Application Of Reverse Genetics Manipulation System Of NDV-Construction Of L Genes Expression Vector

The reverse genetic system(RGS)is to get the genome of virues, construct cDNA clone which inserted the promoter of RNA polymerase , transcript into virus's RNA in vitro, then infect host cells or sensitive cells and a mount of virus as parent were proliferated.Reverse genetics manipulation technique or rescue of virus is the technique of reverse genetic system. The reverse-genetic technique is to recover viruses from cloned cDNA provides a way not only to investigate the function of virus proteins and genetic elements but also to express additional proteins by the insertion of new genes into the viral genome. This technique provides a new method to generate improved vaccines and vaccine vectors.Newcastle disease (ND) is a serious infectious disease of poultry caused by Newcastle disease virus(NDV). NDV is a member of the genus Avulavirus in the family Paramyxoviridae. NDV strains are classified on the basis of their pathogenicity for chickens into highly pathogenic(velogenic),intermediate(mesogenic)and athogenic(lentogenic) strains. Lentogenic strains like NDV strain LaSota are used as live vaccines to protect poultry against Newcastle disease. NDV strain LaSota is used as a vaccine vector for its express foreign gene stably in vivo, and NDV do not show a measurable rate of homologous RNA recombination. It is stability, safety and low-cost. Live NDV vaccines are widely used in the poultry industry with a proven track record of efficacy and safety; expressing foreign proteins at high levels. NDV naturally infects the respiratory tract, leading to he induction of both mucosal and syste micimmunity. Thus, NDV as a vector has a number of applications and attracted more attentions than ever.NDV is a non-segmented, negative-stranded RNA virus. To constructing its reverse-genetic system need full-length genome cDNA recombinant expression plasimd and recombinant helper plasimd inserted sequences which coding RNA replicase and nucleoprotein to cotransfecte the host cells. Between the 6 kinds of proteins coding by NDV genome, protein L is the polymerase of RNA virus, and the complex of protein L and protein P has the activity of polymerase. In the NDV reverse-genetic system, nucleocapsid protein must be interact with Auxiliary protein which Manipulate RNA synthesis, with the help of protein L and P, NP and RNA Packaged into infectious ribonucleoprotein complex (RNP), for NDV transcription and translation, and starting NDV replication. So, to constructing L expresstion vector is the basic of the NDV reverse-genetic system.In this research, NDV LaSota strain was grown in sensitive vero ce1ls, vira1 RNA was extracted from the infected cells using Trizol according to the manufacturer's protocol. After extraction of virus RNA , the approximate 6.6 kb full-length cDNA of L gene is amplified by long RT-PCR. The L gene was cloned in the plasmid pGEM-T easy. The fragment L from pGEM-T easy vector was subcloned into pcDNA3.1 (+) expression vector. RT-PCR, restriction digestion and sequencing analyses, immunoenzymatic method were used to identification. The result shows that the L gene has been transcript effectively, and the recombinant protein L expression vector was successfully constructed. This research will helpful for further study of NDV reverse-genetic system, and the optimization of long RT-PCR will helpful for simplify the construction of clones.