| Adenoviral (Ad) vectors are being extensively investigated. Since the first use of Ad expression vectors in the early 1980s, Ad vectors have shown great promise as vaccine vectors. In addition, Ad vectors are being extensively explored for their applications in gene therapy. Ad vectors are also becoming a therapeutic tool for cancer therapy, by virtue of their own oncolytic nature. Several preclinical and clinical trials have demonstrated therapeutic potential of Ad vectors in the treatment of a variety of cancers. Ad vectors are also being explored for genetic immunotherapy of tumors, as a clinical modality in conjunction with conventional chemo-and radiation-therapy.Ad vectors offer several advantages as vaccine vectors and meet the most important criteria of an ideal vaccine vector in terms of efficacy, safety, and stability. Ad can infect a broad range of both actively dividing as well as post-mitotic quiescent mammalian cells. Transgene expression with Ad vectors is usually robust and can be further enhanced by driving gene expression with strong heterologous promoters. Generation of various Ad vectors is fairly straightforward and the vectors can be easily grown to large scale in tissue culture. Since Ad vectors are replication-defective, there is no serious threat of horizontal transmission. The vector genome does not integrate into the host chromosome and mostly remains epichromosomal, thus making their use safe without a potential risk of insertional mutagenesis. Ad vectors induce strong immunity when administered via parenteral (subcutaneous, intravenous, intramuscular, or intraperitoneal) or mucosal (oral or intranasal) routes. The latter is often desired as most infections occur at mucosal surfaces. Furthermore, Ad vectors appear to be safe for use in humans as human Ad (HAd) serotype 4 and 7 have been used as live oral vaccines in military recruits in the USA for several decades without any known adverse effects Clinical use of HAd vectors is hampered by vector immunity that is prevalent in the majority of the human population, and also develops rapidly after first use of the vector. Nonhuman Ad are not prevalent in humans, and are not cross-neutralized by HAd neutralizing antibodies. Significant progress has been made in the understanding of the biology of these vectors. These vectors efficiently transduce human cells in culture and circumvent preexisting HAd immunity. An ever increasing number of preclinical and clinical studies demonstrating the utility of HAd vectors for therapeutic or prophylactic gene delivery have provided further momentum to the research efforts in the area of developing nonhuman Ad vectors. In addition, these nonhuman Ad vectors also present a platform for designing improved vaccines for veterinary use.Package of the ad vector depending on a special cell line which counld provide protein that essential for the package process of the virus.In the reserch of human adenovirus vector, several special 293 cell lines have been constructed in which E1 and a number of pre-genes had been packed. Accordingly, because DK cell is sensitive to CAV, Nowdays an auxiliary DK cell line named DK/E1 which contian E1 region for reserching CAV vector has been established to speed up the research process of CAV vector, we have establishted a strain of DK cell line that contains more function genes of CAV. The strain will definitely provide useful platform for further reserch.We cloned the gene of anti-hygromycin to the prepared Ad vecor pPolyⅡ-CAV-2-ΔE3(NF). The processes about the clone is detailed as following: Digesting pSilencer2.1-U6 hygro with SspI, we got the expressing cassette of anti-hygromycin gene, then, clone it into pBS-NN vector at EcoRV site which contain two NotI sites located at the two ends. Digested the new plasmid pBS-NHN vector with NotI , the anti-hygromycin gene contains two NotI sites was obtained,finally,we clone the fragment into pPolyⅡ-CAV-2-ΔE3(NF), got a new plasmid named pPolyII-CAV-2-Hygro. Under the Pressure of hygromycin B added in the culture medium with 100μg/ml, transfected DK cell with pPolyII-CAV-2-Hygro, then, got 12 clones. Identified the cell clones through the method of PCR. The primers used in PCR test is designed according to the CAV-2 sequences published in the database of GeneBank.Finally, amone the 12 clones we got a best DK strain named DK/CAV-2.Seven pairs of primers were designed to detect the transcription of CAV gene in the cell line. The result revealed that all seven genes could be amplified normally. To detect the expression of the CAV gene by western-bloting, we got the result that the protein of the hexon was expressed. Through detecting the virus titre generated by DK/CAV-2 and normal DK cell line with TCID50 and hemagglutination experiment, we got the titer of HA were 24 and 23, respectively. Transfection experiment with the whole genome of CAV-2-GFP into the two cell lines, revealed that there were 6/15 wells of DK/CAV-2 cell appeared the typical pathological changes during the 5~7days after transfection, however, only 2/15 wells of normal DK cells were observed in the same condition. From all experiments above, it proved that DK/CAV-2 was more effectively in virus packaging than normal DK. In addition, DK/CAV-2 grew faster than normal DK in the same serum circumstance, and even could grow with no serum. We could still detect the CAV gene transcription from the 10th cells and 30th generation of cells. Based on the RT-PCR experiment. It indicated that we have successfully established a strain of helper Dk cell line which can also stable inherited. Our cell will make for the further reserch about replication defective CAV vector which contains more exogenous gene and more effective.
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