Observation Of Effect Of Juvenile Worms From Schistosoma Japonicum Infected With Retrovirus Containing HTERT Gene

To date, the technologies used for schistosomiasis control still have many deficiencies. Especially, there is no ideal measure in controlling the spread of schistosomiasis japonica. Thirty years ago, some scholars have suggested that the establishment of a stable and continuous passage cell line from schistosome may provide foundation and conditions for finding new schistosomiasis prevention and control technologies. Through decades of exploration for cell culture techniques on Schistosoma japonicum (Sj), however, a continuous passage schistosome cell line has not been achieved. In this study, by an inspiration from transduction of exogenous gene with immortalization function into mammalian somatic cells and successful establishment of immortalized cell lines, we carried out exploration of a biological theory on retroviral vector used to introduce exogenous gene into schistosomula cells, prepared amphotropic retrovirus and pantropic retrovirus containing human telomerase reverse transcriptase gene (hTERT) and observed the integration, transcription, expression and cell proliferation following transduction of exogenous genes into cells or worms from Sj with the two retroviral vectors. Our purpose is to provide an ideal vector for transduction of exogenous gene in the study of schistosome cell immortalization, verify the integration and expression of hTERT gene within schistosome worm bodies and explore the feasibility of inducing cell proliferation. [Objective] To explore biologically theoretical and experimental foundation for introduction of exogenous gene into Sj cells with amphotropic retrovirus vector. [Methods] Amino acid sequence of the receptor for amphotropic murine retroviruses of Rattus norvegicus (rRam-1) was collected from GenBank and its sequence similarity search was performed with Blastp program at NCBI (the National Center for Biotechnology Information) website. Afterwards, the homology analysis were carried out for higher similarity sequences with Cluster W2 program. Conserved regions of rRam-1 receptor and its homologous amino acid sequences were analyzed with RPS-blast and InterproScan tools. Subsequently, protein secondary structures, hydrophobicity, transmembrane regions, signal peptides, subcellular localization signal and post-translation modification sites of Sj proteins homologous to rRam-1 were analyzed and forecasted with several online analysis tools. Based on bioinformatics prediction, Sj-12d cell cultures were infected with amphotropic retrovirus vector containing short segment of exogenous gene and integration and expression of the foreign gene within Sj cells were detected with PCR and RT-PCR analysis. [Results] Similarity search and homology analysis of rRam-1 showed that its amino sequence was highly homologous with amino sequences of Sodium-dependent phosphate transporter from vertebrates and the identities were all more than over 59%. Among them, the identities of amino sequences to the receptor for amphotropic murine retroviruses of Cricetulus griseus (cRam-1) and the receptor for amphotropic murine retroviruses of Homo sapiens (hRam-1) were 93%, respectively. In addition, relatively high homologies with phosphate transporter families from numerous invertebrate species were also found, and the identities were all above 42%. Two proteins of Sj, SJCHGC09605 and SjCHGC05362, were highly homologous to rRam-1 with identities of 54% and 61% and positives of 74% and 72%, respectively. The two Sj proteins were at evolution branches parallel to hRam-1, rRam-1 and cRam-1. Conservation analysis indicated that there were same conserved domain PH04 Superfamily among two Sj proteins, hRam-1, rRam-1 and cRam-1 and all proteins were members of phosphate transporter superfamily. Secondary structure analysis showed that a helixes in SJCHGC09605 and SJCHGC05362 were 68.97% and 39.22%, respectively, and transmembrane prediction showed 7 and 5 possible regions, respectively, which was exactly consistent with hydrophobicity prediction. Subcellular localization and post-translation modification site analysis indicated that the two proteins did not contain signal peptide sequence and subcellular localization signal, and that, lycosylation, phosphorylation and lipid acylation were not found. After infection of cell cultures from Sj juvenile worms with amphotropic retrovirus containing exogenous gene E77.43, the presence and expression of target gene in cells were detected by PCR and RT-PCR analysis and the amplicon of predicted size of 330 bp was generated, which is corresponding with theoretical size. [Conclusion] There were high homology between rRam-1 and either transmembrane protein SjCHGC09605 or SjCHGC05362 on Sj cellular membrane acting as ion transport channel or receptor protein. Furthermore, cell cultures from Sj juvenile worms could be successfully infected with amphotropic retrovirus containing exogenous gene E77.43, indicating the two proteins homologous to rRam-1 may play important roles during infection of Sj cells by retroviruses. These findings provided a biologically theoretical and experimental foundation for transduction of immortal gene into Sj cells with amphotropic retrovirus vector. [Objectives] To establish a cell strain which stably produce amphotropic retro virus containing immortal hTERTgene and observe the integration and expression of hTERT gene in cell cultures from Sj juvenile worm following transduction and its influence on cell multiplication. [Methods] Plasmid pBABE-puro-hTERT was identified by enzyme digestion, PCR and sequencing. The highest puromycin-resistant concentration of PA317 and NIH3T3 cells was determined by gradient dilution method. After transfection of plasmid into PA317 cell with liposome, puromycin-resistant clones were obtained following screening with puromycin and one of clones was randomly selected and cultured for continous passage. PCR, sequencing, immunofluorescence, Western-blot and transmission electron microscopy were performed to identify the puromycin-resistant cell strain and the titer of retrovirus was determined with NIH3T3 cell. Sj-12d