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Recombinant chimeric protein carrying hepatitis B virus epitope and preparation thereof

An antigen epitope, hepatitis B virus technology, applied in the field of medical biology, can solve problems such as large side effects

Active Publication Date: 2012-08-22
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the existing drugs that can effectively treat hepatitis B all have the problem of large side effects. Therefore, vaccine immunization is still the main control measure

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] The plasmid pETP6F carrying the recombinant rotavirus VP6 carrier protein coding gene of Sac I site, BspT104 I site, Kpn I site, Bln I site, Sac II site and Xho I site was constructed by the following steps:

[0155] 1） The following primer pairs were manually designed:

[0156] P174F: CAT GAGCTC ATGGGTACAATGTGG;

[0157] P174R: CAT GAGCTC ATGCGCAGGTTGTGA;

[0158] P197F: GATTA TTCGAA CATACAGCAATTTGAGCAT;

[0159] P197R: TATG TTCGAA TAATCAAATCCAGCAAC;

[0160] P244F: GAT GGTACC ACATGGTATTTTAATCCA;

[0161] P244R; TGT GGTACC ATCAGCTGAATTAATTAC;

[0162] P275F: GCA CCTAGG TTTGGTACAATTGTA;

[0163] P275R: AAA CCTAGG TGCCTGATAAGTATTTAT;

[0164] P298F: AGA CCGCGG ACCCCATCAGTCGCA;

[0165] P298R: GGT CCGCGG TCTCATCAATTGAAATGA;

[0166] P308F: GCA CTCGAG CATCATGCTACTGTA;

[0167] P308R: ATG CTCGAG TGCTGCGACTGATGG;

[0168] and the following primers:

[0169] PETL5:AT CATATG GAGGTTCTGTACTC;

[0170] PVP6-3: TA GGATCC TTATCATTTAACAAGCATG...

Embodiment 2

[0213] The recombinant chimeric protein carrying the hepatitis B virus S82 antigen epitope provided by the present invention is prepared by the following steps:

[0214] A. According to the amino acid sequence of the antigenic epitope, design the following primers respectively:

[0215] S82F: CTTCCTCTTCATCCCTGCTGCTATGCCT G GAGCT

[0216] S82R: CCAGGCATAGCAGCAGGATGAAGAGGAAGAGCT

[0217] B. The primers in step A are paired as follows:

[0218] Two artificially synthesized oligonucleotides of the S antigen epitope S82, that is, the positive strand S82F and the negative strand S82R, were formulated as a primer pair S82F / S82R;

[0219] C. Anneal the primer pair in step B under the following conditions. The annealing mixture contains 2 μl of forward primer, 2 μl of reverse primer, 7 μl of PCR buffer and 59 μl of water; After reacting in a water bath at ℃ for 5 minutes, put it in a water bath at 37℃ and react for 45 minutes. Add 400 μl of TE to the reaction system and mix it ...

Embodiment 3

[0226] A. Primers are designed as:

[0227] C19F: CGAATCATTTTTGCCTTCTGACTTCTTTCCTTCCGTCCGGGATTT

[0228] C19R: CGAAATCCCGGACGGAAGGAAAGAAGTCAGAAGGCAAAAATGATT

[0229] B. The primer pairing is:

[0230] The C antigen epitope C19, whose amino acid sequence is: Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp two artificially synthesized oligonucleotides, positive strand (C19F) and negative strand (C19R), are used as primers For C19F / C19R;

[0231] C, with embodiment 2

[0232] D. 、 The recombinant rotavirus VP6 carrier protein coding gene plasmid pETP6F carrying the BspT104 I site was digested with BspT104I under the same conditions as in step D of Example 2 to obtain a digested plasmid fragment: BspT104I digested plasmid fragment;

[0233] E. 、 Pair the restriction plasmid fragment obtained in step D with the complementary double strand obtained in step C as follows:

[0234] BspT104 I restriction plasmid fragment / C19 complementary double strand;

[0235] After mixing...

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Abstract

The invention discloses a recombinant chimeric protein carrying a hepatitis B virus epitope and the preparation thereof, particularly a recombinant chimeric protein respectively carrying a hepatitis B virus S82, C19, S125, S2[8], S1[93] or C117 epitope, and a recombinant chimeric protein simultaneously carrying hepatitis B virus S82, C19, S125, S2[8], S1[93] and C117 epitopes, and through the immunology WesternBlot detection, each recombinant protein not only has the antigen reactivity and the immunogenicity of a VP6F protein, but also has the antigen reactivity and the immunogenicity of the HBV (hepatitis B virus) epitope, does not have infectivity and the side effects of virulence atavism, the intussusception and the like, and has important value for developing new generation RV (Rota Virus) / HBV recombinant chimeric vaccine and reagent.

Description

technical field [0001] The invention relates to a recombinant chimeric protein, in particular to a recombinant chimeric protein carrying hepatitis B virus antigen epitope with rotavirus VP6 protein as a carrier and its preparation, belonging to the field of medical biotechnology. Background technique [0002] Rotavirus (RV) is the main pathogen of infantile diarrhea. There are more than 600,000 RV infection deaths worldwide each year, 80% of which occur in developing countries. RV belongs to Reoviridae ( Reoviridae ), rotavirus ( Rotaviruses ). The RV genome consists of 11 fragments of double-stranded RNA, encoding 6 structural proteins (VP1-VP4, VP6 and VP7) and 6 non-structural proteins (NSP1-NSP5 / NSP6), respectively. [0003] According to the different antigenicity of the antigenic protein VP6 of the RV group (subgroup), the rotavirus RV discovered so far can be divided into seven groups A to G, and group A is the main pathogen of infantile diarrhea. The 12 proteins...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70
Inventor 陈元鼎
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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