新城疫病毒V_4株NP基因的克隆及测序

Cloning and nucleotide sequence analysis of the nucleocapsid protein gene of NDV strain V_4

目的 研究新城疫病毒 (NDV)核衣壳蛋白 (NP)基因的生物学作用及其抗人喉鳞癌的作用。方法 以提纯的NDVV4 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cDNA ,得到 1条 1.5kb的DNA带 ,与NDVNP基因大小一致 ,平端连接克隆到pUC119质粒中。结果 阳性克隆经酶切鉴定及序列分析表明已获得NDVV4 株NP基因克隆。结论 将NDVV4 株NP基因碱基序列与已发表的NDVBeaudetteC株、LaSota株、D2 6株的NP基因的碱基序列比较 ,同源性分别为 90 .6 4%、90 .17%、98.0 3% ,氨基酸序列差异率是 4.5 0 %、5 .93%、2 .45 %。NDVV4 株NP基因与已发表的NDVBeaudetteC株、LaSota株、D2 6株的NP基因有所不同 ,但具有高度同源性。

Objective To study biochemical function and therapy of the NP(nucleocapsid protein) gene of NDV(new castle disease virus).Methods The purified genome RNA of NDV were used for template.According to the reported NP gene sequence of NDV strain Beaudettec C,a pair of 19 mer primers was designed and synthesized.Then 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,a specific fragment about 1.5 kb as expected appeared.The RT PCR product of strain V 4 was cloned into the pUC119,then the positive clone was tested by restriction endonuclease analysis and it was sequenced by Sanger's method.Results The result suggested that it was the NP gene of NDV strain V 4.Conclusion The NP gene sequence of NDV V 4 as compared with published NP gene sequence of NDV Beaudettec C,La Sota and D 26 ,affinal are 90.64%, 90.17 %,98.03%;difference rate of the sequence of amino acid of the NP gene are 4.50%,5.93%,2.45%.