Karyopherin alpha 2 (KPNA2) is associated with the natural resistance to Schistosoma japanicum infection in Microtus fortis.

Microtus fortis is a naturally vertebrate host resistant to Schistosoma japonicum infection. In order to understand the molecular mechanism and identify the molecules related to the natural resistance to S. japanicum infection of M. fortis, we screened a gene pool named gE76 by expression cloning and proved it to have high anti-schistosomula effects in our previous work. In this study we identified a clone named gE76.44. We found that the conditioned medium of pcDNA1.1-gE76.44 caused 14.0% schistosomula death rate in 96 h, which was significantly higher than that of negative control (P<0.05). The gE76.44 was sequenced and the full-length cDNA was 2008 bp with ORF of 1590bp encoding a polypeptide of 529 amino acid residues. Bioinformatics analysis indicated it was the homologue of karyopherin alpha 2 (KPNA2). To further confirm its anti-schistosome activity, we inserted full length of Mf-KPNA2 (KPNA2 of M. fortis) gene into a retroviral expression vector pLXSN and packaged the recombinant virus with PA317 cells. Mice infected with S. japanicum cercariae were administrated by intravenous injection through tail vein and treated with pLXSN-KPNA2. Adult worms and egg reduction were counted after heart perfusion of mice 42 d after infection. We found that compared with the control, mice injected with Mf-KPNA2 had 39.42% worm burden reduction and 76.50% reduction in LEPG (liver eggs per gram) (P<0.01), indicating its anti-schistosome effect of Mf-KPNA2 in vivo. Taken together, the results suggested Mf-KPNA2 as a novel anti-schistosome molecule in vitro and in vivo.

Identification of the resistance of a novel molecule heat shock protein 90alpha (HSP90alpha) in Microtus fortis to Schistosoma japonicum infection.

Microtus fortis is a naturally resistant vertebrate host of Schistosoma japonicum by preventing completion of parasite's life cycle. Sera of M. fortis were found to have anti-schistosome effect in vitro and in vivo. In order to identify genes associated with the anti-schistosome effect of M. fortis, we screened a M. fortis marrow cDNA expression library by expression cloning and identified a 331-bp clone gC14.75. It was the homologue of heat shock protein 90alpha (HSP90alpha). Full-length of M. fortis HSP90alpha gene, Mf-HSP90alpha, was amplified according to gC14.75 and Cricetulus griseus HSP90alpha. To test the potential anti-schistosome function of Mf-HSP90alpha, we prepared conditioned medium of Mf-HSP90alpha and added it to schistosomula cultured in vitro. It caused 27.0% schistosomula death rate in 96h, which was considerably higher than that of negative control. We transferred Mf-HSP90alpha by retroviral expression vector pLXSN into mice to investigate its anti-schistosome effect in vivo. Compared with those of DMEM injection control, mice injected with Mf-HSP90alpha recombinant retrovirus had 40.8% worm burden reduction and 57.9% reduction in liver eggs per gram (LEPG) indicating its anti-schistosome effect in vivo. Taken together, our results suggested Mf-HSP90alpha as a novel anti-schistosome molecule in vitro and in vivo.

[In vitro killing activity of the fractional proteins from microtus fortis serum to Schistosoma japonicum juveniles].

OBJECTIVE: To explore the killing effect of different fractional proteins from Microtus fortis (Mf) serum to S. japonicum juveniles, and to find possible association of the proteins with the natural resistance to schistosome infection. METHODS: The proteins in Mf serum were separated by means of ion-exchange column chromatography and molecular sieve column chromatography. After desalted by dialysis and lyophilized, the proteins were dissolved in DMEM medium which contained 300 U/ml penicillin and 300 microg/ml streptomycin, and the two-day old schistosomula were added in for in vitro cultivation (100 +/- 20/well). The killing activity of the fractional proteins to the juvenile worms was defined by mortality rate. RESULTS: 58 fractional proteins were separated from Mf serum, in which six proteins were confirmed to have a significant killing activity to schistosomula. The mortality of schistosomula all reached 37% and above, and the highest mortality (87.5%) was observed in the fraction 18.1, while the negative control was 25.09% (P < 0.01). CONCLUSION: Some fractional proteins in Microtus fortis serum show an effect in the natural resistance to schistosome infection.

