摘要
目的： 在家蚕细胞和幼虫中表达日本血吸虫脂肪酸结合蛋白（ Ｓｊ１４） 基因。方法： 将该基因克隆于昆虫病毒转移载体ｐ Ｂａｃ Ｐ Ａ Ｋ Ｈｉｓ１ 中， 再与修饰的家蚕核型多角体病毒（ Ｂｍ Ｎ Ｐ Ｖ） 线性化 Ｄ Ｎ Ａ 共转染家蚕细胞，经体内重组得重组病毒 Ｂｍ Ｓｊ１４ ， 再将 Ｂｍ Ｓｊ１４ 感染家蚕细胞和幼虫以表达 Ｓｊ１４ ， 用 Ｗｅｓｔｅｒｎ ｂｌｏｔ 和间接 Ｅ Ｌ Ｉ Ｓ Ａ测定表达产物的抗原性。结果： Ｂｍ Ｓｊ１４ 感染家蚕细胞和幼虫后 Ｓｊ１４ 获得较高表达； 表达产物ｒ Ｓｊ１４ （ Ｈｉｓ） 为１８ｋ Ｄａ 的融合蛋白， 在家蚕细胞中的产量约为１００ μｇ／１ ×１０６ 细胞， 在培养上清中产量为３３ μｇ／ｍｌ； 在家蚕血淋巴中得量为４ ｍｇ／ｍｌ； 在家蚕幼虫组织的得量为４６ ｍｇ／ｇ 组织。 Ｗｅｓｔｅｒｎ ｂｌｏｔ 和间接 Ｅ Ｌ Ｉ Ｓ Ａ 显示ｒ Ｓｊ１４ （ Ｈｉｓ） 具有较高抗原性。结论： Ｓｊ１４ 在家蚕细胞和幼虫中获得高效表达且表达产物具有较高抗原性。
AIM: To express the fatty acid binding protein ( Sj14FABP) gene of Schistosoma japonicun in the silkworm cells and larvae. METHODS: A 600 bp DNA fragment containing Sj14FABP gene was cloned into baculovirus transfer vector of pBacPAK His1 to construct recombinant transfer vector Sj14\|pBac PAK His1 . Coinfection was accomplished with this vector and Bombyx mori nuclear polyhedrosis virus (BmNPV) DNA in BmN cells. The recombinant virus of Bm\|Sj14 was screened using dot\|blotting. The BmN cells and silkworm larvae were infected with Bm\|Sj14 to express Sj14FABF gene. Western blotting and ELISA were used to identify the antigenicity of the recombinant protein. RESULTS: Sj14FABP gene was successfully expressed in the BmN cells and silkworm larvae infected with Bm\|Sj14. The product was a 18 kDa fusion protein. The yield in BmN cells was about 100 μg/1×10 6 cells and 33 μg/ml cell supernatant. In silkworm larvae, the product yield was 4 mg/ml haemolymph as well as 4\^6 mg/g silkworm tissue. The recombinant protein could be recognized by Western blotting and ELISA using the sera from mice immunized with SWAP. CONCLUSION: Sj14FABP gene has been successfully expressed in BmNPV system and the product has high antigenicity.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
1999年第4期218-221,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家科委863 高技术计划资助
关键词
日本血吸虫
脂肪酸结合蛋白
基因表达
疫苗
Schistosoma japonicum
fatty acid binding protein
gene expression
recombinant BmNPV
vaccine
