The Construction And Identification Of Full-length CDNA Vector Of The NDV Lasota Strain Genome And The Helper Plasmid Of NP And P Gene

Newcastle disease virus (ND) is negative-strand RNA viruses with non-segmented genomes,ND is contagious disease that many poultry can be infected,which has caused huge losses in poultry industry. Newcastle disease vaccines have been used since the last century, but the disease haven’t eradicated yet. Currently, Newcastle disease virus used as a vaccine vector was carried out extensively research in humans and animals. As a vaccine vector, NDV has many advantages, such as easy to vaccination, high-titer, low-cost; only one serotype, relative stability in hereditary, less possible to restructure and virulence back in strains; express foreign proteins at high level.That is revolutionary for RNA virus by reverse genetics basic and application studying, the reverse genetics study of NDV is very extensive. Currently, the main research is that the Newcastle disease virus to express foreign genes as a vaccine vector. In order to start virus transcription and replication, it’s needed that NP protein and virus genomic RNA to form ribonucleoprotein complex (RNP)in addition to viral genomic RNA, then P protein and L protein form large polymerase complex to start virus replication with co-expression. Therefore, not only the full-length cDNA of the viral genome was required, construction of the helper plasmid is essentialto save the Newcastle disease virus. The results were summarized as follows:1. Construction of full-length vector of the virus genome9or10day-old chicken embryos was vaccinated NDV, incubated7days, viral RNA in allantoic fluid was extracted, and was reversed transcription into cDNA. According to the analysis of the virus genome sequence and vector restriction enzyme sites, viral genes were divided into seven fragments to amplify. The vector pBR322was selected as a transcription vector, the hepatitis D virus synthase sequence and T7terminator were connected into the pBR322vector, then vector p3TH was obtained. High-fidelity DNA polymeras used to amplified the seven gene fragments by PCR, the resultes is correct as expected, the blunt-end vector was connected, recombinant plasmids were identified by enzyme cutting, PCR and sequencing analysis. The sequence showed that cloned genes has the homology99%with GenBank accession number JF950510.1after BLAST. NDV2and NDV3were linked into pVAX vector in due order, NDV6. NDV5and NDV4were linked into pBluescript Ⅱ SK vector one by one, then NDV1、NDV7、NDV23、 NDV456were connected to p3THin turn, so the vector of full-length cDNA of viral genome was successfully constructed. 2. Construction and Expression of the helper plasmid pCl-NP and pCl-PNP、P gene was amplified by RT-PCR, the result was correct as expected, NP、P gene was connected into the eukaryotic expression vector pCl-neo, the recombinant plasmid pCl-NP、pCl-P was obtained. Plasmid pCl-NP、pCl-P were identified by PCR, restriction-enzyme analysis and sequencing, the sequence result showed that cloned genes has the homology99%by BLAST. Recombinant plasmids were transfected into BHK-21cells respectively, then the expression of NP、P genes were detected and confirmed by RT-PCR, WB analysis and indirect immunofluorescence analysis. Specific bands were detected by RT-PCR; a53KD NP protein and a44KD P protein were detected by WB; more fluorescent can be detected in NP and P protein by indirect immunofluorescence. For this reason, the helper plasmid pCl-NP and pCl-P was constructed successfully.In this study, the vector of full-length cDNA of viral genome and the helper plasmid pCl-NP and pCl-P was successfully constructed, which laid the foundation of studying reverse genetics operating system of NDV in our laboratory.