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鸡新城疫病毒F蛋白部分表位基因的克隆表达及其免疫反应性
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摘要:
为研究NDV-F蛋白抗原表位在动物免疫应答中的作用特点,作者克隆和表达了NDV-F蛋白基因的部分片段,并检测其免疫反应性.首先根据F基因和载体pGEX-4T-1的多克隆位点自行设计了1对引物,以已克隆的NDV(F48E9株)的F基因为模板,通过PCR扩增获得长度为594 bp的片段.接着在电泳和酶切鉴定后将其定向插入pGEX-4T-1,构建重组质粒pGEX-4T-1-F594,并经PCR、双酶切及测序鉴定后,进一步将其转化到大肠杆菌BL21中.最后用IPTG诱导表达,提取物用SDS-PAGE和Western-blotting分析.结果表明,该基因片段表达的融合蛋白大小约为46 000,以包涵体形式存在,并具有良好的免疫反应性.
关键词:  新城疫病毒  F基因  原核表达  免疫反应性
DOI:
投稿时间:2007-04-24
基金项目:国家自然科学基金
Cloning and expression of a fragment and its immunity of newcastle disease virus (NDV) F gene
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Abstract:
To study character of the NDV-F protein epitope in the animal immune response,we cloned and expressed a gene fragment of NDV-F partial protein,and examined its immune reactivity.Firstly,a pair of specific primer was designed according to the multi-clones position spot of the F gene and carrier pGEX-4T-1 and then a DNA fragment of 594bp in length was amplified from the NDV(F48E9) F gene,which was cloned and kept in our laboratory.Secondly,this fragment was inserted into pGEX-4T-1 after its identification by test of electrophoresis and enzyme digestion,the recombinant plasmid was named pGEX-4T-1-F594,and then it was transformed into a strain of E.coli(BL21).Finally,the DNA fragment was expressed in BL21 induced by IPTG.and the products in bacterium culture extract was determined by SDS-PAGE and Westernblotting.The results showed that the expressed protein was about 46000 in a form of inclusion bodies,and had good immune reactivity.
Key words:  NDV  F gene  prokaryotic expression  immune reactivity

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