schistosomule cells were prepared as described before and cell proliferation was measured with BrudU-ELISA method. Absence of contamination from host cells was confirmed by detection of special rabbit mitochondrium gene with PCR. The highest puromycin-resistant concentration of Sj cells was determined by gradient dilution. After infection of Sj-12d cells with concentrated amphotropic retrovirus and successive screening in puromycin-containing medium, puromycin-resistant Sj cell clones were obtained and selected for continuous cultivation. The integration and expression of exogenous genes hTERT and puror within these cells were observed by PCR, RT-PCR and Western-blot. Proliferation of puromycin-resistant Sj-12d cells was measured by 3H-TdR incorporation, and growth curve plotted by cell count and telomerase activity monitored with TRAP-ELISA method. [Results] The authenticity of pBABE-puro-hTERT plasmid was identified by enzyme digestion, PCR and sequencing. The highest puromycin-resistant concentrations of PA317 and NIH3T3 cells were 6μg/ml and 3μg/ml, respectively. The integration, transcription and expression of exogenous genes hTERT and puror could be confirmed in puromycin-resistant PA317 cell strain by PCR, sequencing, immunofluorescence and Western-blot. Retrovirus particles could be found in culture supernatant and cytoplasm of puromycin-resistant PA317 cells by transmission electron microscopy and the titer of concentrated retrovirus particles was 2×105cfu/ml following infection of NIH3T3 cell line with these virions. After cultivation for 3 days, cell division phase appeared in Sj-12d schistosomule cells and more cell division phases appeared following 10-14 days. Furthermore, DNA synthesis and proliferation were verified by BrdU-ELISA analysis in Sj schistosomule cells cultured for 14 days and the highest puromycin-resistant concentration of Sj cells was 0.5μg/ml. After successive screening for 21 days with puromycin-containing medium, puromycin-resistant clones could be found in Sj-12d cells infected with amphotropic retrovirus and one of clones was randomly selected for successive cultivation. The integration, transcription and protein expression of exogenous genes hTERT and puror within puromycin-resistant Sj-12d cells could be detected by PCR, RT-PCR and Western blot, respectively. However, the copies and transcription level of foreign genes were low within schistosomule cells.3H-TdR incorporation experiment showed that both puromycin-resistant Sj-12d cells and normal cultured Sj-12d cells exhibited some extent proliferation ability but no significant difference was found between them (P>0.05). Telomerase activity could not be detected in puromycin-resistant Sj-12d cells using TRAP-ELISA method. Within 4 weeks, cultivated cells grew fast and then slowly. The number of dead cells and degenerative cells gradually increased and, finally, the cells all died. [Conclusion] After Transfection of PA317 cell with retroviral plasmid pBABE-puro-hTERT, a cell strain, PA317/hTERT, stably producing amphotropic retoviral particles containing hTERT gene was constructed successfully. Integration, transcription and expression of exogenous genes hTERT and puror could be detected in Sj-12d cells infected with amphotropic retrovirus, but the telomerase activity within them could not be activated and the cell proliferation ability could not also be improved.[Objectives] To enhance the ability of retrovirus to infect schistosomes, explore the feasibility of preparation of pantropic retrovirus by cotransfection into packaging cell line with plasmids pVSV-G and pBABE-puro-hTERT and observe integration, transcription, expression and protein locations of exogenous genes in worm bodies post infection of Sj juvenile worms with pantropic retrovirus. [Methods] GP2-293 package cells were cotransfected with plasmids pVSV-G and pBABE-puro-hTERT, and the supernatants of cell cultures were collected 48h post transfection and then NIH3T3 cell line was infected with the concentrated supernatants mixed with polybrene. Puromycin-resistant clones were obtained through successive screening for 12d with puromycin-containing media and the numbers of clones were counted for virus titers. Thereafter, puromycin-resistant clones were randomly selected for continuous culture, integration of exogenous genes hTERT and puror in cells was detected with PCR and expression of hTERT gene was determined with immunocytochemistry stain. Pantropic retrovirus were added to in vitro cultured Sj-12d schistosomules. Medium was replaced 24h after infection and worms were successively cultured for 6d. Integration of exogenous hTERT gene in worm bodies was confirmed by PCR and Southern hybridization analysis, and, simultaneously, RT-PCR, Western blot and immunohistochemical staining were also adopted for study of transcription, expression and protein location of exogenous hTERT gene in worm bodies. [Results] After counting, the titer of pantropic retrovirus was 3.2x 108. Two fragments of predicted sizes of 145 bp and 204 bp of exogenous genes hTERT and puror were amplified from puromycin-resistant NIH3T3 cells with PCR. Protein expression of hTERT gene in cells could be detected with immunocytochemical staining and the expression location was mainly in nucleus. Specific amplicons of exogenous genes hTERT and puror in size of 145 bp and 204 bp were generated from virus-treated schistosomule genomic DNAs using PCR and RT-PCR. Southern hybridization showed that multi-copies of exogenous hTERT gene were integrated into worm chromosomes. Western blot displayed that exogenous hTERT gene was expressed in worm bodies post infection with virus and most expressions were found in subtegumental regions of oral sucker, ventral sucker and the posterior of Sj larval worms by immunohistochemical staining. [Conclussion] Pantropic retrovirus was successfully prepared following cotransfection into packaging cell line with plasmids pVSV-G and pBABE-puro-hTERT and multicopy integration, transcription and protein expression of exogenous hTERT gene within subtegumental regions of suckers and the posterior of Sj larval worms was confirmed post infection of live Sj juvenile worms with this virus.