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用灭活的甲氧西林耐药金黄色葡萄球菌免疫小鼠后,取出小鼠脾脏提取总RNA,以RT-PCR扩增小鼠抗体重链(VH)和轻链(VL)可变区基因,以重叠延伸PCR(SOE-PCR)法将VH和VL拼接成ScFv基因,克隆入噬菌粒载体pCANTAB-5E中,转化大肠杆菌E.coli TG1,构建鼠源抗金黄色葡萄天然噬菌体抗体库。研究结果表明,构建的鼠源性抗甲氧西林耐药金黄色葡萄球菌抗体库库容为3.2×106,多样性良好,共筛选出8个阳性克隆。说明已成功构建抗甲氧西林耐药金黄色葡萄球菌抗体库,为筛选有效治疗甲氧西林耐药金黄色葡萄球菌奠定了基础。
After immunizing mice with inactivated methicillin-resistant Staphylococcus aureus, the spleen of the mice was taken out to extract total RNA, and the mouse antibody heavy chain (VH) and light chain (VL) variable region genes were amplified by RT-PCR , VH and VL spliced into ScFv gene by overlap extension PCR (SOE-PCR) method, cloned into the phagemid vector pCANTAB-5E and transformed into E. coli TG1 to construct a mouse anti-golden grape natural phage antibody library . The results showed that the constructed rat anti-methicillin-resistant Staphylococcus aureus antibody library capacity of 3.2 × 106, good diversity, a total of 8 positive clones were screened. This indicated that the anti-methicillin-resistant Staphylococcus aureus antibody library has been successfully constructed, which lays the foundation for screening effective methicillin-resistant Staphylococcus aureus.