Ag85b-卡介苗重组疫苗的构建及鉴定

目的构建结核分枝杆菌Ag85b-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和治疗性疫苗。方法通过基因工程重组技术将结核分枝杆菌保护性抗原Ag85b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Ag85b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测序鉴定表明成功地构建了Ag85b基因pYU295重组质粒。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-Ag85b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：Ag85b-卡介苗重组疫苗构建正确,Ag85b蛋白在卡介苗中分泌表达。结论成功构建了Ag85b-卡介苗重组疫苗。

The construction and identification of ag85b-recombinant BCG vaccine

Objective To construct ag85b-recombiant BCG vaccine to find an improved vaccine to replace BCG so that TB can be prevented more effectively. Methods The gene of ag85b 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 electreporation. Results The gene of ag85b was amplified by PCR. Ag85b-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 electreporation. The pYUB295 recombinant BCG was identified by amplified recombinant BCG genome DNA and showed ag85b gene was trans- formed into BCG successfully. The SDS-PAGE result of ag85b recombinant BCG culture filtrate showed that the extrinsic ag85b gene could be expressed in BCG. Conclusion The ag85n recombinant BCG were constructed successfully.