猪瘟病毒E2基因的克隆与鉴定  

CLONING AND IDENTIFICATION OF ENVELOPE CLYCOPROTEIN E2 GENE OF HOG CHOLERA VIRUS(HCV)

作　　者：王琴[1] 郎洪武[1] 王在时[1] 丘惠深[1] 李博[1] 

机构地区：[1]中国兽药监察所,北京100081

出　　处：《畜牧兽医学报》2001年第6期548-555,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家攀登计划B类项目专题编号 ( 85 4 4 0 1 0 3 )

摘　　要：根据猪瘟病毒Brescia株全基因序列设计合成特异性引物对P1/P2对 ,采用异硫氰酸胍一步法 (略加修改 ) ,从PK15细胞培养物中提取石门株、兔化弱毒疫苗株、野毒 0 3株及野毒 0 7株猪瘟病毒的总RNA。应用RT PCR方法成功地扩增出E2全基因组约 12 73bp的cDNA片断 ,经电泳证明其大小与推测相符。分别将石门株、兔化弱毒疫苗株、野毒 0 3株及野毒 0 7株的E2基因片段克隆到 pGEM -T载体质粒 ,通过对这 4个重组质粒的EcoRI酶切鉴定、直接与套式PCR扩增 ,并对E2主要抗原区域进行了 2 2 4bp的序列测定 ,表明E2全基因克隆成功 。Specific oligonucleotides were designed according to the nucleotide sequence of genome of Brescia strain of hog cholera virus(HCV)and synthesized chemically.HCV viral RNA of shimen strain(SM),rabbit adapted lapinized(Chinese)strain(HCLV),field strain 03(F03)and field strain 07(F07)were isolated from purified HCV extracts of the culture supernatants of PK-15 cells.Four cDNA fragments of envelope glycoprotein E2 gene of SM strain,HCLV strain,F03 strain and F07 strain were amplified respectively from viral RNA extracts with RT-PCR method.The amplified E2 fragments of four HCV strains were all 1273bp in length by agarose gel electrophoresis.Four E2 fragments were cloned respectively into pGEM-T easy vector and identificated by digestion of restriction ednonuceleases EcoRI,direct PCR and nest PCR.Four E2 major genes were identificated by sequencing respectively and their 75 residues amino acid sequences were deduced.

关 键 词：猪瘟病毒 E2基因 RT-PCR 克隆 

分 类 号：S852.651[农业科学—基础兽医学]