Ganapathi T R, Sunil Kumar G B, Srinivas L, Revathi C J, Bapat V A
Plant Cell culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Plant Cell Rep. 2007 Sep;26(9):1575-84. doi: 10.1007/s00299-007-0379-7. Epub 2007 May 30.
Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.
利用携带pHBS/pHER构建体的根癌农杆菌对大豆细胞悬浮培养物进行转化，以表达乙型肝炎表面抗原（HBsAg）。对转化后的菌落进行筛选，并通过聚合酶链反应（PCR）、逆转录（RT）PCR、蛋白质免疫印迹法和酶联免疫吸附测定（ELISA）分析来检测HBsAg的表达。在pHER转化的细胞中，观察到最高表达量为700 ng/g鲜重。选取表达量最高的菌落来启动细胞悬浮培养，并定期评估HBsAg的表达。与在半固体培养基上培养的菌落相比，细胞悬浮培养物中的表达水平急剧下降。研究了各种参数以最大化细胞生长并维持表达水平。在培养基中添加蛋白酶抑制剂硫酸化亮肽素可使细胞悬浮培养物中的HBsAg表达恢复高达50%。这是首篇研究植物细胞悬浮培养物中重组蛋白表达水平丧失的可能原因及解决方案的报告。
