水泡性口炎病毒核蛋白基因的克隆和序列分析

目的　对水泡性口炎病毒编码群特异性抗原的N基因进行PCR扩增、克隆和序列分析。方法　将水泡性口炎病毒编码群特异性抗原的N基因片段克隆至pMD18 T载体中 ,构建N基因重组质粒载体。进行PCR及限制性内切酶分析 ,筛选获得N基因插入的阳性克隆。经核苷酸序列分析 ,并与Genbank中的VSV编码核衣壳蛋白N基因序列进行比较。结果　该序列与VSVNewJersey型的 0 9/ 82 HD B株同源性最高 ,核苷酸和推测氨基酸的同源性分别为 98.9%和 98.8% ,有 13个核苷酸差异 ,伴有 5个氨基酸改变 ,第 72位的酪氨酸变为组氨酸 ,第 89位的缬氨酸变为异亮氨酸 ,第 183位的天冬氨酸变为天冬酰胺 ,第 2 0 5位的苯丙氨酸变为亮氨酸 ,第 4 18位的丙氨酸变为缬氨酸 ;与In diana型各株的同源性较低 ,与Glasgow株相比 ,核苷酸和氨基酸的同源性分别为 6 7.9%和 71.6 %。结论　已成功地克隆了水泡性口炎病毒N基因 ,为水泡性口炎免疫血清学诊断试剂的制备和分子生物学研究打下了坚实基础

Cloning and Sequencing of N Gene Encoding Nucleocapsid Protein of Vesicular Stomatitis Virus

Objective To amplify,clone and sequence the N gene encoding the nucleocapsid protein of vesicular stomatitis virus(VSV).Methods Design a pair of primers according to the VSV N gene sequence published in Genbank and amplify VSV N gene by RT PCR.Construct a recombinant plasmid by inserting the amplified N gene fragment into pMD18 T vector and identify by PCR and digestion with restiction enzyme.Select the positive clones with N gene inserted for nucletoide sequencing.Compare the sequence with the VSV N gene sequence published in Genbank.Results The homologies of nucleotide and deduced amino acid sequences of the clones to those of 09/82 HD β strain of VSV New Jersey type were 98.9% and 98.8% respectively.However,13 nucleotides of the clones were different from those of 09/82 HD β strain.The Tyr,Val,Asp.Phe and Ala at resides 72,89,183,205 and 418 were changed into His,Ile,Asn,Leu and Val respectively.The clones showed low homology to the strains of Indiana type.The homologies of nucleotide and deduced amino acid to those of Glasgow strain were 67.9% and 71.6% respectively.Conclusion VSV N gene was successfully cloned.It laid a foundation of development of serological diagnostic kit and study on molecular biology of vesicular stomatitis.