Recombinant P. falciparum HRP II (full length) [GST]

Malaria is an endemic infectious disease caused by infection with Plasmodium parasites. It is mainly prevalent in tropical and subtropical regions. Typical clinical manifestations include periodic chills, fever, sweating, and other symptoms, which may be accompanied by signs such as splenomegaly and anemia. Falciparum malaria has irregular fever and a high mortality rate. Human malarial parasites are divided into Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, plasmodium ovale, and Plasmodium knowlesi, which is common to humans and monkeys (Plasmodium knowlesi is mainly transmitted from monkeys to mosquitoes to humans). The life history of several malarial parasites is basically the same, and their complete life history requires two stages of development in the human body and in the mosquito body. Plasmodium falciparum (P. falciparum) is the pathogen that causes falciparum malaria in humans. With the development of molecular biology technology, the structures of more than 30 genes of Plasmodium falciparum have been studied. Among them, the histidine-rich protein II (HRP-II) gene is the application gene of the dipstick method recommended by WHO for diagnosing malaria, and it has great potential in malaria diagnosis. The characteristic of HRP gene is that the content of histidine (His) is as high as 73%. The HRP gene is a typical small exon gene, with a single insertion sequence after the signal peptide, and a polypeptide mainly composed of repeating units after a short hydrophilic amino acid sequence. In addition to HRP-II, there are HRP-I and HRP-III, and the gene structures of the three are very similar. HRP-1, also known as KAHRP, is related to the cell scaffold of red blood cells infected with Plasmodium and is located in the infected red blood cell membrane nodules. This type of nodules includes HRP-I and its binding receptor, namely P.f. infected red blood cell membrane protein 1. They play an important role in the adhesion process of infected red blood cells to microvascular endothelial cells. This process is a major pathogenesis of cerebral malaria caused by P.f. infection, so some people speculate that HRP-I is an important pathogenic factor for cerebral malaria. Unlike HRP-I, HRP-II does not participate in the formation of nodules. From the sequence point of view, the two genes HRP-II and HRP-III show a high degree of homology (85%-90%), and they can even be expressed simultaneously in some asexual malarial parasites, suggesting that both may have evolved from homologous ancestral genes from replication to mutation. The cDNA clone and genomic DNA sequencing found that the His and Ala contents of HRP-III were 30% and 29%, respectively, and the tandem repeat sequence was AHHANN, not AH-HAAD of HRP-II.

Figure 1. Histidine-Rich Protein 2 (HRP2)-Based Malaria Rapid Diagnostic Tests (mRDTs): Normal Operation and the Prozone Phenomenon. (Sources: Poti KE, et al. 2020)

HRP-II is synthesized throughout the asexual period of P.f. The amino acid sequence deduced from the gene contains 34% histidine, 37% alanine and 10% aspartic acid, and the open reading frame of HRP-1 (nucleotides 141-941) is mainly composed of many tandem repeats of tripeptides (AHH)1 and hexapeptides (AHHAAD)s. The hexapeptide sequence near the 3' end of the repeat region is replaced by three pentapeptides (AHHAA). The tripeptide and pentapeptide are usually identical to the first three and first five amino acids in the hexapeptide sequence, indicating that the bases in the repeat region are inserted and/or deleted and extended. The coding gene of HRP-II contains a hydrophobic region signal peptide sequence consisting of 13 amino acid residues, located between the 5' end of the mRNA and the splice site, and the promoter Met is upstream of the hydrophobic region. Immediately after the signal peptide is an intron, followed by the main coding region without introns. The histidine-rich sequence starts at the 25th amino acid at the N-terminus of the region, that is, 75 nucleotides at the 5' end of the coding region. In addition, HRP-II has a succinate terminator at 39-41 amino acids. The difference between the sequences of HRP-II genomic DNA and cDNA is that the 3' end splicing point of the former contains a polypyrimidine region and an A-G dinucleotide eukaryotic consensus sequence. HRP-II appears in the supernatant of synchronized cultures 2-8 hours after the development of the P.f. ring body, indicating that it is actively excreted by infected erythrocytes. HRP-II is rich in His, suggesting that it has a tendency to bind to divalent metal ions. Endogenous HRP-H in human serum and rabbit serum is called histidine-rich glycoprotein (HRG), which contains 12 tandem repeat sequences, each of which is composed of 5 amino acids in the same sequence (Gly-His-His-Pro-His). It determines whether HRP-1 is released into the serum and can inhibit the formation of rosettes. P.f. is rich in His, which can cause erythrocytes to agglutinate and increase the permeability of erythrocytes, causing erythrocytes to invaginate and facilitate the invasion of merozoites. In addition, His is also involved in the formation of hemozoin. The digestive vacuoles of P.f. are the sites of red blood cell degradation, heme aggregation to form crystalline hemozoin, and accumulation of antimalarial drugs. HRP-H is captured and recognized by the corresponding antibodies in the digestive vacuoles. Both recombinant and natural HRP-II promote the formation of hemozoin, but chloroquine can inhibit this process. It is speculated that when P.f. secretes HRP-II into the host serum, HRP-II is transported to the acidic digestive vacuoles along with hemoglobin. Once hemoglobin is decomposed, HRP-II binds to it and participates in the formation of hemozoin.

The detection of malarial parasites includes routine blood tests and etiological examinations. Among them, etiological examinations include peripheral blood smear microscopy, rapid malarial antigen detection, and malarial gene detection. Among these methods, malarial antigen rapid diagnostic test strips (RDT) are characterized by simple and rapid detection. The sensitivity and specificity of different rapid diagnostic test strips vary greatly. Among them, the diagnostic test strips with histidine-rich protein (HRP) 2/HRP3 as target antigens have high sensitivity and specificity for the detection of falciparum malaria, but cannot detect other malarial parasite strains; the diagnostic test strips with lactate dehydrogenase as target antigen can detect falciparum malaria parasites or non-falciparum malaria parasites, but cannot distinguish between vivax malaria parasites, ovale malaria parasites and malaria parasites, and the sensitivity of detection of low parasite density is slightly poor. RDT cannot monitor the response to antimalarial treatment.

PfHRP2
P. falciparum HRP2
Histidine-rich protein 2
Malaria HRP2 antigen