[Comparison of specific genomic DNA fragment between Microtus fortis calamorum and Microtus fortis fortis].

Microtus fortis(Taxonomy ID: 100897), also named as reed vole, is classified as Microtus, Micotinae, Cricetidae, Rodentia, Mammalia on taxonomy. Microtus fortis mainly distributes in China. Some areas of Russia, North Korea and Mongolia close to Northeast borderland of China also have a small number of Microtus fortis in distribution. Microtus fortis in China has principally 4 subspecies, and most of them live is the drainage area of Yangtse River. Schistosoma japonicum (one of commonly parasites in China) can infect about 40 kinds of mammalian animals, including the human being, but could not infect Microtus foris. It is known as the only animal in Dongting Lake region of China which has the ability of natural resistance to Schistosoma japonicum. The Microtus fortis domesticated in laboratory has the same biological characteristics as the wild one and these characteristics could be inherited to its progeny steadily. We got a specific DNA fragment from genomic library of Microtus fortis. This DNA fragment in genomic DNA of human beings, Kunming mice, Balb/c mice and C57BL/6J mice could not be detected by dot blot hybridization and PCR, apart from genomic DNA of Microtus fortis. In this report, the differences of genomic DNA in 34 Microtus fortis were compared between Microtus fortis calamorum(Dongting Lake region of southern China) and Microtus fortis fortis (Ningxia province of northern China). The residing localion of these two subspecies is far away about 1,200 kilometers from each other. The genomic DNA of Microtus fortis calamorum and Microtus fortis fortis were extracted and amplified by PCR according to the specific genomic DNAs sequence of Microtus fortis reported previously (Accession number in GenBank: AF277394). The amplified DNA fragments were inserted into pGEM-T easy vector and sequenced. The DNA fragment sequencing results from the two subspecies were compared to detect whether there was any difference. 19 alleles were found from Microtus fortis (20 of Microtus fortis calamorum and 14 of Microtus fortis fortis). The results of multiple sequence alignment showed that 25 single nucleotide polymorphism (SNP) sites were existed in the different Microtus fortis individuals, including transition (G-->A,A-->G,T-->C,C-->T), transversion(G-->T,A-->T,T-->A,C-->A), insertion (CA) and deletion (TGTTTT). The difference of genomic DNA from two subspecies were obvious, especially on the sites of 146, 192, 223, 224 and 235. The insertion of CA on the sites of 223 and 224 as well as A-->G transition on the site of 235 was only occurred in Microtus fortis fortis. They are not found in Microtus fortis calamorum so far. They could be divided into two groups according to phylogenetic tree analysis results, one was the genomic DNA of Microtus fortis calamorum and the other one was that of Microtus fortis fortis. However, the homologues reached up to 98% between two subspecies. These results are very important for us to further understand the genetic background, biological characteristics, evolutionary rule and the anti-schistosoma japonicum mechanism of Microtus fortis at the molecular levels. The specific base changes of the DNA fragment between the two subspecies are probably correlative with the animal immigration, survival conditions, and species evolution.

[Genetic modification of porcine organs for human in xenotransplantation].

Xenotransplantation (XP) from pig into human has been considered as means to overcome the great lack of donor organ available in transplantation surgery. In order to weaken rejection between human and pig,approaches of gene targeting have been proposed to eliminate "rejection gene"alpha-1,3GT from porcine cells directly and accurately.alpha-1,3GT knockout pigs can be produced by nuclear transfer cloning with the porcine cells(knocking out alpha-1,3GT). Besides the genetic modification of alpha-1,3GT in porcine cells,there is another technical way to interdict activity of complement in series for human by XP. However, porcine endogenous retroviruses (PERV) during XP has been thought to not be negligible in being transmitted with the xenograft to the human recipient. Therefore, it is importance task that we should not only knockout alpha-1,3GT and relative molecules from pigs, but also ensure safety in public health of XP from PERV.