Oral recombinant
The invention relates to a recombinant protein used for immunoprophylaxis of human Helicobacter pylori infection and a degradable slow-releasing microsphere-encapsulated oral vaccine preparation prepared from the same, and the preparation method thereof. Said recombinant protein is composed of A2 subunit and B subunit of the LT of enterotoxigenic Escherichia coli and urease B subunit. The vaccine provided in the invention used in human Helicobacter pylori infection is safe and effective and convenient for oral intake.
1. A recombinant protein, wherein said recombinant protein is formed by fusion of an A2 subunit and a B subunit of heat-labile enterotoxin of enterotoxigenic Escherichia coli and a urease B subunit of Helicobacter pylori , wherein the amino acid sequence of the recombinant protein is presented in SEQ ID NO:2.
2. A slow-release microsphere-encapsulated oral preparation, wherein said preparation encapsulates the recombinant protein of claim 1 .
3. The microsphere-encapsulated oral preparation of claim 2 , wherein said encapsulation substances include sodium alginate, vegetable oil, calcium chloride, and chitosan.
4. The microsphere-encapsulated oral preparation of claim 2 or 3 , wherein the particle diameter of said microsphere-encapsulated preparation is 3.33 μm.
5. The microsphere-encapsulated oral preparation of claim 2 or 3 , wherein said encapsulated preparation is a freeze-dried preparation.
6. The freeze-dried preparation of claim 5 , wherein the excipient of the freeze-dried preparation is 8% mannitol, stabilizing agent is 0.05% EDTA-Na 2 , and the optimal pH value is 10.0.
7. A nucleotide sequence encoding the recombinant protein of claim 1 , wherein said nucleotide sequence is ltA2B-ureB formed by fusion of the coding gene of an A2 subunit and the coding gene of a B subunit of heat-labile enterotoxin of enterotoxigenic Escherichia coli , and the coding gene of a urease B subunit of Helicobacter pylori , wherein the nucleotide sequence is presented in SEQ ID NO: 1.
8. A recombinant plasmid, wherein said plasmid is formed by ligation of the nucleotide sequence of claim 7 and the plasmid pET-11c.
9. A method for preparing a recombinant protein wherein said recombinant protein is formed by fusion of an A2 subunit and a B subunit of heat-labile enterotoxin of enterotoxigenic Escherichia coli and urease B subunit of Helicobacter pylori , wherein said method comprises the following steps:
1) respectively cloning the coding nucleotide sequences of urease B subunit UreB or Helicobacter pylori and subunit of heat-labile enterotoxin LTA2B of enterotoxigenic Escherichia coli , wherein the amino acid sequence of the recombinant protein is presented in SEQ ID NO: 2;
2) ligating the nucleotide sequences obtained by cloning in the step 1) by the method of overlapping PCR to form fusion gene ltA2B-ureB;
3) constructing the fusion gene ltA2B-ureB on a vector, transforming a host, and expressing recombinant protein LTA2B-UreB;
4) separating and purifying the recombinant protein obtained from step 3).
10. A method for preparing an oral recombinant Helicobacter pylori vaccine, which comprises the follow steps:
(1) proportioning the recombinant protein obtained in claim 9 with sodium alginate, vegetable oil, calcium chloride, and chitosan, and preparing into a degradable slow-releasing microsphere-encapsulated preparation; and
(2) optionally preparing the slow-releasing microsphere-encapsulated preparation into a freeze-dried product.