CAJ | 학술논문

目的：评价一种改进型A sia 1型口蹄疫多表位重组疫苗的免疫效能。方法根据A sia 1型口蹄疫病毒的基因序列设计多表位基因，构建重组表达质粒（pRE-oIgG）。用大肠杆菌细胞分别表达靶标蛋白RE-oIgG和口蹄疫病毒3D蛋白，并用组氨酸亲和层析柱纯化。纯化的RE-oIgG ，3D和RE-oIgG与3D的混合物分别经油性佐剂IAS206乳化后配制成相应的疫苗。25只雌性豚鼠被随机分成5组，每组5只豚鼠，经肌肉途径分别注射RE-oIgG、3D、RE-oIgG与3D的混合物、Asia 1口蹄疫灭活疫苗和PBS ，所有动物进行两次免疫。采用ELISA、病毒微量中和试验、攻毒试验和流式细胞术分别测定免疫前后抗口蹄疫特异性抗体、中和抗体、保护率以及淋巴细胞增殖反应。结果 RE-oIgG与3D的混合物能够诱导高效价的抗Asia 1型口蹄疫病毒的特异性抗体，与RE-oIgG单独免疫豚鼠相比较具有明显的差异性（P＜0．05）。除了阴性对照组PBS外，来自所有免疫动物的外周血淋巴细胞经过体外培养刺激后均产生了明显的淋巴细胞增殖反应。单独免疫3D蛋白和安慰剂PBS实验组既没有测到抗口蹄疫病毒特异性抗体，也没有测到中和抗体。而RE-oIgG与3D的混合物不仅诱导机体产生了高水平的保护性中和抗体，而且诱发了明显的淋巴细胞增殖反应。更为重要的是，该蛋白质混合物针对1000 GPID50强毒攻击后，所有动物没有出现症状完全保护。值得一提的是，单独免疫3D蛋白的5只豚鼠中，其中2只完全保护；另外3只虽然出现了临床症状，但相对PBS对照组来说发病时间大约推迟2～3 d。因此，笔者认为RE-oIgG与3D的混合物不仅能够诱导体液免疫反应，而且能够诱发细胞免疫反应，是一种高效能的新型疫苗，将来可用于我国口蹄疫的防控。
목적：평개일충개진형A sia 1형구제역다표위중조역묘적면역효능。방법근거A sia 1형구제역병독적기인서렬설계다표위기인，구건중조표체질립（pRE-oIgG）。용대장간균세포분별표체파표단백RE-oIgG화구제역병독3D단백，병용조안산친화층석주순화。순화적RE-oIgG ，3D화RE-oIgG여3D적혼합물분별경유성좌제IAS206유화후배제성상응적역묘。25지자성돈서피수궤분성5조，매조5지돈서，경기육도경분별주사RE-oIgG、3D、RE-oIgG여3D적혼합물、Asia 1구제역멸활역묘화PBS ，소유동물진행량차면역。채용ELISA、병독미량중화시험、공독시험화류식세포술분별측정면역전후항구제역특이성항체、중화항체、보호솔이급림파세포증식반응。결과 RE-oIgG여3D적혼합물능구유도고효개적항Asia 1형구제역병독적특이성항체，여RE-oIgG단독면역돈서상비교구유명현적차이성（P＜0．05）。제료음성대조조PBS외，래자소유면역동물적외주혈림파세포경과체외배양자격후균산생료명현적림파세포증식반응。단독면역3D단백화안위제PBS실험조기몰유측도항구제역병독특이성항체，야몰유측도중화항체。이RE-oIgG여3D적혼합물불부유도궤체산생료고수평적보호성중화항체，이차유발료명현적림파세포증식반응。경위중요적시，해단백질혼합물침대1000 GPID50강독공격후，소유동물몰유출현증상완전보호。치득일제적시，단독면역3D단백적5지돈서중，기중2지완전보호；령외3지수연출현료림상증상，단상대PBS대조조래설발병시간대약추지2～3 d。인차，필자인위RE-oIgG여3D적혼합물불부능구유도체액면역반응，이차능구유발세포면역반응，시일충고효능적신형역묘，장래가용우아국구제역적방공。
The potency of an improved recombinant multi-epitope vaccine against FMDV type Asia1 was evaluated in this study .A multi-epitope gene based on FMDV type Asia1 was designed and a recombinant expression plasmid (pRE-oIgG) was constructed .The proteins ,RE-oIgG and 3D were expressed in E .coli cells and purified with Ni-NTA agarose resin by affinity chromatography .The proteins ,RE-oIgG ,3D and RE-oIgG plus 3D ,were emulsified in an oil adjuvant ISA 206 .Twenty-five female guinea pigs were randomly divided into five groups and intramuscularly vaccinated for with RE-oIgG ,3D ,RE-oIgG plus 3D ,an inactivated FMDV vaccine (type Asia1) ,and PBS .All animals were vaccinated for two times .Anti-FMDV specific an-tibodies ,neutralization antibodies ,protection potency ,and lymphoproliferation assay were detected by ELISA ,virus neutrali-zation assay ,challenge test ,and flow cytometry ,respectively .Results showed that RE-oIgG plus 3D elicited significant high-level anti-FMDV specific antibodies compared to RE-oIgG alone (P<0 .05) .All the vaccinated animals induced higher level lymphoproliferation responses in vitro except PBS .Both 3D alone and PBS produced the negligible neutralizing antibodies and anti-FMDV specific antibodies .RE-oIgG plus FMDV 3D not only elicited high levels of anti-FMDV neutralizing antibodies ,but also induced significant lymphoproliferation responses .More importantly ,RE-oIgG plus 3D conferred complete protection to guinea pigs against challenge with 1 000 GPID50 .Interestingly ,two of five vaccinated animals with 3D alone were full protected against challenge ,and other three animals significantly showed a delay of 2-3 days in the onset of clinical signs .Therefore ,we considered that RE-oIgG plus 3D induces strong humoral and cellular immune responses ,which may be used for control and prevention of FMD in the future .