences of VP8* and VP5* in sequence:

[0056] GCA AGT TTG ATT TAT CGT CAG CTG TTA ACC AAT TCA TAT TCT GTT GAT CTG CAC GAT GAA ATC GAA CAA ATC GGT AGC GAA AAG ACC CAG AAC GTG ACG ATT AAT CCG GGC CCA TTT GCG CAA ACC CGT TAT GCC CCG GTT AAC TGG CGC CAT GGT GAA ATT AAT GAT TCA ACC ACG GTT GAA CCA GTG CTG GAT GGC CCG TAT CAG CC...

Embodiment 2

[0064] Embodiment 2: Construction of antigenic protein or vaccine adjuvant protein expression cassette

[0065] The expression and localization of antigenic protein or vaccine adjuvant protein is realized through the combined use of multiple expression control elements. These expression control elements include promoters, signal peptide sequences, antigen-encoding genes, anchor subsequences, and linking sequences between each expression control element (see appendix figure 1 ).

[0066] 1) In the present invention, the expression of the antigenic protein is ultimately controlled by a constitutive promoter. The constitutive promoter is derived from the Lactobacillus rRNA promoter, and is one of the following sequences, or one of the sequences with a similarity of more than 85% to the following sequences:

[0067] a) GTC GGT CGA GTT GTT GAC AGG ATA AAG GTC GCC TGG TAT GGT CTC AATATA GCG

[0068] b) GTC GTG GCA GTT GTT GAC AAC CTG TGG GCG GTT TGA TTT GTT CTT GCTATA GCG

[0069]...

Embodiment 3

[0096] Example 3: Human rotavirus P12 serotype VP7 protein expression cassette inserted into Lactobacillus fermentum chromosome, and antibiotic resistance gene removed

[0097] The Lactobacillus described in this example specifically refers to Lactobacillus fermentium.

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Abstract

The invention discloses food grade lactic acid bacteria active carrier Group A rotavirus vaccine and a preparation method thereof. The food grade lactic acid bacteria active carrier Group A rotavirus vaccine is characterized in that VP6 antigen protein from Group A virus, common serotype VP7 antigen protein (P serotype) and VP4 antigen protein (G serotype-expressed separately in the form of VP5* and VP8* protein subunit), vaccine adjuvant escherichia coli thermal unstable toxin B (LTB) and cholera toxin subunit B (CTB) are expressed and secreted by thyA gene deletion lactic acid bacteria cell or shown by the cell wall. Expressions of antigen protein and vaccine adjuvant protein are controlled by inducible or constitutive promoter, protein expression cassette is integrated onto the chromosome of the expression host lactic acid bacteria strain, and external antibiotics resistance gene introduced in gene manipulation is removed. The lactic acid bacteria active carrier rotavirus vaccine has the advantages of having wide serotype covering range, being easy to produce in large scale, and being safe and convenient to use without a refrigerator and a needle tubing.

Description

technical field [0001] The present invention relates to a kind of food-grade lactic acid bacteria live carrier A group rotavirus vaccine and its preparation method, more specifically, the present invention relates to use food grade lactic acid bacteria living cells as the carrier prepared for the prevention of human or animal group A rotavirus A multivalent vaccine against diarrhea, and its vegetative approach. technical background [0002] Rotavirus-infected diarrhea is the second leading cause of emergency visits and death in infants and young children. Almost all children under the age of 3 in the world experience rotavirus infection and disease. Every year, rotavirus diarrhea causes about 130 million people with diarrhea, about 18 million people with severe diarrhea, and about 450,000 deaths. The deaths were mainly distributed in South Asia, Africa, and East Asia. [0003] In my country, nearly 40,000 children die from rotavirus diarrhea every year, accounting for abou...

Claims

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Application Information

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IPC IPC(8): A61K39/15A61P1/12A61P31/14
Inventor 刘占良刘战征刘占术
Owner 刘占良
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