猪O型口蹄疫病毒强弱毒株VP_1基因的克隆与序列分析

Cloning and Sequence Analysis of the VP_1 Gene of Virulence and Vaccine Strains of Foot-and-mouth Disease Virus Type O in Swine

本研究根据口蹄疫病毒 (FMDV)VP1基因的序列 ,设计并合成了 1对用于扩增整个VP1基因的引物 (5P、P6 )。从细胞培养液或组织中提取总RNA ,通过PT_PCR扩增 ,从F2 9株、O3I3株和T5 0 9株中均获得了 1条约 740bp的DNA电泳带。将PCR产物双酶切后电泳回收 ,插入到相应双酶切的pUC18质粒中 ,获得了重组质粒。通过PCR鉴定 ,证明重组质粒pUCVP1/F2 9、pUCVP1/O313、pUCVP1/T5 0 9均插入了VP1基因。对上述 3个重组质粒进行测序后分析 ,F2 9强毒株与O3I3、T5 0 9弱毒株相比 ,其核苷酸序列同源性分别为 98.75 %和 99.0 6 % ;因F2 9株核苷酸发生 3个碱基缺失与 1个碱基替换 ,故推导的氨基酸序列同源性分别仅为 44 .13%和 41.32 % ;而T5 0 9株与O3I3株相比 ,其核苷酸、氨基酸序列同源性分别为 99.37%和 95 .31%。通过序列分析发现 ,本研究的 3个毒株与国内大多数毒株 (包括 1997年台湾暴发FMDV所分离的毒株 ,除O/A/ 5 8株外 )均属于同一基因型 ,核苷酸序列同源性为 85 %～ 94% ;而与国外毒株相比 ,属不同的基因型 ,核苷酸序列同源性仅为 81%～ 82 %。其推导的氨基酸序列 ,除F2 9株与O/HK/ 93株及 1997年台湾暴发FMDV分离的少数毒株的氨基酸序列同源性仅为 45 %～ 6 3%外 ,本研究的

One pair of primers amplified the whole VP 1 gene of foot_and_mouth disease virus(FMDV)was designed and synthesized.The predicted 740bp VP 1 fragment had been isolated by RT_PCR from FMDV F29,O313 and T509 strains.After cleaved with restriction endonucleases,those fragments had been inserted into the pUC18 vector.The recombinant plasmids of pUCVP 1/F29,pUCVP 1/O313 and pUCVP 1/T509,which contained the VP 1 gene of FMDV F29,O313 and T509 strains,respectively,were obtained by restriction endonuclease digestion,PCR method and sequencing.The sequences of the VP 1 gene of FMDV three strains were obtained by Sanger's sequencing technique with their amino acid sequence being deduced.Compared with these of the O313 and T509 strains,the homology of the sequences of the F29 strain VP 1 gene was 98.75% and 99.06%,respectively.However,the homlolgy of the deduced amino acid sequences was only 44.13% and 41.32%,respectively,because deletion of three nucleotides and substitution of one nucleotide occurred within the VP 1 gene of the F29 strain.Homlolgy between O313 and T509 strains is 99.37% for nucleotide sequence and 95.31% for amino acid sequence.The results of sequence analysis showed that mostly strains of FMDV in China belong to the same genetype.Homology among the mostly strains in China are 85%～94% for nucleotide sequence(except O/A/58 strain)and 87%～95% for amino acid sequence(except F29 and O/HK/93 strains).However,compared with the foreign strains,they belong to the different genetype.Homology between those three strains and foreign strains is 81%～82% for nucleotide sequence.These results might be contributive to the selection of vaccine strains and investigation in molecular epidemiology.