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目的评价重组亮氨酸氨基肽酶(rSjLAP)和重组果糖二磷酸醛缩酶(rSjFBPA)抗原用于诊断人血吸虫感染以及疗效考核的价值。方法异丙基-β-D-硫代半乳糖苷(IPTG)诱导pET-28a-rSjLAP/BL21和pET-28a-rSjFBPA/BL21表达目的蛋白,组氨酸标签亲和纯化柱纯化rSjLAP和rSjFBPA蛋白。88只BALB/c雌性小鼠随机分为4组,A、B和C组(各21只小鼠),D组(25只小鼠)。A、B和C组分别感染5、15和25条日本血吸虫尾蚴。D组为不感染对照组,在实验的第1天全部处死。A、B和C组在感染后第3、7、10、14、20、30和60天,分别处死小鼠3只,采眼球血制备血清,检测其抗体水平。采用单独或联合rSjLAP和rSjFBPA为抗原,ELISA法检测小鼠血清、急性血吸虫病(38份)和慢性血吸虫病患者(96份)血清中的抗体,以健康人(90份)血清为对照,同时检测华支睾吸虫病(33份)、卫氏并殖吸虫病(40份)和钩虫病患者(37份)血清,并检测急性血吸虫病患者吡喹酮治疗(60 mg/kg,2次/d×2 d)后1年的血清(36份)、慢性血吸虫病患者吡喹酮治疗(剂量,疗程同前)后1年(36份)和2年(64份)的血清。结果 BALB/c小鼠在感染后第10天,rSjLAP和rSjFBPA单独或联合使用均可检测到小鼠血清中的IgG抗体;B组(0.535±0.053,0.595±0.033,0.696±0.104)和C组(0.548±0.060,0.608±0.063,0.621±0.090)早期抗体水平明显高于A组(0.415±0.038,0.455±0.056,0.498±0.077)(P<0.05)。用rSjLAP为抗原可检测急性血吸虫病和慢性血吸虫病患者血清,阳性率分别为97.4%(37/38)和87.5%(84/96)(P>0.05);用rSjFBPA为抗原检测,其阳性率分别为94.7%(36/38)和88.5%(85/96)(P>0.05);用rSjLAP和rSjFBPA联合为抗原检测,则其阳性率分别为94.7%(36/38)和85.4%(82/96)(P>0.05)。rSjLAP或联合抗原的特异性均为96.7%(87/90),而rSjFBPA的特异性为97.8%(88/90)。给予吡喹酮治疗后,rSjLAP和rSjFBPA单独或联合使用检测急性血吸虫病血清(0.236±0.212,0.287±0.191,0.235±0.120)和慢性血吸虫病患者(0.266±0.124,0.261±0.143,0.265±0.140;0.204±0.074,0.176±0.074,0.176±0.073),抗体滴度普遍下降,与对照组(0.188±0.056,0.173±0.045,0.184±0.051)相比,差异无统计学意义(P>0.05)。rSjLAP和rSjFBPA单独为抗原检测华支睾吸虫病患者血清,交叉反应率为15.2%(5/33)和12.1%(4/33),两者联合则为9.2%(3/33)。rSjLAP检测卫氏并殖吸虫的交叉反应率为15.0%(6/40),rSjFBPA为12.5%(5/40),两种抗原联合检测为15.0%(6/40)。上述抗原单独或联合检测钩虫的交叉反应率均为8.1%(3/37)。上述各组阳性率与健康人群之间差异有统计学意义(P<0.05),显示该重组抗原在其他蠕虫检测中存在一定的交叉反应。结论用rSjFBPA和rSjLAP作为抗原的ELISA法诊断血吸虫病具有良好的敏感性和特异性。
Objective To evaluate the value of recombinant leucine aminopeptidase (rSjLAP) and recombinant fructose diphosphate aldolase (rSjFBPA) antigen in the diagnosis of human schistosomiasis infection and efficacy evaluation. Methods The expressed protein of pET-28a-rSjLAP / BL21 and pET-28a-rSjFBPA / BL21 were induced by isopropyl-β-D-thiogalactopyranoside (IPTG) . Eighty BALB / c female mice were randomly divided into 4 groups, groups A, B and C (21 mice each) and group D (25 mice). Groups A, B and C were infected with 5, 15 and 25 S. japonicum cercariae respectively. D group was not infected control group, all died on the first day of experiment. Groups A, B and C were sacrificed on the 3rd, 7th, 10th, 14th, 20th, 30th and 60th day after infection respectively. Antibodies in sera of mice, acute schistosomiasis (38) and chronic schistosomiasis (96) were detected by ELISA alone or in combination with rSjLAP and rSjFBPA as antigens, and serum from healthy persons (90) as control Clonorchis sinensis (33), Paragonimus westermani (40) and hookworm (37) sera were tested for serum levels of praziquantel in patients with acute schistosomiasis (60 mg / kg twice daily) One year (36 doses) and two years (64 doses) serum of patients with chronic schistosomiasis after one year of treatment (d / d × 2 d) Results BALB / c mice on day 10 post infection, rSjLAP and rSjFBPA alone or in combination could detect IgG in mice serum; group B (0.535 ± 0.053, 0.595 ± 0.033, 0.696 ± 0.104) and group C (0.548 ± 0.060,0.608 ± 0.063,0.621 ± 0.090), the level of early antibody was significantly higher than that of group A (0.415 ± 0.038,0.455 ± 0.056,0.498 ± 0.077) (P <0.05). The positive rates of rSjLAP as antigens were 97.4% (37/38) and 87.5% (84/96) in patients with acute schistosomiasis and chronic schistosomiasis, respectively (P> 0.05). The positive rate of rSjFBPA was (94.7%, 36/38) and 88.5% (85/96), respectively (P> 0.05). The positive rates of rSjLAP and rSjFBPA were 94.7% (36/38) and 85.4% /96)(P>0.05). The specificity of rSjLAP or combination antigen was 96.7% (87/90), while the specificity of rSjFBPA was 97.8% (88/90). After administration of praziquantel, rSjLAP and rSjFBPA alone or in combination detected sera from patients with acute schistosomiasis (0.236 ± 0.212,0.287 ± 0.191,0.235 ± 0.120) and chronic schistosomiasis (0.266 ± 0.124,0.261 ± 0.143,0.265 ± 0.140; 0.204 ± 0.074,0.176 ± 0.074,0.176 ± 0.073). The antibody titers generally decreased compared with the control group (0.188 ± 0.056,0.173 ± 0.045,0.184 ± 0.051), the difference was not statistically significant (P> 0.05). The results showed that rSjLAP and rSjFBPA detected the serum of patients with Clonorchiasis sinensis by antigen alone. The cross-reactivity rate was 15.2% (5/33) and 12.1% (4/33) respectively. The combined rate was 9.2% (3/33). The cross-reactivity of rSjLAP to Paragonimus westermani was 15.0% (6/40), rSjFBPA was 12.5% (5/40), and the combined detection of two antigens was 15.0% (6/40). Cross-reactivity of these antigens alone or in combination with hookworms was 8.1% (3/37). There was a significant difference between the positive rate of the above groups and the healthy population (P <0.05), indicating that there was some cross-reaction between the recombinant antigens and other worms. Conclusion The ELISA method using rSjFBPA and rSjLAP as antigen has good sensitivity and specificity for the diagnosis of schistosomiasis.