Ag85a-卡介苗重组疫苗的构建及鉴定

目的 构建结核分枝杆菌ag85a-卡介苗重组疫苗,研究其免疫原性及抗结核作用,以期获得结核病预防性或治疗性疫苗.方法 通过基因工程重组技术将结核分枝杆菌保护性抗原Ag85a的编码基因与穿梭质粒载体pYUB295重组,通过电穿孔技术导入卡介苗中,应用PCR扩增、PAGE电泳鉴定重组卡介苗.结果 通过PCR扩增获得约800 bp的ag85a基因,与穿梭表达载体pYUB295重组后,通过PCR扩增、限制性内切酶酶切、DNA测序鉴定表明ag85a基因正确插入到pYUB295质粒中.将重组质粒通过电穿孔导入卡介苗,重组卡介苗在抗性培养基上生长良好.pYUB295-ag85a重组卡介苗基因组DNA的PCR扩增以及培养上清液的PAGE电泳表明:ag85a-卡介苗重组疫苗构建正确,Ag85a蛋白在卡介苗中分泌表达.结论 成功构建了ag85a-卡介苗重组疫苗.

Construction and identification of Ag85a-recombinant BCG vaccine

Objective] To construct Ag85a-recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively. Methods] The gene of Ag85a was amplified by PCR with primers designed according to the sequences publicized in Genebank and the vector used. The gene was recombined with shuttle plasmids pYUB295. The recombinant shuttle plasmid was identified by PCR, enzyme digestion and DNA sequencing and then transformed into BCG by electroporation. Results] The gene of Ag85a was amplified by PCR. Ag85a-pYUB295 recombinant plasmid was constructed through recombinant technology and identified by PCR, enzyme digestion and DNA sequencing. The recombinant plasmid was transformed into BCG by electroporation. The pYUB295 recombinant BCG was identified by amplified recombinant BCG genome DNA and showed Ag85a gene was transformed into BCG successfully. The SDS-PAGE result of Ag85a recombinant BCG culture filtrate showed: the extrinsic Ag85a gene could be expressed in BCG. Conclusion] The Ag85a recombinant BCG were constructed successfully.