蛔虫抗原基因ALAg表达载体的构建及其重组蛋白诱导小鼠的免疫保护研究

目的 目的 确定蛔虫基因工程疫苗候选基因。方法 方法 连接经BamH I和EcoR I酶切的pMD18?T?ALAg和pET?28a(+)质粒的纯化回收产物, 将连接产物pET?28a(+)?ALAg表达载体转化表达菌株BL21(DE3)进行诱导表达, 并纯化重组蛋白 (rALAg)。30只小鼠分成免疫组、 佐剂组和对照组, 每组10只, 各组分别接种重组蛋白与弗氏完全佐剂(FCA)混合物、 FCA以及磷酸盐缓冲液 （PBS） 后, 采用蛔虫感染性虫卵进行攻击(3 600个/只), 观察各组小鼠体内虫体数, 并采用间接ELISA法检测小鼠血清中抗体IgG。结果 结果 rALAg能被兔抗蛔虫阳性血清所识别。免疫组小鼠的肝脏和肺脏中的蛔虫幼虫数量（25.30±4.55） 比对照组 （57.60±5.76） 减少了69.26%, 与对照组和佐剂组比较差异有统计学意义(P < 0.01); 免疫组IgG抗体水平检测血清吸光度值A450 （0.858±0.003） 与对照组 （0.149±0.004）、 佐剂组比较 （0.134±0.004） 差异有统计学意义(P < 0.01)。结论 结论 ALAg基因可作为蛔虫基因工程疫苗的候选基因。

Construction of expression vector of ALAg and immune protection of its recombinant protein induced in mice

Objective To determine the candidate genes for engineering vaccine of Ascaris lumbricoides. Methods pMD18-T-ALAg and plasmid expression vector pET-28a(+) were digested with BamH I and EcoR I and linked to each other. The resultant plasmid pET-28a(+)-ALAg was transferred into E. coli BL21 (DE3) and its expression was induced with IPTG, and the recombinant ALAg(rALAg) was purified. A total of 30 mice were equally divided into 3 groups, the mice in each group were injected with rALAg-FCA, FCA and PBS respectively, then they were attacked by infectious eggs of Ascaris (3 600 per mouse). The IgG levels in sera of mice in each group were detected by indirect ELASA. Results rALAg was recognized by the sera from repeatedly Ascaris lumbricoides inoculated rabbits. The numbers of larvae of Ascaris lumbricoides from liver and lung of mice were 25.30±4.55 in the rALAg-FCA group and 57.60±5.76 in the PBS group, respectively, the former being the reducing rate of 69.26%, and the difference among the 3 groups showed statistical significances (P < 0.01). The IgG levels(A450 value)of the rALAg-FCA, FCA and PBS groups were 0.858±0.003, 0.149±0.004 and 0.134±0.004, respectively, there were statistical differences among them (P < 0.01). Conclusion ALAg can be used as a candidate gene of genetic engineering vaccine of Ascaris lumbricoides.