Cloning and Nucleotide Sequence Analysis of the Nucleocapsid Protein Gene of NDV Strain V_4 and Construction of Eukaryotic Vector

Cloning is necessary for the study of biochemical function of the nucleocapsid protein gene of Newcastle disease virus(NDV).The purified genome RNA of NDV was used as template.According to the reported NP gene sequence of NDV strain Beaudette C,a pair of 19 mer primers were designed and synthesized.The NP gene of NDV strain V 4 was amplified by reverse transcription polymerase chain reaction(RT PCR).The amplified products were analyzed by agarose gel electrophoresis,and there appeared a specific fragment about 1.5 kb as expected.The RT PCR product of strain V 4 was cloned into the pUC119,then the positive clone was tested by restriction endonuclease analysis and sequenced by Sanger method.The result suggested that it was the NP gene of NDV strain V 4.The NP gene sequence of NDV V 4 was compared with the published NP gene sequence of NDV Beaudettec C,La Sota and D26,the affine are 90.64%, 90.17% and 98.03% respectively.Difference rates of the sequence of amino acids of the NP gene were 4.50%,5 93% and 2.45% respectively.Cloned plasmids which contained the nucleocapsid protein genes of NDV,strain V 4 and pcDNA 3 were used for construction of eukaryotic vector which contained CMV promoter and signal sequence of BGH polyA,which expressed the nucleocapsid protein.