Cloning and characterization of a novel gene encoding keratin-like protein from nematode Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis (Nb) is one of the most important parasites in studying Th2 immune response of the host, but little is known about its antigenic structures of the excretory-secretory or structural proteins of the parasite. Here we report cloning and characterization of a novel antigenic gene from cDNA library of Nb adult worm by immunoscreening. The positive clone, KLP-Nb, had an open reading frame of 612 bp that encodes a 203-amino-acid protein and was homologous to 'similar to keratins in a glycine-rich region' of Caenorhabditis elegans. Its expression was confirmed by Northern blotting and IgG enzyme-linked immunosorbent assay. This protein seems to be one of the components of cuticle that covers the nematode body.

Effects of DNA vaccine in murine malaria using a full-length cDNA library.

In an attempt to develop a novel malaria vaccine, we constructed a full-length cDNA library from the erythrocytic-stage parasites of Plasmodium berghei ANKA strain using the plasmid vector pCE-FL, which is driven by an EF321 promoter and a CMV-IE enhancer. Here we report the initial trial to screen this library for DNA vaccine candidates against malaria parasite infection in mice. The library of P. berghei was divided into five groups, each representing 2,000 independent clones. Eight female BALB/c mice were injected with these subsets, with an initial injection directly into the spleen, followed by two subsequent intramuscular injections at 1-week intervals. As a control, the plasmid vector without any insert was used. Two weeks after the last injection, 50,000 infected erythrocytes were injected intraperitoneally. Unexpectedly, the survival rate of the vaccinated groups was lower than that of the control (p = 0.053, by Kaplan-Meyer method), suggesting that these DNA vaccines had adverse effects. There was no difference in parasitemia between the two groups. There was no difference between antibody titers before and after immunization in either group. Accelerated deaths in immunized mice occurred from 7 to 10 days after infection, when fur bristling, shivering and convulsions were observed. These observations suggested the possibility that the vaccination had an adverse effect on the cellular immunity that resulted in the development of severe malaria in BALB/c mice, which do not usually develop cerebral malaria.

Cloning, expression analysis, and chromosomal localization of a novel butyrophilin-like receptor.

We isolated a cDNA clone which shows a similarity with human butyrophilin from a human colon mucosa cDNA library. The cDNA is 1964 bases long, with one open reading frame encoding a protein of 433 amino acids. The deduced amino acid sequence shows an overall homology of 36.5% with the human butyrophilin protein. This gene is mainly expressed in small intestine, colon, testis, and leukocytes. The chromosomal location of the gene was determined on the chromosome 5q35 region by polymerase chain reaction-based analysis with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Isolation and chromosomal mapping of a novel human gene showing homology to Na+/PO4 cotransporter.

We isolated a cDNA clone which shows a significant similarity with the renal Na+/phosphate cotransporter (NPT) from a human intestine mucosa cDNA library. The cDNA is 2626 bases long, with one open reading frame encoding a protein of 497 amino acids. The deduced amino acids sequence shows an overall homology of 48% with the human renal NPT1 protein. This gene is expressed in intestine, colon, liver, and pancreas. Thus, this gene may code for intestinal type NPT or closely related proteins. The chromosomal location of the gene was determined on the chromosome 6p21.3-p22 region by polymerase chain reaction-based analysis with both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel.

Selective production of transgenic mice using green fluorescent protein as a marker.

The low efficiency of transgenic animal production by microinjection has been a serious problem especially for the production of transgenic livestock. We developed a method to selectively produce transgenic mice using green fluorescent protein (GFP) as a marker. Using this method, we obtained eight fetuses and four live-born mice derived from 55 GFP-positive blastocysts. PCR analysis showed 11 out of 12 mice (fetuses and newborn mice) were transgenic. Southern blot analysis showed that 8 out of 12 were transgenic. GFP expression was also observed in bovine blastocysts, suggesting that this method should contribute to the efficient production of transgenic livestock.