Recombination plasmodium falciparum circumsporozoite surface protein and its preparation method and use
A technology of Plasmodium falciparum and recombinant protein, applied in the field of DNA recombination technology and genetic engineering vaccine, which can solve the problems of short duration, instability and low vaccine efficacy.
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Embodiment 1
[0124] Example 1 Synthesis of PfCSP-C and PfCSP-RC genes
[0125] 1. Design of Recombinant Proteins
[0126] The sequences of the PfCSP-C and PfCSP-RC recombinant proteins of the present invention are derived from the amino acid sequence of the circumsporozoite surface protein of Plasmodium falciparum 3D7 strain, the amino acid sequence of which is shown in GenBank accession number: X15363 (SEQ ID NO: 8).
[0127] The PfCSP-C recombinant protein is composed of the C-terminal region of PfCSP (without anchor region), and the PfCSP-RC recombinant protein is composed of the RI region, central repeat region and C-terminus (without anchor region) of PfCSP. Each functional region directly connected. figure 1The schematic diagrams of the protein structures of native PfCSP, PfCSP-C and PfCSP-RC constructed in this example are shown.
[0128] In addition, an XhoI cloning site (Leu-Glu) and a Saccharomyces cerevisiae α-factor signal peptide cleavage sequence (Lys-Arg) were added to the...
Embodiment 2
[0147] Example 2 Secretory expression of PfCSP-C and PfCSP-RC genes in Pichia pastoris
[0148] 2.1 Construction of expression vector
[0149] PfCSP-C and PfCSP-RC genes were inserted into pPIC9 yeast expression plasmid (purchased from Invitrogen) through XhoI and Eco RI sites. In this way, the N-terminus of the recombinant protein is fused to the C-terminus of the Saccharomyces cerevisiae α-factor signal peptide on the vector. Because the N-terminus of the recombinant protein molecule contains three specific amino acid Glu-lys-Arg sequences of the signal peptide cleavage point. Therefore, the target protein released by secretory expression does not contain a signal peptide sequence.
[0150] The basic elements of the pPIC9K vector are the same as those of pPIC9, but it carries the kanamycin resistance gene, and G418 can be used to screen for high-copy inserts. In this way, the fragment containing PfCSP-C and PfCSP-RC was excised from the pBluscript vector with BamH I and E...
Embodiment 3
[0159] Example 3PfCSP-C and PfCSP-RC fermentation and purification
[0160] After the optimization of expression conditions, the expression level of shake flask can reach 100mg / L. Fermentation expression was carried out with 15L irrigation. During the whole fermentation cycle, the cells in the early stage grow exponentially, and the cell density reaches OD 600 =100. In the growth phase of glycerol addition, the cell growth increases linearly, and the cell density reaches the OD. 600 = 560. However, at the stage of methanol-induced expression, the total cell density remained basically unchanged, and the expression of the target protein started 3-7 hr after methanol was added, and with time, the expression yield increased rapidly and was continuously secreted into the fermentation broth. The expression level of each recombinant protein can reach more than 1g / L.
[0161] The purification of PfCSP-C and PfCSP-RC expression products was carried out in two steps: since the isoe...
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