摘要
【目的】验证安氏隐孢子虫卵囊壁蛋白(COWP)的免疫原性,为建立安氏隐孢子虫病免疫学诊断方法奠定基础。【方法】根据Gen Bank已公布的安氏隐孢子虫COWP同源基因序列(DQ060431)设计引物,克隆COWP基因并构建原核表达载体,经IPTG诱导表达后,采用蛋白印迹法检测其免疫原性。【结果】扩增获得的COWP基因片段为549bp,其序列与Gen Bank已公布COWP同源基因序列(DQ060431)的同源性达100%,将其插入p ET-32a载体可构建p ET-32a-COWP表达载体。重组菌在30℃下经0.5 mmol/L IPTG诱导5 h后,其表达形式以可溶性表达为主;NTA-Ni亲和层析柱层析时用200 mmol/L咪唑洗脱液洗脱,即能得到纯化的重组蛋白,重组蛋白分子量约40 k Da。以抗His标签鼠单克隆抗体或鼠抗安氏隐孢子虫高免血清为一抗进行Western blotting检测,均能获得预期的目的条带。【结论】重组COWP蛋白具有良好的免疫原性,可作为安氏隐孢子虫病免疫学诊断的候选抗原。
【Objective】The present study was conducted to verify immunogenicity of Cryptosporidium andersoni oocyst wall protein（COWP）in order to lay the foundation for immunodiagnosis of cryptosporidiosis. 【 Method 】 According to the COWP sequence（DQ060431） was published in Gen Bank, a pair of primers was designed to amplify the COWP sequence by PCR method. The cloned COWP sequence was connected to p ET-32 a, and to construct p ET-32a-COWP expression vector. Then recombinant strain was induced with IPTG,and its immunogenicity was determined by Western blotting. 【Result 】The cloned COWP sequence was 549 bp in length and had genomic homology of 99.9 % with ref-erence sequence（ DQ060431）. The recombinant strain was induced with 0. 5 mmol / L IPTG for 5 h at 30 ℃, and expressed in the form of soluble protein mostly. The recombinant protein was purified by NTA-Ni affinity chromatography with 200 mmol/L imidazole eluent, and its molecular weight was 40 k Da. Anti-His rat monoclonal antibody or rat anti-Cryptosporidium andersoni serum treated as the first antibody detected by Western blotting could be found a clear target band respectively. 【Conclusion】The recombinant protein COWP has high immunogenicity and can be used as a potential candidate antigen for immunodiagnosis of cryptosporidiosis.
出处
《南方农业学报》
CAS
CSCD
北大核心
2014年第10期1870-1875,共6页
Journal of Southern Agriculture
基金
公益性行业(农业)专项项目(201103008)
广西基本科研业务费专项项目(桂科专项12-2)
广西水产畜牧兽医局科技项目(桂渔牧科1304525
桂渔牧财[2011]52号)
