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采用ＰＣＲ方法，从ＨＰＶ１６型质粒中分离出ＨＰＶ１６Ｅ７基因片段，用ＥｃｏＲＩ和ＳａｌＩ双酶解后，定向插入到表达质粒ｐＹＡ３３３２（ａｓｄ＋）的Ｐｔｒｃ启动子下游，构建成ｐＹＡ３３３２－ＨＰＶ１６－Ｅ７重组表达质粒。在大肠杆菌Ｘ６２１２上筛选出重组质粒后，通过中间菌株Ｘ３７３０的转化过渡，将重组质粒导入鼠伤寒沙门氏减毒疫苗菌株Ｘ４０７２（ａｓｄ－），构建成宿主和载体之间具有平衡致死结构的ＨＰＶ１６型重组疫苗。ＳＤＳ－ＰＡＧＥ染色和Ｗｅｓｔｅｒｎｂｌｏｔ法分析证实，该重组疫苗能特异地表达ＨＰＶ１６Ｅ７融合蛋白
The HPV16E7 gene fragment was isolated from HPV16 plasmid by PCR and double digested with EcoRI and SalI and then inserted into the downstream of Ptrc promoter expressing plasmid pYA3332 (asd +) to construct pYA3332-HPV16-E7 recombinant expression plasmid. After the recombinant plasmid was screened on Escherichia coli X6212, the recombinant plasmid was introduced into Salmonella typhimurium attenuated vaccine strain X4072 (asd-) through transformation and transformation of the intermediate strain X3730 to construct a balanced lethal structure of HPV16 between the host and the vector Recombinant vaccine. SDS-PAGE staining and Western blot analysis confirmed that the recombinant vaccine can express HPV16E7 fusion protein