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目的构建HBV表面抗原(HBsAg)的植物表达载体并在胡萝卜细胞中表达。方法以限制酶切M13/HB获得940bp含PreS2的HBsAg基因片段,将其插入到植物表达载体pBPC55,新质粒命名为pBPC91。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒pBPC93共同经基因枪(PDS-1000/He)转化胡萝卜悬浮细胞。经含除草剂(Bio-laphos)的MS液体培养基筛选,获得除草剂抗性胡萝卜细胞。结果纯化质粒pBPC91经酶切及序列分析表明HBsAg基因正确插入到E35S启动子下游且无密码子移位。对除草剂抗生胡萝卜细胞的组织学检测证实GUS基因有效表达;该细胞内DNA提取物PCR有940bp扩增带,蛋白萃取物的ELISA检测证实分别有HBsAg及PreS2-Ag蛋白表达。结论构建质粒pBPC91的PreS2及HBsAg基因可在转化胡萝卜细胞内表达。提示以植物细胞生产医用疫苗具有可行性。
Objective To construct a plant expression vector of HBV surface antigen (HBsAg) and express it in carrot cells. Methods The 940bp HBsAg gene fragment containing PreS2 was obtained by restriction enzyme digestion of M13 / HB, which was inserted into plant expression vector pBPC55. The new plasmid was named pBPC91. This was transformed into carrot suspension cells by gene gun (PDS-1000 / He) together with screening plasmid pBPC93 containing herbicide resistance gene and GUS protein gene. The herbicide-resistant carrot cells were obtained by selection with Bio-laphos-containing MS liquid medium. Results The purified plasmid pBPC91 was digested by restriction enzyme and sequence analysis showed that the HBsAg gene was correctly inserted into the downstream of E35S promoter and had no codon shift. Histological examination of the herbicide-resistant carrot cells confirmed that the GUS gene was efficiently expressed. The intracellular DNA extract PCR amplified 940bp. The protein extracts were tested by ELISA for the presence of HBsAg and PreS2-Ag protein, respectively. Conclusion The construction of plasmid pBPC91 PreS2 and HBsAg genes can be expressed in transformed carrot cells. It is feasible to use plant cells to produce medical vaccine.