日本血吸虫卵壳蛋白基因的克隆与表达

CLONING AND EXPRESSION OF SCHISTOSOMA JAPONICUM EGGSHELL PROTEIN GENE

目的：在体外高效表达具有生物学和免疫学活性的日本血吸虫卵壳蛋白，探讨其作为抗卵免疫靶抗原的可能性。方法：采用ＰＣＲ技术扩增日本血吸虫卵壳蛋白基因，将其克隆到ｐｃＤＮＡ３质粒，在ＨｅＬａ细胞中高效表达，对表达产物进行鉴定和免疫原性研究。结果：特异扩增日本血吸虫卵壳蛋白基因编码序列（６１８ｂｐ），构建了真核表达质粒ｐｃＤＮＡ３／ＥＳＧ，在ＨｅＬａ细胞中高效表达卵壳蛋白，其表达量高达２４．４％，分子量约为２２ｋＤａ，ｄｏｔ－ＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ分析显示，表达蛋白能与感染兔血清及虫卵免疫血清产生较强的免疫反应；此外，表达蛋白还能特异刺激经虫卵免疫后的ＢＡＬＢ／ｃ小鼠脾细胞增殖，其转化率达４４．５７％。结论：成功地构建重组质粒ｐｃＤＮＡ３／ＥＳＧ，并在ＨｅＬａ细胞中高效表达卵壳蛋白基因。

AIM: To construct an eukaryotic expression system pcDNA3/ESG HeLa cell and explore the possibility of whether the eggshell protein might be used as potential targets for vaccine development. METHODS: The eggshell protein gene(ESG)was amplified using PCR technique and inserted into vector pcDNA3 to construct recombinant vector pcDNA3/ESG. The expression of the eggshell protein was performed in HeLa cell line.SDS PAGE,dot ELISA and Western blotting were used to identify the antigenicity of the expressed product. RESULTS: The amplification of ESG(618)bp was achieved and high level expression of the eggshell protein was obtained in HeLa cells and the amount of total expressed products was up to 24 4%.Dot ELISA and Western blot analysis demonstrated that the expressed protein could be recognized by sera from rabbits infected with S japonicum and from female worm or egg immunized rabbits. In addition, the expressed protien could specifically stimulate proliferative response of the immunized BALB/c mouse spleen cells, the transformation rate being up to 44 57%.CONCLUSION: The eukaryotic expression system pcDNA3/ESG was successfully established and high level expression of the eggshell protein of S japonicum was achieved in HeLa cell line. The expressed recombinant protein exhibited immunological activities.