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构建了O型口蹄疫病毒China99株结构蛋白P1-2A、非结构蛋白3C以及部分2B基因(P1-2X3C)的植物双元表达载体pBin438/P1-2X3C,通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得40余株抗性植株,对得到的抗性植株进行分子生物学检测,65%的再生植株PCR检测阳性;RT-PCR结果证实P1-2X3C基因在转基因番茄中能够有效转录;ELISA和Western blot检测表明转基因植株中表达的目的蛋白具有免疫反应性。转基因番茄叶片蛋白粗提液经肌肉途径免疫豚鼠,于第3次免疫后28d用100ID50/0.2mL的同源强毒攻击,结果表明口蹄疫病毒P1-2X3C基因的转基因番茄表达产物具有良好的免疫原性,豚鼠3免后血清效价可达1:641:128,攻毒后两组免疫豚鼠保护率分别达3/5和5/5。
Abstract：The plant constitutive expression vector pBin438/P1-2X3C with multi-genes of foot-and-mouth disease virus(FMDV) including the gene fragments coding structural polyprotein P1-2A,non-structural protein 3C and part of 2B of FMDV O/China99 strain was constructed.P1-2X3C gene was transformed into tomato plants via Agrobacterium tumefaciens strain GV3101.After selected by kanamysin,about forty resistant lines were obtained.The regenerated resistant plants were identified by molecular biological methods,65% of them were positive by PCR;their specific transcription was verified by RT-PCR;and the recombinant protein expression was confirmed by sandwich-ELISA and Western blot analyses.Guinea pigs,immunized intramuscularly with a soluble crude extract of transgenic tomato leaves expressing the structural polyprotein P1-2A and the protease 3C were found to produce specific antibody responses to FMDV,and ELISA antibody titers reached respectively 1:64 and 1:128.The results showed that expression product of transgenic plants with P1-2X3C gene of FMDV had immunogenicity.Additionally,after three immunizations,all of the animals of group B were completely protected against experimental challenge with 100ID50/0.2mL FMDV,three out of five animals of group A were totally protected against FMD challenge.In conclusion,this study demonstrates the possibility of using transgenic plants to express polyprotein P1-2A and protease 3C of FMDV and their utilization as effective experimental immunogens.
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基本信息:
中图分类号:S852.4
引用信息:
[1]潘丽,张永光,王永录,等.口蹄疫病毒结构蛋白P1-2A及蛋白酶3C基因的转基因番茄对豚鼠免疫反应的初步研究[J].病毒学报,2006(04):297-303.
基金信息:
863国家高技术研究发展计划(2001AA2130712003AA241110)
2006-07-30
2006-07-30