CAJ | 학술논문

为研究猪源牛病毒性腹泻病毒（Bovine viral diarrhea virus,BVDV）生物学特性,将本实验室分离得到的SD0803毒株在马-达氏牛肾细胞（mardin-darby bovine kidney,MDBK）上连续传40代,提取第40代病毒基因组RNA,设计扩增及测序引物,用RT-PCR方法分5段扩增第40代病毒基因片段,并进行全长测序,利用DNASTAR软件进行序列拼接及分析,与亲代病毒SD0803序列比对分析;用Real-time PCR方法测定第1、10、20、30和40代细胞上清中的病毒复制效率,并绘制多步生长曲线。测序结果表明,第40代病毒与亲代病毒核苷酸序列的同源性为99.8%,氨基酸序列的同源性为99.6%,其中有23处发生核苷酸突变,14处为有义突变,氨基酸变化主要集中在E2和NS5B区域。多步生长曲线显示,传代病毒和亲本病毒均能在MDBK细胞上获得较高的复制效率,并有着相似的生长特性,复制效率基本一致。本次研究为研制猪源BVDV疫苗做好了物质储备,为下一步研究其致病性和抗原性奠定基础。
위연구저원우병독성복사병독（Bovine viral diarrhea virus,BVDV）생물학특성,장본실험실분리득도적SD0803독주재마-체씨우신세포（mardin-darby bovine kidney,MDBK）상련속전40대,제취제40대병독기인조RNA,설계확증급측서인물,용RT-PCR방법분5단확증제40대병독기인편단,병진행전장측서,이용DNASTAR연건진행서렬병접급분석,여친대병독SD0803서렬비대분석;용Real-time PCR방법측정제1、10、20、30화40대세포상청중적병독복제효솔,병회제다보생장곡선。측서결과표명,제40대병독여친대병독핵감산서렬적동원성위99.8%,안기산서렬적동원성위99.6%,기중유23처발생핵감산돌변,14처위유의돌변,안기산변화주요집중재E2화NS5B구역。다보생장곡선현시,전대병독화친본병독균능재MDBK세포상획득교고적복제효솔,병유착상사적생장특성,복제효솔기본일치。본차연구위연제저원BVDV역묘주호료물질저비,위하일보연구기치병성화항원성전정기출。
To develop a Bovine viral diarrhea virus（BVDV） vaccine,the BVDV strain SD0803 of pig origin was passaged in MDBK cells for 40 times.Viral RNA was extracted from the 40th passage virus and 5 segments were amplified in RT-PCR.The complete genomic sequence was megaligned and compared with its parental virus using DNASTAR software.The results showed that the homology between the 40th passage and parental virus was 99.8% in nucleotides and 99.6 % in amino acids.Twenty three nucleotide mutations were identified,of which 15 were sense mutations.Amino acid mutations were mainly located on E2 and NS5B.To compare the growth characteristics between the parent virus and passaged viruses,supernatants were collected from infected MDCK cells at passages 1,10,20,30 and 40,and measured the amounts of released viral RNAs in RT-PCR.The multi-step growth curves showed that the parent virus and passaged viruses had high replication efficiency in MDBK cells,and shared similar growth properties.Some mutations that occurred during virus passages had no effect on the virus titers as determined by titration.