Optimization And Reformation Of Gene Transfer Vectors Derived From Equine Infectious Anemia Virus

Equine infectious anemia virus (EIAV) is a non-primate and the genetically simplest lentivirus, which has the abilities to integrate efficiently their genome into the chromosomal DNA of host cells and be stably replicated and transmitted to all of the progeny of these cells offering the potential for long-term expression, can be as gene transfer vectors to introduce the foreign target gene into host cells.Chinese equine infectious anemia virus donkey leukocyte attenuated strain (EIAV-DLA) is a unique lentiviral vaccine strain used widely for prevention and control of equine infectious anemia (EIA) in China up to now. A series of replication-deficient EIAV gene transfer vector plasmids, envelope gene-deleted gag/pol expression plasmids and VSV-G gene expression plasmids have been constructed and the gene transfer vector systems of these three plasmids were established based on an infectious molecular clone, pOK8266, derived from the EIAV-DLA.This study was a continuous job after the construction of EIAV three-plasmid gene transfer vector systems. A series of optimization and reformation methods were done to improve the gene transfer efficiency, trying to construct EIAV four-plasmid gene transfer vector systems. The results were as follows:1.The EIAV vector pcPPTWPRE was constructed by inserting the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) into pcPPTPRE(+) by sense. Results of transfection showed that the pcPPTWPRE could express the EGFP more efficiently than pcPPTPRE(+) in HEK293 cells, while a little efficiently in DF-1 cells.2.The core CAG promoter was amplified by polymerase chain reaction(PCR)from the plasmid pCAGG-MCS and subcloned into pcPPTWPRE to obtain recombinant plasmid pcPPTEIACAGP. Results of transfection showed that the core CAG promoter could express the EGFP more efficiently than CMVIE promoter in DF-1 cell,while the equal efficiency with CMVIE promoter in HEK293 cell.3.The EIAV vectors pWCAGP0.8 containing part of chickenβ-actin intron 1 and pWCAGP1.6 containing the full intron were constructed by Restriction Enzyme digestion to investigate the influence of the intron on the EGFP expression of EIAV vectors. Results of transfection showed that the chickenβ-actin intron 1 could not increase the EGFP expression, but decrease the expression in HEK293 cell and DF-1 cells. 4.The sequences of RRE and RU5 were amplified by PCR from the plasmid pOK8266T and subcloned into the EIAV gag/pol eukaryotic expression vector pCDGP in order. It was found that the R-U5 area of the EIAV 5`-LTR was critical to the transcription of the gag/pol gene. It was also demonstrated that the Rev protein and Rev-response element (RRE) were essential to the expression of the EIAV gag/pol expression vectors. The reformed EIAV vector pSRU5GPR could express Gag/Pol protein detected by IFA after cotransfection with pCDREV expression vector.The work provides the theoretical basis and technical supports for the construction of EIAV four-plasmid gene transfer vector systems. It also offers a new approach for gene therapy, preparation of transgenic animals and the study of gene function.