Studies On Minigenome Of Human Respiratory Syncytial Virus

Objective: Human Respiratory Syncytial Virus (RSV) is the most important pathogen of pediatric respiratory tract disease worldwide, till now no effective preventive way is available. The genetically engineered attenuated living RSV through the use of reverse genetics technology is of good safety and immunogenicity, and is a potential RSV vaccine candidate, which can be rescued by transfecting infectious cDNA of the RSV genome into host cells. Because such investigation needs full understanding of the characteristic of RSV genome, consisting of a single strand negative RNA, and the technology relating to RSV RNA rescue, we plan to construct minigenome of RSV as the initial step, with reporter gene or the partial viral genome located between the RSV gene start signal and gene end signal, flanked with the cis-acting elements necessary for RSV replication and transcription such as the leader and trailer sequences. In order for the minigenome to function properly, it is necessary to transfect minigenome into host cells together with recombinant plasmids expressing requisite proteins (N, P, L, M2-1) or with RSV as helper virus. At present, T7 RNA polymerase (T7 RNP) usually be used to synthesize minigenome cDNA into viral genome RNA. Here in this paper, we intend to study the function of T7 transcriptional system, construct the minigenome containing Enhanced Green Fluorescent Protein (EGFP), and then transfect the minigenome into cell line BSR T7/5 expressing T7 RNP infected with RSV. Exploiting EGFP as reporter gene, we can conclude if the constructed minigenome function well, which will settle a basis for further investigation on RSV infectious cDNA.Methods: According to the complete sequence of T7 RNP, a pair of primers containing EcoR V and Xho I were designed and synthesized. The genome of E.coli BL21(DE3)containing T7 RNP gene was extracted and amplified by PCR, and then the PCR product was subcloned into the eukaryotic vector pcDNA3.1(+). Meanwhile, TER and EGFP cut from vector px8δt and pGEM-T easy/EGFP, respectively, were cloned into pcDNAII sequentially with EGFP located exactly between T7 promoter and TER. The resulting two recombinant vectors were co-transfected into BHK cells and observed in 48 hours after transfection under fluorecent microscope. In the same time, all RSV requisite cis-acting elements including gene start (GS) signal, gene end (GE) signal, genomic promoter leader and antigenomic promoter trailer sequences together with multiple clone sites were synthsized sequentially and disignated as GSGE. Then two pair of specific primers, each containing T7 promoter at upstream and downstream, respectively, were used to obtain GSGE1 and GSGE2 from the above GSGE by PCR. Following that, these two genes and EGFP were cloned further into vector px8δT and named as px8δT/GSGE1/EGFP and px8δT/GSGE2/EGFP. In order to make sure the minigenomes function well, they were transfected into BSR T7/5 by using lipofectamine 2000 and RSV as helper virus to provide the necessary trans-acting elements supporting minigenome's replication and transcription. After 72h, the expression of reporter gene EGFP was detected under fluorescent microscope.Results: The eukaryotic expressive vector pcDNA3.1(+)/T7 RNP and the recombinant vector pcDNAII/EGFP/TER were successfully constructed and the fluorescence of EGFP can be observed in the transfected BHK cells under fluorescent microscopy. The minigenomes expressed EGFP and functioned well in the BSR T7/5 with RSV as helper virus.Conclusions: The eukaryotic vector pcDNA3.1 (+) /T7 RNP can express T7 RNP, which interacts with T7 promoter and TER in the vector of pcDNAII, leading to EGFP expression. The minigenomes have been constructed successfully, which put a firm solidation for further investigation of RSV reverse genetics.