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发明授权
CN105132461B 共表达SAL-2和hLYZ双抑菌基因重组腺病毒的构建方法及重组腺病毒和应用 有权

共表达SAL-2和hLYZ双抑菌基因重组腺病毒的构建方法及重组腺病毒和应用
摘要：
本发明公开了一种共表达SAL‑2基因和hLYZ双抑菌基因重组腺病毒载体的构建方法及重组腺病毒和应用，采用从金黄色葡萄球菌噬菌体基因组和人乳体细胞总RNA中分别扩增出的SAL‑2基因和hLYZ基因，将获得的目的基因克隆到pShuttle‑IRES‑hrGFP‑2穿梭载体中，线性化重组穿梭载体后与骨架载体pAdeasy‑1共转染大肠杆菌BJ5183感受态细胞，使其在BJ5183内进行同源重组，构建了包含上述目的基因的重组腺病毒质粒Ad‑SAL‑LYZ；将重组腺病毒质粒由PacⅠ酶切后，经脂质体介导转染HEK293细胞，包装获得高滴度的重组腺病毒；评价SAL‑2基因和hLYZ基因在腺病毒介导下对奶牛乳房炎主要致病菌的联合抑制作用。
摘要（英）：

The invention discloses a recombinant adenovirus capable of co-expressing double antibacterial genes of SAL-2 and hLYZ, and a construction method and application thereof. The construction method includes amplifying the SAL-2 gene and the hLYZ gene from a staphylococcus aureus phage genome and a human milk somatic cell total RNA (ribonucleic acid) respectively, cloning the obtained target genes into a shuttle vector pShuttle-IRES-hrGFP-2, co-transfecting an escherichia coli BJ5183 competent cell by the shuttle vector and a framework vector pAdeasy-1 after linear recombination of the shuttle vector to achieve homologous recombination in BJ5183, and constructing a recombinant adenovirus plasmid AD-SAL-LYZ containing the target genes; transfecting an HEK293 cell through liposome mediation after Pac I digestion of the recombinant adenovirus plasmid, and packing to obtain the high-titer recombinant adenovirus; evaluating the joint inhibiting effect of the SAL-2 gene and the hLYZ gene on main pathogenic bacteria of cow mastitis under adenovirus mediation.

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