【摘要】 自行设计和合成可转录mRNA不能翻译功能性蛋白分子的全长Ag85A编码基因突变体(Ag85A-M),克隆至真核表达载体pIRES2-EGFP,用于后续制备Ag85A-M疫苗,研究双股Ag85 DAN疫苗是否能通过RNA传感器提呈抗原信息,诱导固有和适应性免疫应答。设计Ag85A-M基因,将编码Ag85A基因(891 bp)中的第114位和230位的碱基C突变为G,使之含有TAG和TGA双重翻译终止子。合成Ag85A(M)全基因片段,利用普通PCR方法按照设计将Ag85A(M)全基因序列分别拆分成332 bp(A)、332 bp(B)、264 bp(C)三个小片段和488 bp(D)、423 bp(E)两个小片段进行扩增合成。构建pIRES2-EGFP-Ag85A(M)载体,将Ag85A(M)全长基因片段通过pIRES2-EGFP载体多克隆位点(multiple clone site, MCS)中双酶切位点EcoR I/BamH I插入载体,再经双酶切和基因测序鉴定,DC2.4中基因表达确认。结果显示,采用普通PCR法分段扩增合成获得与预先设计的长度一致的A、B、C、D和E小片段,合成的Ag85A-M的全部序列(891 bp)、突变位置和碱基正确。三片段拆分(A、B和C小片段)阳性克隆率平均值为87.67%,基因保真度为90.33%。二片段拆分(D和E小片段)阳性克隆率平均值为81.5%,基因保真度为23%。结果表明,本研究获得了可用于后续研究的含有114和230位点突变的全长Ag85A-M DNA片段,三片段基因拆分方法获得的332 bp及以下片段PCR法合成保真度显著高于二片段拆分方法获得的423 bp及以上片段。更多还原

【Abstract】 A self-designed and synthesized mRNA transcribable and untranslatable functional protein molecule Ag85 A DNA of full-length fragment encoded genes was cloned into eukaryotic vector pIRES2-EGFP used for follow-up preparation of Ag85 A-M vaccine, and for studying double strand Ag85 DNA vaccine whether it could present antigen information, and induce inherent and adaptable immune responses. Ag85 A-M gene was designed and replaced 114 th and 230 th base C mutated into G in encoded Ag85 A gene(891 bp), to make it contain double translation terminator TAG and TGA. The Ag85 A(M) full-length gene fragment was demerged common PCR into 332 bp(A), 332 bp(B), and 264 bp(C) three small fragments respectively, and 488 bp(D), 423 bp(E) two small fragments and carried out proliferation synthesis. And constructed pIRES2-EGFP-Ag85 A-M vector through EcoR I/BamH I double restriction site to insert pIRES2-EGFP vector, and confirmed the gene expression in DC2.4 by double enzyme cutting and identified with gene sequencing. The results showed that the synthesized Ag85-M complete sequence(891 bp) mutation sites and bases adopted by common PCR method amplified, synthesized and obtained fragment by fragment accorded with the length of A, B, C, D, and E small fragments were corrected. And the average positive clone ratio of three demerged fragments(A, B, and C small fragments) was 87.67%, the gene fidelity was 90.33%. And the average positive clone ratio of two demerged fragments(D and E small fragments) was 81.5%, the gene fidelity was 23%. The results suggested that this study had obtained that could be used in the follow up study on full length Ag85 A-M DNA fragment containing 114 and 230 mutation sites, the fidelity of 332 bp obtained by three-fragment demerging method and below gene obtained by PCR synthesis was significantly higher than those 423 bp and above fragments obtained by two-fragment demerging method.更多还原