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目的建立乙肝病毒表面抗原(HBsAg)的人参细胞表达系统。方法构建含有HBsAg基因的植物细胞表达载体质粒pBIBSb。采用农杆菌感染人参细胞的方法,经农杆菌培养、农杆菌与受体细胞共培养、受体细胞的脱菌培养以及转化体细胞的选择培养后,筛选在G418抗生素培养基上正常生长的细胞株。应用ELISA方法检测抗性细胞株的HBsAg表达;提取基因组DNA进行PCR扩增反应。结果质粒pBIBSb的酶切结果正确。经G418抗生素筛选得到8株正常生长的细胞株,其中5株能够表达HBsAg,提取基因组DNA进行PCR扩增反应,得到大小约700 bp的扩增片段。结论获得了整合HBsAg基因,并能够稳定表达HBsAg的人参细胞表达系统。
Objective To establish the ginseng cell expression system of hepatitis B virus surface antigen (HBsAg). Methods The plant cell expression plasmid pBIBSb containing HBsAg gene was constructed. Agrobacterium tumefaciens was used to infect Ginseng cells. After Agrobacterium tumefaciens cultivation, Agrobacterium tumefaciens co-culture with recipient cells, desensitization of recipient cells and selection of transformed somatic cells, the cells that grew normally on G418 antibiotic medium were screened. Strain. HBsAg expression in resistant cell lines was detected by ELISA and genomic DNA was extracted for PCR amplification. Results The result of plasmid pBIBSb digestion was correct. Eight normal cell lines were screened by G418 antibiotics. Five of them could express HBsAg, genomic DNA was extracted and amplified by PCR. The amplified fragment was about 700 bp in size. Conclusion Ginseng cell expression system that integrates HBsAg gene and can stably express HBsAg was obtained.