日本血吸虫SjRAD23基因的克隆表达及基因重组抗原的免疫保护效果

Cloning, expression and protective efficacy evaluation of radiation sensitive protein 23 (RAD23) from Schistosoma japonicum

摘要:

辐射敏感蛋白23具有核苷酸切除修复功能，在泛素蛋白酶体途径中起到重要作用。本研究利用PCR技术克隆了日本血吸虫辐射敏感蛋白23 (SjRAD23) 编码的cDNA序列，成功获得SjRAD23的基因序列，其ORF为1 053 bp。构建SjRAD23基因重组表达质粒pET28a(+)-SjRAD23，并在大肠杆菌BL21中成功诱导表达，重组蛋白在上清和沉淀中都有存在。利用免疫组化技术检测该蛋白在虫体的分布情况，该蛋白广泛分布在日本血吸虫虫体被膜。用重组蛋白免疫BALB/c小鼠后，免疫小鼠血清中检测到较高水平的特异性IgG、IgG1和IgG2a。Western blotting分析显示重组蛋白能够被日本血吸虫成虫可溶性抗原免疫小鼠血清所识别。用重组蛋白rSjRAD23免疫结果与206佐剂对照组比较，rSjRAD23在BALB/c小鼠中诱导了35.94%减虫率，40.59%肝脏减卵率。结果表明SjRAD23具有作为疫苗候选分子的潜力。

Abstract:

Radiation sensitive protein 23 (RAD23) is a nucleotide excision repair (NER) protein that plays an important role in Ubiquitin-proteasome pathway (UPP). Schistosoma japonicum radiation sensitive protein23 (SjRAD23) cDNA sequences were amplified by PCR and cloned into pET28a (+) vector to construct recombinant expression plasmid pET28a (+)-SjRAD23. The recombinant protein was expressed as both inclusion bodies and the supernatant in Escherichia coli BL21 (DE3) cell. Immunofluorescence observation shows that SjRAD23 was mainly distributed on the tegument surface of the worms. ELISA assay reveals that specific IgG, IgG1 and IgG2a antibodies could be detected in the sera of rSjRAD23 immunized mice. Western blotting analysis shows that the recombinant SjRAD23 could be recognized by serum specific to soluble adult worm antigen of S. japonicum. BALB/c mice vaccinated with rSjRAD23 combined with 206 adjuvant revealed 35.94% worm reduction and 40.59% liver egg reduction when compared with that of the adjuvant control group .This study suggests that rSjRAD23 has the potential as a vaccine against schistosome infection.