真核表达编码Ag85A基因重组体的构建

抗原85复合体（Ag85）是BCG合成的能够刺激机体产生细胞免疫和体液免疫的多种成分之一，Ag85A是抗原85复合体组成成分之一，可显著刺激细胞免疫功能增强。为研究经口接种Ag85A的DNA疫苗的免疫效应，根据结核分枝杆菌Ag85A的基因序列自行设计了一对PCR引物，以人型结核杆菌H37Rv标准毒力株的DNA为模板，经过PCR扩增出Ag85A目的基因，纯化PCR产物TA克隆入载体pUCm-T载体，蓝白斑筛选将回收的PCR产物用限制性核酸内切酶Xhol和BamHI双酶酶切后，经T4DNA连接酶作用，与真核表达载体pCDNA3．1^＋连接，筛选得到的阳性克隆经DNA测序鉴定证实为Ag85A基因，且被克隆到载体pCDNA3．1^＋中的CMV启动子的下游，成功构建并鉴定的真核表达载体pCDNA3．1^＋携带Ag85A基因的重组体，命名为pCDNA3．1^＋／Ag85A。将其转化大肠埃希菌并使之大量扩增，并采用无内毒素提取质粒方法收集此重组质粒DNA．即为可经口途径喂饲小鼠的结核杆菌Ag85A的DNA疫苗，为口服DNA疫苗的临床应用研究奠定基础。

Construction of an Oral Recombinant DNA Vaccine Encoding Tuberculosis Ag85A Gene

Antigen 85 complex is one of the multi-components that could stimulate organism to produce cell immunity and humour immunity synthesized by BCG. Ag85A is one of the components of antigen 85 complexes that could stimulate cell immunity function to strengthen. In order to study the immunity effect of Ag85A DNA vaccine inoculate orally, a pair of primers were self-designed according to Ag85A sequence of Mycobacterium tuberculosis and took standard strain of virulence as template, and amplified the aimed gene of Ag85A through PCR, then purified the PCR products TA and cloned into a vector pUCm-T, blue-white spot screening and recovered the PCR products, after digested both with restriction enzymes of XhoI and BamHI, under the reaction of T4DNA ligase and linked to eukaryotic expression vector pCDNA3.1 ; obtained and proved to be Ag85A gene through screening and DNA sequencing to be positive clone and it was on the lower reaches site of CMV promoter. The successful constructed and identified to be cukaryotie expression vector pCDNA3.1 carrying Ag85A gene recombinant body was named as pCDNA3.1/Ag85A. Transformed it into E. coli to amplify it in great amount, and adopted endotoxin free extraction method of plasmid to collect recombinant plasmid DNA, and immediately could feed the mice orally as vaccine of Ag85A gene of M. tuberculosis to provide foundation of oral DNA vaccine for clinical application research.