摘要
以PCR技术从日本血吸虫成虫mRNA反转录制备的cDNA中克隆日本血吸虫锌指蛋白编码基因Sjzfp1以及181～786bp的DNA片段,分别以pGEX-4T和pET-28a(+)为表达载体制备重组抗原rSjzfp1/GST和rSjzfp1-1/His。将重组抗原与206佐剂混合后免疫小鼠,观察免疫预防效果。利用实时荧光定量PCR法检测Sjzfp1在不同发育期虫体中的表达情况。结果显示,获得了日本血吸虫Sjzfp1基因全长序列,其ORF为1017bp,编码338个氨基酸,推导的理论分子质量为38.5ku。生物信息学分析显示Sjzfp1为日本血吸虫环形锌指蛋白或PHD型锌指蛋白。以rSjzfp1/GST和rSjzfp1-1/His免疫小鼠,诱导的减虫率分别为50.43%和17.85%,肝虫卵减少率分别为72.64%和15.96%。实时荧光定量PCR分析表明Sjzfp1基因在各发育期虫体中均有表达,其中在尾蚴和毛蚴中表达量较高,在终末宿主体内各发育期虫体中,以42d成虫表达量最高。结果表明,Sjzfp1基因在抗血吸虫疫苗研究中具有进一步研究的潜力。
The objectives of the present study were intend to clone and express the zinc finger protein encoding gene Sjzfp1 of Schistosoma japonicum,evaluate the immunoprotection of the recombinant antigen in mice against schistosomiasis,and analyze the Sjzfp1 mRNA expression profiles at different stages of S.japonicum.The full-length cDNA and 181 to 786bp partial cDNA of Sjzfp1 were amplified by PCR using cDNA generated from the mRNA of S.japonicum as the template,then the purified PCR product was subcloned into pGEX-4T and pET-28a（＋） vectors to express recombinant protein rSjzfp1/GST and rSjzfp1-1/His,respectively.The purified rSjzfp1/GST and rSjzfp1-1/His were used to vaccinate BALB/c mice using ISA206 as adjuvant.The expression profiles of Sjzfp1 were determined by real-time PCR using the cDNAs isolated from parasites in different stages.The full length gene of Sjzfp1 was successfully cloned.Its ORF was 1017 nucleotides（nt）,encoding a 338-amino acid protein with a calculated molecular mass of 38.5ku.Bioinformatics analysis indicated that Sjzfp1 protein was a ring zinc finger protein or PHD zinc finger protein.In the challenge experiments in BALB/c mice,vaccination with rSjzfp1/GST and rSjzfp1-1/His induced reductions of 50.43% and 17.85% for worm burden,72.64% and 15.96% for hepatic egg counting,respectively.Real-time PCR analysis revealed that there were significantly higher expressions in cercariae and miracidia.In addition,in the different developmental stages in the definitive host,the highest expression occurred in 42d-old adults.In conclusion,the Sjzfp1 gene had good potential for the development of vaccines against schistosoma.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第8期835-841,共7页
Chinese Veterinary Science
基金
国家高技术研究发展计划(863)项目(2006AA10A207)
公益性行业(农业)科研专项(200903036)
