CAJ | 학술논문

为制备Asia 1型口蹄疫病毒（FMDV）双层结构病毒样颗粒，选择牛病毒性腹泻病毒（BVDV）流行株BVDV JZ05-1核心蛋白p14的第169～270位氨基酸和 FMDV Asia 1/JS/CHA/05结构蛋白 VP1的第1～211位氨基酸，两者之间设计柔性肽连接，人工合成 p14-柔性肽-VP1融合蛋白编码序列，全基因长990 bp。合成基因与枯草芽胞杆菌表达载体 p7257-P43连接，构建成表达质粒 p7257-P43-P14-VP1。转化枯草芽胞杆菌 WB800，经筛选、鉴定，获得表达 p14-VP1融合蛋白的枯草芽胞杆菌。该重组表达菌在含有40μg/mL氯霉素的 LB中培养后，菌体超声破碎可检测到目的蛋白。Western blot 结果显示，该蛋白能特异性识别Asia 1型FMDV抗体，具有良好的反应原性。电镜观察显示，融合蛋白组装为直径约50 nm大小的病毒样颗粒（VLP）。
위제비Asia 1형구제역병독（FMDV）쌍층결구병독양과립，선택우병독성복사병독（BVDV）류행주BVDV JZ05-1핵심단백p14적제169～270위안기산화 FMDV Asia 1/JS/CHA/05결구단백 VP1적제1～211위안기산，량자지간설계유성태련접，인공합성 p14-유성태-VP1융합단백편마서렬，전기인장990 bp。합성기인여고초아포간균표체재체 p7257-P43련접，구건성표체질립 p7257-P43-P14-VP1。전화고초아포간균 WB800，경사선、감정，획득표체 p14-VP1융합단백적고초아포간균。해중조표체균재함유40μg/mL록매소적 LB중배양후，균체초성파쇄가검측도목적단백。Western blot 결과현시，해단백능특이성식별Asia 1형FMDV항체，구유량호적반응원성。전경관찰현시，융합단백조장위직경약50 nm대소적병독양과립（VLP）。
In order to prepare the double-layer structure virus-like particles of foot-and-mouth disease virus (FMDV)type Asia 1,the 169-270 amino acids of bovine viral diarrhea virus(BVDV)JZ05-1 strain,its core protein P14,and the 1-211 amino acids of FMDV Asia 1/JS/CHA/05,its structural protein VP1 ,were se-lected out,respectively.Between the two peptides,a flexible motif sequence was inserted into as a linker. The P14-flexible motif-VP1 coding sequence was synthesized and inserted into Bacillus subtilis expression vector p7257-P43 to build up the shuttle plasmid p7257-P43-P14-VP1,and its positive clones were screened out and confirmed by sequence analysis.The shuttle plasmid was transformed into Bacillus subti-lis WB800 cells,and SDS-PAGE analysis showed that expression product consists in the WB800 cells in LB broth with 40μg/mL chloromycetin (Cm),and the fusion protein P14+VP1 could specifically react with the bovine positive sera against FMDV type Asia 1 in Western blot test.By electron microscopy observa-tion,the fusion protein can produce VLPs,which diameter is 50 nanometer aproximately.It provided a new way to explore new VLP vaccine.