四川省猕猴桃病毒A外壳蛋白基因的序列分析、原核表达及抗血清制备  

Sequence analysis, prokaryotic expression and antiserum preparation of coat protein gene of Actinidia virus A in Sichuan province

作　　者：吕蕊 彭期定 杨婷 林宏辉[1] 董家红[2] 席德慧[1] LüRui;PENG Qiding;YANG Ting;LIN Honghui;DONG Jiahong;XI Dehui(Key Laboratory of Bio-resources and Eco-environment of Ministry of Education/College of Life Sciences,Sichuan University,Chengdu 610065,Sichuan,China;Biotechnology and Germplasm Resources Institute,Yunnan Academy of Agricultural Sciences/Yunnan Provincial Key Lab of Agricultural Biotechnology/Key Lab of Southwestern Crop Gene Resources and Germplasm Innovation,Ministry of Agriculture,Kunming 650205,Yunnan,China)

机构地区：[1]四川大学生命科学学院·生物资源与生态环境教育部重点实验室,成都610065 [2]云南省农业科学院生物技术与种质资源研究所·云南省农业生物技术重点实验室·农业部西南作物基因资源与种质创制重点实验室,昆明650205

出　　处：《果树学报》2019年第9期1121-1129,共9页Journal of Fruit Science

基　　金：国家自然科学基金(31772131);四川省应用基础研究项目(2019YJ0135)

摘　　要：【目的】研究猕猴桃病毒A(Actinidia virus A,AcVA)的发生及其多样性,为我国猕猴桃产业中AcVA病毒的快速检测、科学防控以及猕猴桃品种选育提供科学依据。【方法】通过巢式PCR从四川省猕猴桃感病叶片中扩增AcVA CP基因序列,进行序列分析和同源性比对;构建AcVA CP基因的原核表达载体,诱导目的蛋白表达后制备抗血清,用Western blot检测效价。【结果】克隆到3个AcVA四川分离株的完整CP基因序列,成功构建了pET28a-AcVA-DJY4-CP重组质粒,原核表达目的蛋白并制备出特异性抗血清,效价达到1∶5 000。【结论】首次获得了3个AcVA四川分离株,丰富了AcVA的序列多样性,并探索了原核表达的最佳条件,制备了高效价抗血清,为今后AcVA病毒的快速检测和防控提供了技术支撑。【Objective】The purpose of the research was to study the occurrence and molecular diversity of Actinidia virus A(AcVA),so as to provide scientific basis for the rapid detection,scientific prevention and control of AcVA and the variety breeding of kiwifruit in China.【Methods】Total RNA was extracted from kiwifruit leaves by CTAB(Cetyl Trimethyl Ammonium Bromide).Nested-PCR was used to amplify AcVA CP gene sequence.The first round PCR amplification was performed by using primers AcVA-1F(5’-ATGAATCGTTCGAGCA TAGGT-3’)and AcVA-1R(5’-TGCGAACATGGTCCCACACTTA-3’),and the pair of primers was designed according to the full length of AcVA sequence,and the amplified fragment was 888 bp that included the complete CP gene sequence.The reaction was conducted under conditions of initial 5 min denaturation at 94℃,34 cycles of 94℃for 60 s,54℃for 60 s,72℃for 60 s and extension for 10 min at 72℃.Then,1μL PCR product was used as template for the second round of PCR amplification with the primers AcVA-2F(5’-ATGGCAAAGAATATCTCAAG-3’)and AcVA-2R(5’-CTATATTTCAACAGCCTGC-3’).PCR was performed using the following parameters:one cycle at 94℃for 5 min,34 cycles at 94℃for 30 s,54℃for 30 s,and 72℃for 40 s,and extension for 10 min at 72℃.The BLAST algorithm was used to search the NCBI GenBank(http://www.ncbi.nlm.nih.gov/)databases for homologous sequences and ascertain the identity of target gene.DNAMAN was used to analyze the AcVA CP gene sequence,and MegAlign was used to analyze the sequence identity.The phylogenetic tree was constructed using the Neighbor-Joining(NJ)method in MEGA 6.0.The restriction enzymes Bam HⅠand SalⅠwere added to the corresponding end of the AcVA CP gene sequence,respectively.The PCR purified products and pET28a vector were digested by Bam HⅠand SalⅠ,after that they were ligated with T4 DNA ligase(TaKaRa)and transferred into E.coli(Escherichia coli)strains DH5á(TaKaRa),and finally plated onto LB(Luria-Bertani)agar containing Kana(Kanamycin).The expression strain BL21 con

关 键 词：猕猴桃病毒A 外壳蛋白 序列分析 原核表达 抗血清制备 

分 类 号：S663.4[农业科学—果树学]