日本血吸虫P7抗原的克隆表达、虫期特异性分析以及早期诊断价值的研究

Cloning,Expression and Stage-specific Analysis of Schistosoma japonicum P7 Antigen and Evaluation of its Value in Early Diagnosis

目的克隆表达日本血吸虫(Schistosoma japonicum)P7抗原片段(GenBank登录号为EU121231),研究各虫期中该目的片段的转录和表达情况,初步评价其作为早期诊断抗原的潜力。方法将日本血吸虫童虫cDNA文库中免疫筛选出的阳性克隆P7进行体外扩增,将目的基因亚克隆至原核表达载体pET28a中。用双酶切的方法鉴定重组质粒,将阳性重组质粒转化至大肠埃希菌(E.coli)BL21(DE3)感受态细胞中,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达并纯化。用蛋白质印迹(Western blotting)分析初步鉴定表达产物的免疫诊断价值。利用逆转录PCR和Westernblotting分析检测P7片段在日本血吸虫各个虫期中的转录和表达情况。以P7重组蛋白为抗原用间接ELISA检测感染日本血吸虫后14 d的兔血清(18份),日本血吸虫病(28份)、华支睾吸虫病(30份)和卫氏并殖吸虫病(20份)患者血清,以及健康人血清(30份),计算特异性和敏感性。结果成功构建了重组表达载体P7/pET28a,并在E.coli中表达。Western blotting分析结果显示P7重组蛋白能被感染日本血吸虫后14 d的小鼠血清和P7重组蛋白免疫的多抗兔血清识别,但不能被感染后42 d的小鼠血清识别,蛋白相对分子质量(Mr)约为20 100。采用逆转录PCR技术在尾蚴、童虫和成虫中均检测到了P7片段的mRNA。Western blotting分析结果显示,仅在童虫阶段检测到目的蛋白。间接ELISA检测感染早期兔血清的检出率为83.3%(15/18),检测日本血吸虫病患者血清的敏感性为75.0%(21/28),特异性为93.8%(75/80),与华支睾吸虫病和卫氏并殖吸虫病患者血清的交叉反应分别为6.7%(2/30)和5.0%(1/20)。结论日本血吸虫P7抗原可能为潜在的日本血吸虫病早期诊断的靶分子。

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 IPTG 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 post-infection,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 schisto-somiasis 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.