Cloning, Expression Of Structural Genes And Construction Of Full Length Genome Of Strain CC Of Newscastle Disease Virus

Newcastle disease virus (NDV) infection results in Newcastle disease (ND), a widespread and highly contagious anthropo zoonosis, which greatly threaten birds and human health. As a kind of paramyxovirus, NDV can infect many birds, such as fowls, Meleagris gallopavos, struthios, mallards, Pigeons, and gooses. Spreading swiftly served as a character to the ND endemic. Vaccine inoculation is a safe and effective way to prevent NDV. For the inactivated vaccine which was widely used currently existed latent danger, researchers wanted to find some new type vaccine to substitute the inactivated vaccine. The current technique of gentic manipulation of RNA virus by reverse genetic system have developed quickly,it has been used in NDV to construct viral vector,and then to develop effective vetor-viral vaccine against NDV.In this study,the L gene,the end sequence of genome and intergenic regions(IG regions) were cloned to make clear the gene background of NDV CC strain thoroughly. Meanwhile, three eukaryotic expression plasmids pCI-NP, pCI-P, pCI-L were constructed and transfected into CEF respectively, the results were detected by Indirect immunofluore- scence assay and RT-PCR. In addition, the full length cDNA was divided into 5 sections cloned and a plasmid pKS(-)-ND15 about 11000bp including the 3′end and 5′end of the genome of NDV CC strain was constructed.Two pairs of special oligonucleotide primers were generated to amplify the L gene of NDV CC strain by RT-PCR which was divided into two fragment named as L1 and L2. And then they were sequenced after being inserted in pMD18-T vector respectively. The results showed The nucleotide sequence of the L gene was 6615bp long encoding 2204 amino acids.By restriction enzymes digestion and sequence analysis,the result showed two sections were obtained successfully. The cloned L gene sequence showed 86.1%～98.5% nucleotide identity and 92.8%～98.4% amino acid identity compared with the L gene sequences from NDV strains B1,Clone30,LaSota,ZJ1,Herts33,Ulster67,IT227,Largo71,HBV4,Mukteswar, highest homology with B1 strain about 98.5% and 98.4%. In addition, at the 51st,63rd,196th,1492nd,1527th and 2139th position of amino acid sequence, the velogenic strain were L,S,V,N,N,R, but the low virulent strain were I,T,A,D,H,K.In order to obtain the the 3′end and 5′end of the genome of NDV CC strain,two primers of the two section were designed,fragment of 3′end and fragment of 5′end were cloned by RT-PCR. And then they were inserted into the pMD18-T vector to construct two recombinant plasmids respectively,and the objective fragments were 55bp and 114bp respectively. By sequence analysis, The cloned 3′end squence showed 87.3%～100% nucleotide identity and 77.8%～100% amino acid identity compared with the 3′end sequences from NDV strains B1,Clone30,LaSota,ZJ1,Herts33,Ulster67,IT227,Largo71,HBV4,Mukteswar, with highest homology of LaSota strain. In addition,at the 16th and 19th position of amino acid sequence, the velogenic strain were S and Y, but the low virulent strain were L and H. By the same way, the nucleotide identity and amino acid identity of 5′end squence both were 100%, highest with B1 strain. In addition, at the 6th and 12nd position of amino acid sequence, the velogenic strain were T and I, but the low virulent strain were L and T.Meanwhile, the intergenic regions were cloned by RT-PCR too, they were sequenced after restriction enzymes digestion. The nucleotide sequence of NP-P was 295bp long, the nucleotide identity and amino acid identity were 98.3% and 96.9%,highest with B1 strain. The nucleotide sequence of P-M was 215bp long, the nucleotide identity and amino acid identity were 99.1% and 97.2%, highest with Clone30 strain. The nucleotide sequence of M-F was 159bp long, the nucleotide identity and amino acid identity were 99.4% and 100%,highest with B1 strain. The nucleotide sequence of F-HN was 206bp long, the nucleotide identity and amino acid identity both were 100%,highest with B1 strain. The nucleotide sequence of HN-L was 235bp long, the nucleotide identity and amino acid identity both were 100%,highest with B1 strain.For the construction of expression plasmids for the NP, P and L protein,the ORFs of NP,P and L gene of CC were acquired by RT-PCR, and were cloned into expression plasmid pCI(Promega)respectively to construct the eukaryotic expression plasmids pCI-NP,pCI-P,pCI-L and transfected into CEF respectively, the results were detected by Indirect immunofluorescence assay and RT-PCR.After analyzing the restriction enzyme sites of genome and transcription vector pKS(-),seven restriction enzymes(XbaI,ClaI,SphI,SpeI,SacI,AflⅡand SalI)were chosen to construct a transcription vector containing the full length genome cDNA of CC.Firstly,two genomic fragments the nucleotide sequence assembly model of which wereXbaⅠ-1-3522(ClaⅠ)-3636-XbaⅠandXbaⅠ-10959-11372-(AflⅡ)-15186- (Sal)respectively were acquired by RT-PCR and cloned into pMD18-T vector.Secondly,the two fragments were ligated by utilizing the XbaI site.Thirdly,by utilizing the unique sites existed in the CC genome or artificially introduced and the artificially synthesized site in PCR products,three remaining genomic fragments the nucleotide sequence assemble model of which were XbaI-3429-3522(ClaI)-6111(SphI)-6410-SalI, XbaI-5415-6111-(SphI)-8390(SacI)-8515-(SalI), XbaI-8194-8390-(SacI)-11372-(AflⅡ) -11792-(Sal) respectively were cloned into transcription vector successively and respectively,and a transcription vector pKS(-)-ND12345 containing the full length genome cDNA of CC will be finally acquired. As a phase, a plasmid pKS(-)-ND15 about 11000bp including the 3′end and 5′end of the genome of NDV CC strain was constructed.Thus, the constructions of pCI-NP,pCI-P,pCI-L and pKS(-)-ND15 were the basis for the reverse genetics manipulation of NDV.