| Inhibition of avian influenza virus (AIV)replication by retrovirus-mediated specificantisense RNA and ribozymePh.D. Candidate: MENG Qingwen Supervisor: Prof. YU KangzhenABSTRACTAvian influenza, classified as one of List A virulent infectious diseases by OIE, is an infection and/or disease syndrome caused by influenza A viruses, which are negative strand RNA viruses belonging to the Orthomvxoviridae family. Avian influenza caused by highly pathogenic avian influenza viruses can cause up to 100% mortality.Avian influenza viruses were thought to be limited in their ability to directly infect humans until 1997, when 18 people infected with avian influenza H5N1 viruses occurred in Hong Kong. In 1999, two human infections with avian influenza H9N2 viruses were also identified in Hong Kong. These implied that AIV was very important for the public health. So far the inactivated vaccine was used for prevention and control of AIV. AIV has many subtypes due to the high rate of antigenic variation of HA and NA antigens. This makes it a great challenge to developing different kinds of vaccines including genetic engineering vaccines.In this study, according to the mechanism of antisense RNA, NP, FBI and PB2, which were conservative genes in replication of AIV, were used as targets to design antisense RNA and ribozymes with specific splice abilities. They could inhibit the replication of AIV in cells, which have potential applications in AI prevention.The full-length cDNA and 250bp of 5'-end of NP gene of AIV A/Chicken/Xinjiang/96 (HUNS), and two fragments of 365bp and 931bp from 5'-end of PB1 gene ofA/Goose/Guangdong/ 1/96 (H5N1) (containing complete untranslation region and partial coding region of 5 '-end) were inserted reversely into the plxSN retrovirus vector and named as plxas-NP, plxas-5'-NP, plxas-5'-PBl and plxas-PBl, respectively. The recombinant plasmids were transferred into PA317 cell lines with lipofectin and calcium phosphate precipitation. Positive colony was screened by G418, and the single colony was cultured. Recombinant retroviruses harvested from the cultural supernatant was titred by infecting NIH3T3. Anti-AIV cell lines were established by infection with recombinant virus and named as Mplxas-NP, Mplxas-5'-NP, Mplxas-5 '-PB1 and Mplxas-PBl, respectively. Genes of interest were proved to be integrated into the genome of MDCK cells by PCR, their expressions were detected by nested RT-PCR. Anti-AIV cells were infected with AIV and detected by HA test. The results demonstrated that these retrovirus-mediated specific antisense RNAs and ribozymes could inhibit replication of AIV.Specific ribozymes targeted to AIV A/Goose/Guangdong/l/96(H5Nl ) PB1 , PB2 mRNA were designed with the aid of softwares according to the "Hammerhead structure"described by Symons. The possible cleavage sites (15! site in PB1 and 441 site in PB2) were searched through analyzing the secondary structures of substrate and ribozymes. The following step was to design and construct self-splicing transcriptional vectors in vitro with 5'-cis-Rz and 3"-cis-Rz, which were named as RzPBl > RzPB2, respectively. At the same time, the substrate splicing fragments were also cloned into in vitro transcriptional T-vectors, and named as Tpbl ^ Tpb2, respectively. The 32P labeled ribozymes and target RNAs were got through m vitro transcription, then the target RNAs were processed with in vitro cleavage reactions with ribozymes. The enzyme kinetics analysis showed that RzPBl and RzPB2 could cleave the target RNAs specifically and efficiently. The self-splicing ribozymes with 5'-cis-Rz and 3'-cis-Rz were obtained through double digesting RzPBl and RzPB2 with Xhol and Sal. Then they were cloned into plxSN serially. So the retrovirus plasmids expressing ribozyme and the shotgun retrovirus plasmids expressing ribozyme were constructed and named as pIxRzPBl, plxRzPB2 and pIxAB, respectively. The recombinant plasmids were transferred into PA317 cell lines with lipofectin. Positive colonies were screened by G418, and the single co...
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