Fusion protein and its use, its anti-malarial vaccine and antibody
A fusion protein and anti-malarial technology, applied in the field of biomedicine, can solve problems such as difficulty in obtaining artificial multi-epitope antigens, and achieve good development prospects, strong immunogenicity, and stable physical and chemical properties
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[0056] According to one embodiment of the present invention, the M.RCAg-1 gene is cloned into the prokaryotic expression vector pDS-ex containing different carrier tags or no carrier tag, expressed and purified to obtain M.RCAg-1 / Exp.V-1 , M.RCAg-1 / Exp.V-2, M.RCAg-1 / Exp.V-3 three kinds of multi-epitope proteins, the amino acid sequences of which are respectively as SEQ ID No.: 1, SEQ ID No.: 3 and Shown in SEQ ID No.: 5; its DNA sequences are shown in SEQ ID No.: 2, SEQ ID No.: 4 and SEQ ID No.: 6, respectively.
[0057] figure 1 Shows the amino acid sequence comparison schematic diagram of M.RCAg-1 / Exp.V-1, M.RCAg-1 / Exp.V-2 and M.RCAg-1 / Exp.V-3 three kinds of multi-epitope proteins, wherein M.RCAg-1 / Exp.V-1 is a multi-epitopic protein with 43 carrier amino acids fused at the N-terminus with an enterokinase digestion recognition site, and M.RCAg-1 / Exp.V-2 is in A multi-epitope protein with 43 carrier amino acids fused to the N-terminus with a PreScission Protease cleavage re...
Embodiment 1
[0063] Embodiment 1 constructs VR1012-312 plasmid and pDS-ex plasmid
[0064] According to the method disclosed in Chinese Patent Application No. 200410080982.6, VR1012-312 plasmid and pDS-ex plasmid were constructed. details as follows:
[0065] According to the method disclosed in Chinese Patent Application No.200410080982.6, the gene fragment m.rcag-1 (that is, the ES312 gene fragment disclosed in Chinese Patent Application No.200410080982.6) was obtained, and the resulting gene fragment m.rcag-1 was connected to pDS- On the ex plasmid (according to the method disclosed in Chinese Patent Application No.200410080982.6, the prokaryotic expression vector pET-30a (+) (obtained from Novagen) was replaced by Bgl II with the Not I and Eag I restriction sites by PCR technology, Kpn I is moved forward, and EcoRV, Bgl II, and Nsp V restriction sites are removed, thereby obtaining the pDS-ex plasmid), its DNA sequence is shown in SEQ ID No.: 10, and its physical map is Figure 7 sho...
Embodiment 2
[0066] Embodiment 2 obtains M.RCAg-1 / Exp.V-1 protein
[0067] 1. The VR1012-312 plasmid and pDS-ex plasmid obtained in Example 1 were subjected to EcoR I and BglII double enzyme digestion respectively, so that m.rcag-1 was connected into the pDS-ex vector, thereby constructing the recombinant plasmid M.RCAg- 1 / pDS-ex.
[0068] The specific steps are as follows:
[0069] 1) The restriction enzyme digestion system of VR1012-312 plasmid and pDS-ex plasmid (taking EcoR I and Bgl II as examples) is as follows:
[0070]
[0071] Digest at 37°C for 2 hours, identify by agarose gel electrophoresis, and recover with a DNA fragment recovery kit to obtain gene fragment m.rcag-1 and pDS-ex plasmid fragment.
[0072] 2) Dephosphorylation of the recovered gene fragment m.rcag-1 and pDS-ex plasmid fragment
[0073] Concrete reaction system is as follows:
[0074]
[0075] 3) In the ligation reaction of the recovered gene fragment m.rcag-1 and the pDS-ex plasmid fragment, the molar ...
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