Cloning and characterization of the merozoite surface antigen 1 gene of Plasmodium berghei.

HAIBING ZHONG 0 1 JIN-YUAN FAN 0 1 SHUTONG YANG 0 1 EUGENE A. DAVIDSON 0 1 0 Department of Biochemistry and Molecular Biology, Georgetown University Medical Center , Washington , District of Columbia 1 Biology, Georgetown University School of Medicine , 3900 Reser- voir Road, Washington, DC 20007 Merozoite surface antigen 1 (MSA1) is a promising candidate for vaccine development against malaria parasites. Here, we report the complete nucleotide sequence of the gene encoding the precursor to this major surface antigen of Plasmodium berghei strain ANKA using cDNA library screening and polymerase chain reaction techniques. A single open reading frame of 5,376 basepairs encoding a protein with a calculated molecular mass of 197 kD was defined. The protein contains a putative signal peptide of 19 amino acids, a membrane anchor sequence of 18 residues, and shows two epidermal growth factor-like domains rich in Cys residues at the C-terminus. There are four repeat sequences of oligopeptides in the molecule: tetrapeptide (Ser-Thr-Thr-Thr), tripeptide (Pro-Thr-Pro and Pro-Ala-Ala), and dipeptide (Ser-Gly). Furthermore, three nine-residue stretches of a motif (Ala-Ser-Asn-Pro-Gly-Ala-Ser-Ala-Ser) are located near each other. All of these repeat sequences are unexceptionally located in the variable regions when compared with other MSA1 molecules. The molecule displays 79% overall identity to the analogous antigen of P. yoelii yoelii strain YM, 70% to that of P. chabaudi chabaudi strain AS, and 38% to that of P. falciparum strain Wellcome. MATERIALS AND METHODS Preparation of parasites. Plasmodium berghei (ANKA strain) was a kind gift from Dr. G. Grau (Geneva, Switzerland). A total of 1 3 104 parasitized mouse erythrocytes were injected into each animal intraperitoneally. Parasitemia was determined by counting the percentage of infected red blood cells after Giemsa-staining. When the parasitemia increased above 70%, the mice were killed and their blood was collected in a heparinized tube. The blood was centrifuged at 650 3 g in a benchtop centrifuge, and the pellet was washed three times with 13 phosphate-buffered saline (PBS; 1mM NaH2PO4, 0.85% NaCl, pH 7.4). The blood cell pellet was then resuspended in PBS for reinfection into naive animals, or for storage in liquid nitrogen. Isolation of RNA and purification of mRNA. Isolation of total RNA was performed using a kit according to the manufacturers instructions (RNA isolation kit; Stratagene, Inc., La Jolla, CA) following lysis of the parasitized erythrocytes. The RNA was resuspended in 500 ml of diethylpyrocarbonatetreated water and its concentration was determined by reading its absorbance at 260 nm (A260). Presumed mRNA was isolated with a biotinylated probe using a purification kit (Promega, Inc., Madison, WI) and concentrated by precipitation with ethanol. Synthesis of cDNA and library construction. A SuperScripty DNA synthesis kit (Gibco-BRL, Gaithersburg, MD) was used for cDNA synthesis. Library preparation was accomplished with this using a lZAPII vector (EcoRIarmed; Stratagene, Inc.) according to the instructions of the manufacturer. To determine the efficiency of construction, 1 ml of the reaction was mixed with 200 ml of the XL1-Blue host cells (Stratagene, Inc.) at an A600 5 0.5 and preincubated at 378C for 15 min, 23 ml of top agar (488C) was added, followed by 15 ml of 0.5 M isopropyl b-thiogalactoside (in water) and 50 ml of 5-bromo-4-chloro-3-indolyl bD-galactopyranoside (250 mg/ml in dimethylformamide), and the mixture was plated onto NZY plates (Gibco-BRL). After incubation at 378C for 68 hr, the ratio of the white recombinant plaques to the background blue ones was determined. Screening the cDNA library. The probe was synthesized according to the previously published partial sequence of the MSA1 gene of P. berghei:9 59-TTAAATAACTTAAATAAATCTGGTTTGGTAGGAGAAGGCGAATCGAAAAAAATATTAGCAAAA-39, and 39-end-labeled with fluoresceinUTP (Amersham, Inc., Arlington Heights, IL). A total of 106 plaques were primarily screened and the putatively positive clones were picked, titered, and secondarily screened. The positive phages coinfected Escherichia coli SOLR strain cells with the helper phages and formed circular phagemids by in vivo excision. The transformed bacterial colonies were screened with a polymerase chain reaction (PCR) procedure. The colony was resuspended in 20 ml of water and boiled for 10 min. Ten microliters of water containing the picked colony was mixed with 10 ml of PCR buffer, AmpliTaqt DNA polymerase (Perkin-Elmer, Norwalk, CT) (2.5 units/ 100 ml), and primers (1.0 mM) each and cycled as follows: denaturation at 948C for 1 min, annealing at 508C for 1 min, and extension at 728C for 2 min for a total of 30 cycles. The clones that produced a 250-basepair (bp) fragment by the PCR were isolated. Identification and sequencing of the cDNA clones. The positive clones were isolated and plasmid DNA was purified by a miniprep procedure.10 The size of the insert was determined by digestion with Eco RI, followed by gel electrophoresis. The inserts were sequenced by the dideoxynucleotide termination method.11 Purification of genomic DNA. Genomic DNA was purified according to the procedure of the manufacturer (Wizardy Genomic Purification Kit; Promega Corp.). The DNA was dissolved by adding 100 ml of DNA hydration solution and the concentration was determined by rapid agarose gel electrophoresis (Promega Corp.). Hot-start PCR. A hot-start PCR was performed according to the procedure of Chou and others with some modifications (the starting temperature was 608C and cycles were 75 sec).12 The 4.3-kb PCR band was excised from the 0.8% agarose gel and purified using a GeneClean IIt Kit (Bio101, Inc., La Jolla, CA) with silica matrix as absorbent and lowsalt solution as recovery reagent.13 Cloning of the PCR product. The PCR product was cloned into the pNoTA/T7 vector using a Prime PCR Clonery cloning system (5 Prime fi 3 Prime, Inc., Boulder, CO). The concentration of purified PCR product was determined by measuring the A260. Competent cells (E. coli JM109) were screened and white clones were selected and analyzed by mini-prep DNA isolation followed by digestion with restriction enzymes. Autosequencing. Sequencing was done using an ABI 377 DNA autosequencer (Applied Biosystems, Foster City, CA) based on fluorescence methodology using the PRISMy Ready Reaction DyeDeoxyy Terminator Cycle Sequencing Kit (Perkin-Elmer). Characteristics of the 39-untranslated region (UTR) of the MSA1 cDNA clones. The cDNA clones were selected based on hybridization with the described probe. Six clones were examined in detail. The insert of one of the cDNA clones was composed of a coding sequence of 1,143 bp and a 39-UTR) of 467 bp (Figure 1). It is apparent that this 39-UTR sequence does not contain a long stretch of a poly(A) tail, although two eukaryotic poly(A) signals (AAUAAA) can be found about 140 and 400 bp downstream of t (...truncated)