关键词: 猪囊尾蚴')">猪囊尾蚴, AgB基因, 重叠延伸PCR, 真核表达, BHK-21

Abstract:

【Objective】 To clone Cysticercus cellulosae AgB gene and express in vitro. 【Method】 The upper and downer fragments of AgB gene were amplified by RT-PCR, respectively. Then, the complete gene of AgB was cloned by splicing overlap extension PCR method using 35 nucleotides overlap between the upper and downer fragments and inserted into the vector pVAX1. This recombinant plasmid pVAX1/B was identified by digestion of endonuclease，PCR and sequencing．Then，it was transfected into BHK-21 cell line mediated by liposome 2000. Specific AgB protein expressed in BHK-21 cell line was detected by SDS-PAGE, Western-blotting and indirect immunofluorescence test. 【Result】 Electrophoresis analysis showed that the specific sequences were amplified and the sizes of products were accord with expectation, respectively. AgB was expressed in BHK-21 cells and was recognized by cysticercosis cellulosae positive serum. 【Conclusion】 Cysticercus cellulosae AgB gene has been cloned using splicing overlap extension PCR method, and expressed in BHK-21 cell line successfully.

Key words: Cysticercus cellulosae')">Cysticercus cellulosae, AgB gene, splicing overlap extension PCR, eukaryotic expression, BHK-21