日本血吸虫P7抗原的克隆、表达及阶段特异性分析及其早期诊断价值评估
[Cloning, expression and stage-specific analysis of Schistosoma japonicum P7 antigen and evaluation of its value in early diagnosis].
作者信息
Xu Bin, Duan Xin-Wei, Lu Yan, Chen Shen-Bo, Feng Zheng, Hu Wei
机构信息
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai 200025, China.
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2011 Jun;29(3):161-6.
OBJECTIVE
To clone and express Schistosoma japonicum P7 antigen (GenBank accession No. EU121231), analyze stage-specific transcription and expression of the antigen, and evaluate its value in early diagnosis.
METHODS
The positive clone (P7) screened from schistosomula cDNA library was amplified by PCR. The PCR product was subcloned into prokaryotic expression vector pET28a. The recombinant plasmids were identified by restrictive enzymes digestion. The positive recombinant plasmids were transformed into E. coli BL21 (DE3), induced by IPG for expression and purified. The diagnostic value of P7 recombinant protein was evaluated by Western blotting analysis. RT-PCR and Western blotting were used to investigate the differential transcription and expression of P7 during the developmental stages. The specific antibodies against P7 recombinant protein in the sera of S. japonicum-infected rabbits at 14 d postinfection, sera of schistosomiasis (28 cases), clonorchiasis (30 cases) and paragonimiasis (20 cases) patients, and sera of healthy people (30 cases) were detected by ELISA, respectively.
RESULTS
The expression vector of p7/pET28a was established and the P7 recombinant protein (about Mr 20 100) was expressed in E. coli. Western blotting analysis showed that the recombinant protein was specifically recognized by immunized rabbit sera, and sera from mice on the 14th day post infection, but was not recognized by the sera of mice at 42 d post-infection. P7 mRNA was detected in cercariae, schistosomula and adult worms, while the protein was only found in schistosomula. The positive rate of rabbit sera collected at 14 d post-infection was 83.3% (15/18). The sensitivity and specificity of ELISA for diagnosis of schistosomiasis japonica were 75.0% (21/28) and 93.8% (75/80), respectively. And the P7 protein showed cross reaction with sera of clonorchiasis and paragonimiasis patients with positive rates of 6.7% (2/30) and 5.0% (1/20), respectively.
CONCLUSION
P7 antigen might be a potential candidate for early diagnosis of schistosomiasis.
目的
克隆并表达日本血吸虫P7抗原（GenBank登录号：EU121231），分析该抗原的阶段特异性转录与表达情况，并评估其在早期诊断中的价值。
方法
从日本血吸虫童虫cDNA文库中筛选出的阳性克隆（P7）经PCR扩增。将PCR产物亚克隆至原核表达载体pET28a。通过限制性内切酶酶切鉴定重组质粒。将阳性重组质粒转化至大肠杆菌BL21（DE3），经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达并纯化。采用蛋白质印迹法分析P7重组蛋白的诊断价值。运用逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法研究P7在不同发育阶段的差异转录与表达。分别采用酶联免疫吸附测定（ELISA）检测日本血吸虫感染后14 d兔血清、血吸虫病患者（28例）血清、华支睾吸虫病患者（30例）血清、肺吸虫病患者（20例）血清及健康人（30例）血清中针对P7重组蛋白的特异性抗体。
结果
构建了p7/pET28a表达载体，P7重组蛋白（约20 100 Mr）在大肠杆菌中得以表达。蛋白质印迹分析显示，该重组蛋白能被免疫兔血清以及感染后14 d小鼠血清特异性识别，但不能被感染后42 d小鼠血清识别。在尾蚴、童虫及成虫中均检测到P7 mRNA，而蛋白仅在童虫中发现。感染后14 d采集的兔血清阳性率为83.3%（15/18）。ELISA诊断日本血吸虫病的敏感性和特异性分别为75.0%（21/28）和93.8%（75/80）。P7蛋白与华支睾吸虫病和肺吸虫病患者血清存在交叉反应，阳性率分别为6.7%（2/30）和5.0%（1/20）。
结论
P7抗原可能是日本血吸虫病早期诊断的潜在候选抗原。
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