新城疫病毒V_4株NP基因的克隆、序列测定及真核表达载体的构建

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

研究新城疫病毒 ( NDV)核衣壳蛋白基因的生物学作用 ,以提纯的 NDV V4 株基因组 RNA为模板 ,化学合成 NP基因的特异核苷酸引物 ,RT- PCR扩增 NP基因 c DNA,得到一条 1 .5kb的DNA带 ,与 NDV NP基因大小一致 ,平端连接克隆到 p UC1 1 9质粒中 .阳性克隆经酶切鉴定及序列分析表明已获得新城疫病毒 V4 株 NP基因克隆 .将 NDV V4 株 NP基因碱基序列与已发表的NDV Beaudettec C株、La Sota株、D2 6株的 NP基因的碱基序列比较 ,同源性分别为 90 .64%、90 .1 7%、98.0 3% ,氨基酸序列差异率是 4.50 %、5.93%、2 .45% .NDV V4 株 NP基因与已发表的NDV Beaudettec C株、La Sota株、D2 6株的 NP基因有所不同 ,但具有高度同源性 .将 NDV V4 株NP基因 c DNA克隆到 pc DNA3 真核表达载体中 ,构建表达

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.