秀丽隐杆线虫C31B8.8基因的克隆、表达及免疫特性分析  

Cloning,Expression and Immunologic Identification of C31B8.8 Gene of Caenorhabditis elegans

作　　者：孙蕊[1,2] 李正宇[1,2] 何汉江[1,2] 吕志跃[1,2] 吴忠道[1,2] 

机构地区：[1]中山大学中山医学院寄生虫学教研室 [2]中山大学中山医学院教育部热带病防治重点实验室,广州510080

出　　处：《中国寄生虫学与寄生虫病杂志》2011年第4期241-246,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家重点基础研究发展规划项目(973计划)(No2010CB530004)~~

摘　　要：目的克隆、表达野生型秀丽隐杆线虫C31B8.8基因,并分析其免疫特性。方法提取人工培养的秀丽隐杆线虫总RNA,逆转录合成cDNA,PCR扩增C31B8.8基因,克隆入pMD-18T载体,测序正确后将C31B8.8基因亚克隆入带有组氨酸标签的表达载体pET-30a,转入大肠埃希菌BL21中,经异丙基硫代-β-D-半乳糖苷诱导,表达目的蛋白C31B8.8。用钴离子柱亲和层析法纯化该蛋白。融合蛋白采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)鉴定和蛋白质印迹(Western blotting)分析。10只BALB/c小鼠随机均分为抗原免疫组和佐剂对照组,抗原免疫组以纯化的重组C31B8.8蛋白免疫小鼠,抗原免疫剂量为40μg/(只·次),共免疫4次,每周免疫1次。佐剂对照组以PBS代替抗原,同法免疫小鼠。每次免疫前取血,末次免疫后1周眼球采血,分离血清,ELISA检测不同时期血清抗体效价。同时以C31B8.8蛋白和广州管圆线虫Ⅳ期幼虫虫体可溶性蛋白作为抗原进行Western blotting。结果 DNA测序结果显示重组质粒构建成功,并诱导出重组C31B8.8蛋白。重组蛋白经质谱鉴定和Western blotting分析证实为目的蛋白。纯化C31B8.8蛋白免疫小鼠后,与对照组相比,能产生较高的抗体水平。Western blotting分析显示,重组C31B8.8蛋白能被免疫血清识别,且免疫血清与广州管圆线虫Ⅳ期幼虫虫体可溶性抗原存在交叉反应。结论秀丽隐杆线虫C31B8.8蛋白具有较好的免疫原性和免疫反应性,重组C31B8.8蛋白免疫小鼠血清与广州管圆线虫Ⅳ期幼虫虫体可溶性抗原存在交叉反应。Objective To clone and express C31B8.8 gene of wild-type Caenorhabditis elegans, and study the immunological characteristic of the recombinant protein. Methods Total RNA was extracted from cultivated C. elegans and reversely transcribed into cDNA. C31B8.8 gene was amplified by PCR and cloned into pMD-18T vector for sequencing. The accurate sequence was subcloned into the expression vector pET-30a with （His） 6-tag. The recombinant plasmid was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was identified by using matrix-assisted laser desorption ionization time of flight mass spectrometry （MALDI-TOF-MS） and Western blotting. 10 BALB/c mice were randomly divided into C31B8.8-immunized group and PBS＋adjuvant group. Mice in C31B8.8- immunized group were immunized with 40 μg of purified C31B8.8 antigen formulated in Freund＇s adjunvant. Mice in PBS＋ adjuvant group received only adjuvant emulsified with PBS. All the mice received four immunizations every week with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization and the antibody titer was analyzed by ELISA. The recombinant C31B8.8 protein and soluble components of Angiostrongylus cantonens/s fourth-stage larvae were identified by Western blotting. Results The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. MALDI-TOF-MS and Western blotting analysis showed that the recom- binant C31B8.8 protein was the target protein. Compared with PBS＋adjuvant group, mice immunized with purified protein C31B8.8 produced higher level of IgG. The anti-C31B8.8 serum recognized recombinant C31B8.8 protein, and reacted with soluble antigens of A. cantonensis fourth-stage larvae Conclusion C elegans C31B88 ＇gene shows certain immunogenicity and ty, and the soluble antigens of A. cantonensis fourth-stage larvae can react with anti-C31B8.8

关 键 词：秀丽隐杆线虫 C31B8.8基因 克隆 表达 免疫特性 

分 类 号：R383.19[医药卫生—医学寄生虫学]