Preliminary Studies On SV40 LARGE T ANTIGEN Gene Delivery To Penaeus Chinensis Lymphoid Cells With Pantropic Retroviral Vectors

Recently, the shrimp aquaculture industry has been seriously hampered by shrimp viral diseases. In order to understand the cellular biology, immune response, and pathogenesis of viral infections in penaeid shrimp, an immortalized cell line is urgently needed. The present studies were carried out to transfer the Simian Virus 40 Large T antigen gene (SV40LT) into penaeus Chinensis lymphoid cells with recombinant pantropic ritrovirals. The aim of our studies was to transform shrimp cells and develop techniques for the in vivo culture of shrimp cells.The recombinant vector was constructed by subcloning SV40LT gene (2.1 Kb) from SV40LT gene-carrying plasmid pLLTSN into the BamH I and Xho I sites of pantropic retroviral expression vector pLXRN. The results of enzyme digestion and DNA sequencing indicated that the recombinant retroviral expression vector pLXRN-SV40LT was constructed correctly.The recombinant vector and pVSV-G plasmid were co-transfected into a packaging cell line GP2-293 by calcium phosphate precipitation method. The viral supernatants were collected at 2 days post-transfection. Subsequently the viral titer was determined by NIH3T3, with the result of 4×106 cfu/ml.The recombinant virals were used to infect lymphoid cells of Penaeus chinensis. The positive population was selected by G418-resistant and then subcultured. Cell culture results showed the transduced cells had transformative features and were subcultured for more than 10 passages. In contrast, the untransduced cells could be subcultured only for 3 passages. The transduced cells were contaminated by fungi when subcultured for 12 passages and had to be discarded. The SV40LT gene in the genome of transduced cells was detected by PCR. The telomerase activities of transduced cells and untransduced cells were assayed respectively using the telomeric repeat amplification protocol (TRAP). PCR results suggested that the genomic DNA of the transduced cells contained SV40LT gene. Moreover, telomerase activity in transduced cells was detected to be distinctly higher than that in untransduced cells.