Recombinant chimeric protein carrying rotavirus antigen epitope and preparation thereof

An antigenic epitope, rotavirus technology, applied in the field of medical biology, can solve the problems of incomplete protection of RV infection, viral nucleic acid contamination, only reduction of mortality and severe diarrhea

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

Since the above-mentioned RV vaccines used in humans have risks such as intussusception and other viral nucleic acid contamination, and all of them can only reduce the mor

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] 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:

[0159] 1) Manually design the following primer pairs:

[0160] P174F: CAT GAGCTC ATGGGTACAATGTGG;

[0161] P174R: CAT GAGCTC ATGCGCAGGTTGTGA;

[0162] P197F: GATTA TTCGAA CATACAGCAATTTGAGCAT;

[0163] P197R: TATG TTCGAA TAATCAAATCCAGCAAC;

[0164] P244F: GAT GGTACC ACATGGTATTTTAATCCA;

[0165] P244R; TGT GGTACC ATCAGCTGAATTAATTAC;

[0166] P275F: GCA CCTAGG TTTGGTACAATTGTA;

[0167] P275R: AAA CCTAGG TGCCTGATAAGTATTTAT;

[0168] P298F: AGA CCGCGG ACCCCATCAGTCGCA;

[0169] P298R: GGT CCGCGG TCTCATCAATTGAAATGA;

[0170] P308F: GCA CTCGAG CATCATGCTACTGTA;

[0171] P308R: ATG CTCGAG TGCTGCGACTGATGG;

[0172] and the following primers:

[0173] PETL5:AT CATATG GAGGTTCTGTACTC;

[0174] PVP6-3: TA GGATCC TTATCATTTAACAAGCATGCTTCTAAT...

Embodiment 2

[0217] The recombinant protein carrying the P[4]223 epitope provided by the present invention is prepared through the following steps:

[0218] A. According to the amino acid sequence of the antigenic epitope, design the following primers:

[0219] P[4] 223F: CCCACCAATTCAAAATACTAGAAATGTAGTTCCAGAGCT;

[0220] P[4]223R: CTGGAACTACATTTTCTAGTATTTTGAATTGGTGGGAGCT;

[0221] B. The primers of step A are paired as follows:

[0222] Two artificially synthesized oligonucleotides of VP4 epitope P[4]223, namely positive strand P[4]223F and negative strand P[4]223R, were formulated as a primer pair: P[4]223F / P[4 ]223R;

[0223]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 for 5 minutes, react in a water bath at 37°C for 45 minutes. Add 400 μl of TE to the reaction system and mix well with an equal volume of isopropano...

Embodiment 3

[0230] A. Primers are designed as:

[0231] P[4]56F: CGAATGATTCAACTACAGTGGAACCAGTTTTAGATGGTCCTTATCAACCTT;

[0232] P[4]56R: CGAAGGTTGATAAGGACCATCTAAAACTGGTTCCACTGTAGTTGAATCATT;

[0233] B. The pairing of primers is as follows: two artificially synthesized oligonucleotides of VP4 epitope P[4]56, that is, the positive strand P[4]56F and the negative strand P[4]56R, are used as a primer pair: P[4] ]56F / P[4]56R;

[0234] C, with embodiment 2

[0235] D. The plasmid pETP6F of the recombinant rotavirus VP6 carrier protein coding gene carrying the BspT104 I site was digested with the BspT104 I site under the same conditions as in step D of Example 2 to obtain a digested plasmid fragment: BspT104 I enzyme cut plasmid fragments;

[0236] E. Perform the corresponding pairing of the restriction plasmid fragment obtained in step D and the complementary double strand obtained in step C as follows:

[0237] BspT104 I restriction plasmid fragment / P[4]56 complementary double strand;

...

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Abstract

The invention provides recombinant chimeric proteins carrying rotavirus antigen epitopes and preparation thereof. The recombinant chimeric proteins are respectively six recombinant proteins carrying rotavirus P[4]223, P[4]56, G1[142], G4[208], P[8]296 and G2[87] antigen epitopes, and recombinant proteins simultaneously carrying P[4]223, P[4]56, G1[142], G4[208], P[8]296 and G2[87] antigen epitopes. The immunologic WesternBlot detection indicates that all the recombinant chimeric proteins can be subjected to specific immune reaction with anti-rotavirus totivirus, anti-rotavirus VP6 and anti-rotavirus VP4/VP7 antibodies. Thus, the recombinant-expressed recombinant chimeric proteins have favorable immunoreactivity for VP6 carrier proteins and inserted rotavirus antigen epitopes, do not have infectivity, do not have virulence reversion, intussusception or any other side effect, and have high application value for further developing rotavirus RV recombinant chimeric vaccines.

Description

[0001] technical field [0002] The invention relates to a recombinant chimeric protein, in particular to a recombinant chimeric protein carrying a rotavirus epitope and its preparation, belonging to the field of medical biotechnology. Background technique [0003] 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. [0004] 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 (genes) of group A rotavirus RV can be divid...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C12R1/19
Inventor 陈元鼎
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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