日本血吸虫特异性抗原基因的筛选和EST序列测定

ISOLATION AND IDENTIFICATION OF SCHISTOSOMA JAPO NICUM CHINESE STRAIN ANTIGEN GENES FROM A cDNA LIBRARY BY IMMUNO LOGY SCREENING AND THE EST STRATEGY

目的 筛选和鉴定日本血吸虫特异性抗原基因。方法 采用日本血吸虫病流行区对再感染具有抵抗力的个体混合血清 ,对 Sjc DNA文库进行免疫学筛选 ,对获得的阳性克隆 c DNA插入片段进行 PCR鉴定和 PCR直接序列分析 ,通过互联网将获得的 EST序列送入 NCBI Gen Bank进行同源性检索 ,并将发现的未知基因 EST序列送入 NCBI db EST,以获得 EST编号和 Gen Bank进入编号。结果 通过对大约 2 .5× 10 4个噬菌体斑的免疫学筛选 ,获得了 5个阳性克隆 ,PCR直接序列分析表明 ,在这 5个阳性克隆中 ,1个为编码 Sj GST的基因克隆 ,另外 4个为 S.j未知抗原基因克隆 ,其 EST序列已被 Gen Bank接受 ,并获得进入编号。结论 应用对再感染具有抵抗力的个体混合血清 ,从 Sjc DNA文库中筛选到 4个日本血吸虫未知抗原基因重组克隆 。

Objective To isolate and identif y expressed Schistosoma japonicum (Chinese strai n) antigen genes by immunological screening and the EST strategy for searching s chistosome vaccine candidates. Methods The cDNA library of S.j (Chinese strain) adult worm was screened with mixed human sera fr om individuals who are resistant to reinfection at an endemic area with S.j , and then the positive cDNA clones were identified using PCR, and PCR directed sequen cing method from 5' end of the sequence, then the ESTs were compared with all se quences in GenBank database. The novel ESTs were submitted to NCBI dbEST and Gen Bank for accession numbers. Results Of the 2.5 ×10 4 phage plaq ues immunoscreened, 5 positive cDN A clones were found. After PCR directed sequencing and homology comparisons with GenBank database, EST sequences of the positive clones showed that one was Sj G ST gene clone, others had only partial homology with schistosome genes or non s c histosome genes and thus classified as “non identified genes” or novel genes. Th e sequences of 4 novel genes ESTs were submitted and obtained their accession nu mbers from GenBank. Conclusion Immunological sc reening using human sera with res istance to reinfection for isolating antigen genes and EST strategy can been use d to discover Schistosoma japonicum antigen gene s with potential value for vaccine candidates.