Plasmodium relictum

Zhang, Xuejin, Meadows, Siobhan N. A., Martin, Tori, Doran, Alex, Angles, Rachel, Sander, Samantha, Bronson, Ellen & Witola, William H., 2022, Plasmodium relictum MSP- 1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus), International Journal for Parasitology: Parasites and Wildlife 19, pp. 89-95 : 90

2.2. Cloning of P. relictum MSP-1 coding sequence

A 1476 bp long truncated coding sequence of the P. relictum isolate SGS1 MSP-1 (starting from the first codon) based on the gene reported in GenBank (Accession number KC969175.1) was synthesized by Integrated DNA Technologies (IDT, USA). The synthesized gene fragment was supplied by IDT cloned in pUCIDT (AMP) vector and lyophilized. Before use, the DNA was reconstituted in sterile molecular-grade nuclease-free water. The primer pair used for PCR-amplification of the truncated MSP-1 coding gene fragment for directional cloning in the pET15b expression vector (Novagen) in-frame with the N-terminal hexahistidine tag (His-tag) was 5 ′ - CTCGAG ATG A- CAAATTCTTTTGGTAAAC-3 ′ (Forward, with the XhoI restriction site italicized and start codon in bold) and 5 ′ - GGATCC TTA TTCTGTA- TATTTGTTAATCT-3 ′ (Reverse, with the BamHI site italicized and stop codon in bold). The truncated MSP-1 gene was amplified by PCR using high fidelity DNA polymerase (Affymetrix), and T/A-cloned into the pGEMT-Easy vector (Promega). After propagation of the recombinant vector in E. coli and plasmid extraction, the MSP-1 coding sequence was excised by dual XhoI/BamHI restriction enzyme digestion and subcloned at XhoI/BamHI site of the pET15b expression vector in-frame with the His-tag at the N-terminal. The recombinant pET15b vector was sequenced to confirm identity of the gene fragment insert, and the recombinant vector transformed into protein expression E. coli, BL21 (DE3) pLysS (Novagen).