摘要
【目的】原核克隆及表达华支睾吸虫乳酸脱氢酶(CsLDH)两个表位aa10-20(E10-20)及aa94-102(E94-102),初步研究两表位与CsLDH免疫学及酶学相互关系。【方法】将E10-20及E94-102克隆至pGEX-4T-1载体,表达纯化重组蛋白,用Western Blotting和间接ELISA法检测CsLDH免疫血清对表位重组蛋白的识别,同时检测两表位重组蛋白的免疫血清对CsLDH蛋白的识别;利用CsLDH标准酶活性反应体系,比较两表位免疫血清与CsLDH免疫血清对CsLDH催化丙酮酸还原成乳酸反应的影响。【结果】成功构建pGEX-4T-1-E10-20及pGEX-4T-1-E94-102重组质粒,SDS-PAGE鉴定表达的重组蛋白,菌体裂解液经亲和层析纯化,获得与GST融合表达的蛋白E10-20及E94-102。两表位重组蛋白均可被CsLDH免疫血清识别,而对E94-102更易于识别;重组CsLDH也能被E10-20及E94-102免疫血清识别,而不能被对照血清识别。E94-102免疫血清能抑制CsLDH催化丙酮酸还原成乳酸的反应。【结论】表位结构和功能研究深化了对CsLDH结构的理解,并为开辟CsLDH疫苗研究新路径奠定基础。
【Objective】 To clone and prokaryotically express the two epitopes aa10-20（E10-20） and aa94-102（E94-102） of CsLDH from Clonorchis sinensis,then to study the relationship between the two epitopes and CsLDH basically. 【Methods】 E10-20 and E94-102 were cloned to pGEX-4T-1 vector,then the recombinant protein were expressed and purified. CsLDH immunized sera was used as the first antibody,two epitopes were identified by Western blotting and IgG-ELISA analysis. E10-20 and E94-102 immunized sera was used as the first antibody,CsLDH was identified by the same methods above. Enzyme activity of recombinant CsLDH was assayed in the standard reaction system by adding different dilution sera. 【Results】 The recombinant plasmids pGEX-4T-1-E10-20 and pGEX-4T-1-E94-102 were constructed successfully. The expression products and purified ones were identified by SDS-PAGE. Western blotting and ELISA both showed that the CsLDH immunized sera could identify the two epitopes and E94-102 more easily. Epitopes immunized sera both could identify CsLDH by Western blotting and ELISA analysis. E94-102 immunized sera could inhibit the CsLDH enzyme activity as the CsLDH immunized sera could do. 【Conclusion】 Construction and function of the two epitopes helps to understand the construction of CsLDH and supply new methods to the vaccine study of the CsLDH.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2010年第4期486-490,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金(815100891000005)
广东省医学科研基金(B2008157)
关键词
华支睾吸虫
乳酸脱氢酶
表位
Clonorchis sinensis
lactate dehydragenase
epitope
