Recombinant malaria vaccine
A kind of composition and technology of malaria parasite, applied in the field of recombinant malaria vaccine and preparation thereof
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Examples
Embodiment 1
[0040] Preparation of p83 / 30 fragment
[0041] The amino acid sequence of the p83 / 30 fragment is shown in SEQ ID NO: 1 (strain F) or SEQ ID NO: 2 (strain D). Cloning the nucleic acid sequence encoding the fragment into figure 1 In the expression vector pZE23D 83 / 30 shown in. The fragment-encoding nucleic acid is operably linked to the IPTG-inducible pA1lacO1 promoter. The Escherichia coli production strain is W3110Z2 (for example, Bacteriol. Ref. 36, (1972), 525-530; Proc. Natl. Acad. Sci. USA 78 (1981), 7069-7072).
[0042] E. coli cells were pre-cultured in LB medium. The preculture was diluted 1:50 with Superbroth and grown in fermentors at 37°C. In optical density (OD 600 ) is 1.5, add doxycycline (200ng / ml). in OD 600 = 4.5, IPTG (1 mM) was added.
[0043] 2.5h after induction with IPTG or at OD 600 At =7, the cells were collected.
[0044] The collected bacteria were disrupted by continuous homogenization at a turnover rate of 10 l / h and a maximum pressure of 1...
Embodiment 2
[0060] Preparation of p38 / 42 fragment
[0061] The amino acid sequence of the p38 / 42 fragment is shown in SEQ ID NO: 3 (strain F) or SEQ ID NO: 4 (strain D).
[0062] Inclusion bodies of the p38 / 42 fragment were prepared in substantially the same manner as described in Example 1 for the p83 / 30 fragment.
Embodiment 3
[0064] Preparation of compositions comprising equimolar amounts of p83 / 30 and p38 / 42 fragments
[0065] The inclusion bodies obtained in Examples 1 and 2 were respectively dissolved by adding a dissolution buffer (6M guanidine hydrochloride, 50 mM sodium phosphate, 10 mM dithiothreitol, 1 mM EDTA, pH 8.0) to the inclusion body (IB) .
[0066] For the p83 / 30 fragment, three different ratios of IB to lysis buffer were used, namely 1 g IB + 2.5 ml buffer, 1 g IB + 4.0 ml buffer and 1 g IB + 8.0 ml buffer. For the p38 / 40 fragment, 1 g of IB was added to 2.5 ml of buffer.
[0067] Filtrates were refolded by incubation overnight at room temperature in 500 mM arginine, 50 mM sodium phosphate, 1 mM reduced L-glutathione, 0.1 mM oxidized glutathione, 1 mM EDTA pH 8.0.
[0068] The resulting protein solution was concentrated 5-fold by ultrafiltration. The buffer was exchanged with 20 mM sodium phosphate, 50 mM NaCl, 0.01% Tween 80, pH 8.0. After filtration with a 0.2 μm filter, the ...
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Description
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Application Information
- IPC
- C07K14/445; A61K39/015
- CPC
- A61K39/015; C07K14/445; A61P33/06; A61P37/04; Y02A50/30
- Inventors
- 赫尔曼·比雅尔; 罗尔夫·卢茨