【摘要】 通过三亲杂交法,构建克隆有阿克苏(Akesu/58) O 型口蹄疫病毒vp1 基因的双元表达载体pBin FMDVVP1。采用农杆菌介导法转化NC89烟草叶盘,经卡那霉素筛选,共获得49株抗性植株。对抗性植株总DNA进行目的基因的PCR检测,有40株阳性植株。对阳性植株总RNA进行目的基因的RT PCR检测,有21株阳性植株。将7株ELISA和Western blot检测阳性植株叶片提取物分别与弗氏佐剂乳化,在0、15、30 和45d 腹膜腔接种Balb/C小白鼠,于第4次免疫后第9d进行血清抗体检测;第12d用104SM50 LD的同源强毒进行攻击;攻毒后24h采血,通过乳鼠病毒血症试验判定攻击Balb/C小白鼠的发病和保护情况。结果表明:双元表达载体pBin FMDVVP1构建正确;vp1基因转入NC89烟草并获得表达;7组中有2组Balb/C小白鼠血清抗体呈阳性,攻毒保护率分别为100%和63%。证明2株转基因烟草表达的VP1蛋白具有较好的免疫原性,所免疫的2 组Balb/C小白鼠对同源强毒攻击有一定的抵抗能力。更多还原

【Abstract】 A binary plasmid was constructed by triparental mating, which contained the vp1 gene of foot-and-mouth disease virus strain Akesu/58 serotype O. The Nicotiana Benthamiana （NC） leaves were transformed by Agrobacterium tumefaciens-mediated method with the binary expression plasmid. After selected by kanamycin, forty-nine resistant lines were obtained. Forty lines were positive through PCR with total DNA and twenty-one lines were positive through RT-PCR with total RNA from fresh leaves tissue. The leave extracts of seven positive lines analyzed with ELISA and western-blot were respectively emulsified in Freund′s adjuvant and the Balb/C mice were immunized intraperitoneally at days 0,15,30 and 45. After the last inoculation, the sera antibody were analyzed at 9 days and the mice were experimentally challenged intraperitoneally with 10⁴ suckling mouse 50% lethal doses (SM₅₀LD) of FMDV at 12 days. Protection was determined by the absence of viremia at 24h post-inoculation. Viremia was tested by intradermal inoculation of peripheral blood into suckling mice. These results showed that the constructed binary expression plasmid was functionary; vp1 gene was transformed into NC plants and expresed affirmatively; the structure protein VP1 expressed in two transgenic plants lines may possibly be used as a source of antigen, the inoculated Balb/C mice developed a specific antibody response; the protection against FMDV challenge in two groups was 100% and 63% respectively.更多还原