【摘要】 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-bot分析,重组噬菌体可展示约26 ku的重组蛋白;其大小与预期相符,具有免疫原性。更多还原

【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 EcoR I restriction fragment of the T4 integrative plasmid pR, the fragment was used to transform E. coli DH5αto obtain the T4 bacte-riophage recombinant integrative plasmid pR-3C, and the success of which was confirmed by digestion with EcoR I restriction, PCR amplification, insert orientation screening and nucleotide sequencing. After further transformation of E. coli E2, the pR-3C was used for homologous combination with bacteriophageφT4-Z1 and, using lysozyme dependence screening, the lysogenic bacteriophageφT4-3C was obtained. Bacteriophage PCR amplification, SDS-PAGE and Western-blotting analyses demonstrated that the recombinant bacteriophage could display a recombination protein of approximately 26 ku in size, and it had immunological reactivity.更多还原