重叠延伸PCR法扩增猪囊尾蚴AgB基因及其在BHK-21细胞中的表达

 【目的】克隆猪囊尾蚴AgB基因并在体外细胞中表达。【方法】采用 RT-PCR法分别扩增AgB基因的上半段和下半段序列。采用重叠延伸PCR法（splicing overlap extension PCR method，SOE-PCR）把具有35个相同碱基的上下半段扩增为全长基因，并构建到真核表达载体pVAX1，将酶切、PCR、测序鉴定的阳性质粒经脂质体转染 BHK-21细胞。通过SDS-PAGE、Western-blotting、间接免疫荧光法，检测细胞中B抗原的表达。【结果】琼脂糖凝胶电泳显示扩增的不同片段大小分别与预期的相同。重组表达载体转染BHK细胞后有荧光出现。表达的蛋白能被猪囊尾蚴病阳性血清所识别。【结论】通过重叠延伸PCR法成功扩增了AgB基因全长并构建到真核表达载体，目的蛋白在BHK-21细胞中表达。

Cloning of Cysticercus cellulosae AgB Gene by Using Splicing Overlap Extension PCR Method and Expression in BHK Cells

【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.