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口蹄疫病毒3C蛋白酶的T4噬菌体表面展示
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田纯见,毕英佐,曹永长,谢青梅
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(华南农业大学 动物科学学院,广东 广州510642)
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摘要：
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对FMDV 3C基因克隆质粒p3C-T进行PCR扩增，获得了口蹄疫病毒3C蛋白酶基因片段，将此片段与T4噬菌体整合质粒pR的EcoRⅠ酶切片段连接，转化E.coli DH5α工程菌，经EcoRⅠ酶切、质粒PCR扩增、插入方向筛选及测序鉴定，成功获得了T4噬菌体重组整合质粒pR-3C。将其转化E.coli E2后，与SOC基因缺失的噬菌体φT4-Z1同源重组，经溶菌酶依赖性筛选，获得了快速溶菌型噬菌体φT4-3C。经噬菌体PCR扩增、SDS-PAGE分析和Western-blot分析，重组噬菌体可展示约26ku的重组蛋白;其大小与预期相符，具有免疫原性。 |
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关键词：
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口蹄疫病毒；3C蛋白酶；T4噬菌体；表面展示 |
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中图分类号：S 852.659.1文献标识码：
A 文章编号：1673-4696(2006)07-0519-04 |
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T4 bacteriophage surface display of foot-and-mouth disease
virus 3 C protease |
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TIAN Chun-jian，BI Ying-zuo，CAO Yong-chang，XIE Qing-mei |
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(College of Animal Science, South China Agricultural University,Guangzhou 510642,China) |
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Abstract: The foot-and-mouth disease virus 3 C protease gene fragment was obtained by PCR amplification of the 3C gene in the cloned p3C-T plasmid. After ligation with the EcoRⅠ restriction fragment of the T4 integrative plasmid pR, the fragment was used to transform E. coli DH5α to obtain the T4 bacte-riophage recombinant integrative plasmid pR-3C, and the success of which was confirmed by digestion with EcoRⅠrestriction, PCR amplification, insert orientation screening and nucleotide sequencing. After further transformation of E. coli E2, the pR-3C was used for homologous combination with bacteriophage φT4-Z1 and, using lysozyme dependence screening, the lysogenic bacteriophage φ T4-3C was obtained. Bacteriophage PCR amplification, SDS-PAGE and Western-blotting analyses demonstrated that the recombinant bacteriophage could display a recombination protein of approximately 26ku in size, and it had immunological reactivity. |
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Key words: foot-and-mouth disease virus; 3 C protease; T4 bacteriophage; surface display |
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