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目的确定蛔虫基因工程疫苗候选基因。方法连接经BamHI和EcoRI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(3600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 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A45(00.858±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 ALAg基因可作为蛔虫基因工程疫苗的候选基因。
Objective To determine the candidate gene of roundworm genetically engineered vaccine. Methods The pMD18-T-ALAg and pET-28a (+) plasmids digested with BamHI and EcoRI were ligated and purified. The recombinant plasmid pET-28a (+) - ALAg was transformed into BL21 (DE3) , And recombinant protein (rALAg) purified. Thirty mice were randomly divided into immunization group, adjuvant group and control group, with 10 mice in each group. The mice were inoculated with FCA mixture, FCA and phosphate buffered saline (PBS) Infected eggs were challenged (3600 cells per mouse). The numbers of parasites in each group of mice were observed, and the serum IgG of the mice was detected by indirect ELISA. Results rALAg can be detected by rabbit anti-roundworm positive serum. The number of ascaris larvae in the liver and lung of the immunized mice (25.30 ± 4.55) was decreased by 69.26% compared with the control group (57.60 ± 5.76), the difference was statistically significant (P <0.01) compared with the control group and the adjuvant group The level of IgG antibody in serum of the patients with A45 (00.858 ± 0.003) was significantly higher than that of the control group (0.149 ± 0.004) and the adjuvant group (0.134 ± 0.004) (P <0.01). Conclusion ALAg gene can be used as a candidate gene for Ascaris genetic engineering vaccine.