Fusion protein for preparing Abeta (Amyloidbeta-peptide) epitope vaccines, and preparation method and application thereof
A fusion protein and epitope vaccine technology, applied in the field of recombinant fusion protein and its preparation, to improve the effect of humoral immunity and overcome immune tolerance
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Embodiment 1
[0054] Embodiment 1: the design of fusion protein
[0055] Fusion protein EcA-PADRE-Aβ for preparing Aβ epitope vaccine designed by the present invention 1-15 (referred to as EPA) consists of Escherichia coli asparaginase EcA at the N-terminal, helper T cell epitope PADRE in the middle and human Aβ at the C-terminal 42 functional B cell epitope Aβ 1-15 It consists of three parts. Among them, the first part EcA is directly connected to the second part PADRE, the second part PADRE is connected to the third part Aβ 1-15 A Linker connection is added in the middle to reduce steric hindrance and facilitate the effective presentation of epitope polypeptides. The structural model of the fusion protein EPA is shown in figure 1 Shown in B.
[0056] In addition, in the implementation process, the present invention has also designed another fusion protein EcA-TTe-Aβ by replacing PADRE with T auxiliary epitope TTe (amino acid sequence such as SEQ ID NO: 17, gene coding sequence such a...
Embodiment 2
[0057] Embodiment 2: Construction and identification of fusion protein EPA expression vector
[0058] 1. EcA, PADRE-Aβ 1-15 Amplification of fusion genes
[0059] The EcA coding gene was amplified by PCR using the plasmid pMD19T-EcA as a template, using primers (see Table 1) SEQIDNO: 11 and SEQIDNO: 12, and introducing restriction sites NcoI and BamHI while designing the primers.
[0060] PADRE-Aβ 1-15 The fusion gene is amplified by direct PCR with primers. At first, primers (see Table 1) SEQIDNO:13 and SEQIDNO:14 are used as templates to amplify the PCR product, and then primers (see Table 1) SEQIDNO:15 and SEQIDNO:16 are used to recover The purified PCR product above was used as a template for the final amplification of PADRE-Aβ 1-15 Fusion gene, when designing primers (see table 1) SEQIDNO:15 and SEQIDNO:16, introduce restriction site BamHI and XhoI simultaneously,
[0061] Table 1: Primer Design
[0062]
[0063] 2. Construction of fusion protein expression vector...
Embodiment 3
[0068] Example 3: Induced expression, extraction, separation, purification and identification of fusion protein EPA in E.coli
[0069] 1. Induced expression of fusion protein in E.coli
[0070] Inoculate the constructed positive BL21(DE3) strain containing the pEPA plasmid into the r In the 2YT liquid medium (1.6% tryptone, 1% yeast extract, 0.5% NaCl, pH=7.0), shake culture at 37°C until OD 600 When it is 0.6-1.0, induce culture with IPTG at a concentration of 1 mmol / L at 25°C for 14 hours. Take 1mL of the bacterial liquid to collect the bacterial cells by centrifugation, resuspend the bacterial cells in the protein electrophoresis sample buffer, boil for 5 minutes, and perform SDS-PAGE detection, and then use Coomassie brilliant blue R-250 staining to observe the purpose, the results (such as Figure 4 Shown in A) The fusion protein with a molecular weight of about 42kDa (theoretical molecular weight is 41657.79Da) was successfully expressed.
[0071] 2. Extraction and se...
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