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

A Preliminary Study on Protective Immune Response of Guinea Pigs against Challenge with Foot-and-mouth Disease Virus by Immunization with Transgenic Tomato Plants Expressing Genes Encoding the Structural Polyprotein P1-2A and the Protease 3C

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-PIER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1：64～1：128，攻毒后两组免疫豚鼠保护率分别达3／5和5／5。

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.