DNA molecule for encoding recombinant circumsporozoite surface protein of plasmodium falciparum

[0125] Example 1 Synthesis of PfCSP-C and PfCSP-RC genes

[0126] 1. Design of recombinant protein

[0127] The sequences of the PfCSP-C and PfCSP-RC recombinant proteins of the present invention come from the amino acid sequence of the circumspore surface protein of Plasmodium falciparum 3D7 strain, and the amino acid sequence is shown in GenBank accession number: X15363 (SEQ ID NO: 8).

[0128] The PfCSP-C recombinant protein is composed of the C-terminal region of PfCSP (without the anchor region), and the PfCSP-RC recombinant protein is composed of the RI region, central repeat region and C-terminal region of PfCSP (without the anchor region). connected.figure 1 A schematic diagram of the protein structures of natural PfCSP, PfCSP-C and PfCSP-RC constructed in this example is shown.

[0129] In addition, an XhoI cloning site (Leu-Glu) and a Saccharomyces cerevisiae α-factor signal peptide cleavage sequence (Lys-Arg) were added at the N-terminal of the fusion antigen. A p...

[0148] Example 2 Secretion and expression of PfCSP-C and PfCSP-RC genes in Pichia pastoris

[0149] 2.1 Construction of expression vector

[0150] PfCSP-C and PfCSP-RC genes were inserted into pPIC9 yeast expression plasmid (purchased from Invitrogen Company) through XhoI and EcoRI sites. In this way, the N-terminal of the recombinant protein is fused with the C-terminal of the Saccharomyces cerevisiae α-factor signal peptide on the carrier. Because the N-terminal of the recombinant protein molecule contains three specific amino acid Glu-lys-Arg sequences of the cutting point of the signal peptide. Therefore, the target protein released by secretory expression does not contain a signal peptide sequence.

[0151] The basic elements of the pPIC9K vector are the same as pPIC9, but it has a kanamycin resistance gene, and G418 can be used for screening of high-copy inserts. Like this, use BamH I and EcoR I to cut out the fragment containing PfCSP-C and PfCSP-RC from pBluscript v...

[0160] Example 3 Fermentation and purification of PfCSP-C and PfCSP-RC

[0161] After optimizing the expression conditions, the expression level in the shake flask can reach 100mg / L. Fermentation expression was performed 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 increased linearly, and the cell density reached OD 600 =560. However, in the stage of methanol-induced expression, the total cell density remained basically unchanged, while the expression of the target protein began 3-7 hours after the addition of methanol, and as time went on, 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.

[0162] The purification of the expression products of PfCSP-C and PfCSP-RC was carried out in two step...