猪繁殖和呼吸综合征病毒基质膜蛋白和核衣壳蛋白基因的克隆与鉴定  

Identification and Cloning of M and N Protein Gene of Porcine Reproductive and Respiratory Syndrome Virus

作　　者：蔡家利[1] 姜平[1] 蔡宝祥[1] 马志勇 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点开放实验室

出　　处：《中国兽医学报》1999年第1期3-6,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金

摘　　要：猪繁殖和呼吸综合征病毒（ＰＲＲＳＶ）的基质膜（Ｍ）蛋白和核衣壳（Ｎ）蛋白是２种重要的结构蛋白。本试验根据ＰＲＲＳＶ美洲毒株的基因序列，设计了能够扩增Ｍ和Ｎ蛋白基因的１对引物，并在２个引物的两端分别加入ＥｃｏＲＩ和ＢａｍＨＩ酶切位点，经ＲＴ－ＰＣＲ技术获得了一约９５０ｂｐ的目的基因片段，并将此基因克隆于ＰＢＶ２２０载体，构建了ＭＮ蛋白基因重组质粒ＰＢＶＭＮ，进一步由重组质粒回收ＭＮ基因片段亚克隆于ｐＳＫ＋（ｐＢｌｕｅｓｃｒｉｐｔＳＫ＋）测序载体，构建了重组质粒ＰＢＳＭＮ，经部分序列分析鉴定，重组质粒含ＭＮ蛋白基因。此基因的克隆为进一步研究该病毒ＭＮ蛋白免疫学特性及其分子基础和基因诊断技术奠定了基础。Matrix membrane (M) protein and nucleocapsid (N) protein of PRRSV are two important structural proteins. Primers for RT PCR were designed on the basis of VR2385 isolate sequence of US PRRSV which amplified the entire protein coding regions of the M and N genes. Unique restriction sites (Eco RI and Bam HI) at the termini of the PCR products were introduced. A PCR product with the expected size of about 950 bp was obtained from a modified live PRRSV. The PCR product of the M and N genes from PRRSV was then digested with Eco RI and Bam HI, purified and cloned into vector PBV220 and one recombinant PBVMN was constructed. The M and N gene of PBVMN was further subcloned into vector pSK + (pBluescript SK +) and one recombinant PBSMN was obtained. A partial sequence of PBSMN containing the full length of M and N genes was identified with an automated DNA sequencer. The report provides some valuable materials for investigation of M and N protein antigenic properties and molecular characteristics as well as genomic diagnostic technique of PRRSV.

关 键 词：猪 基因克隆 MN蛋白 PRRSV 病毒 核衣壳 膜蛋白 

分 类 号：S858.285.3[农业科学—临床兽医学] R852.65[农业科学—兽医学]