【摘要】 用PCR法将乙肝表面抗原基因S2 S片段从乙肝病毒中扩增得到 ,将其插入到蓝藻热休克表达载体 pEUTMT1中 ,构建成表达重组质粒 pES2ST1。将蓝藻Synechococ cussp .PCC794 2的总染色体与质粒 pES2ST1同时进行EcoRI和SacI双酶切 ,再连接构建成为系列含有蓝藻染色体DNA同源片段的供体表达质粒。经转化筛选得到蓝藻Synechococcussp .PCC794 2转化藻株。PCR和Southern杂交证明目的基因已经整合到宿主的染色体中。转化藻通过热诱导后 ,Northern blot结果呈阳性 ,用化学发光检测技术可以检测到微量目的蛋白的表达 ,检测含量约为 0 .78～ 0 .6 4ng/ml,目的蛋白约为可溶性蛋白的 1.1× 10 - 6 ～ 1.5× 10 - 6 。更多还原

【Abstract】 The gene of mid protein of HBsAg (S 2S) was cloned by PCR from HBV according to the known HBV sequence. Then subcloned S 2S gene into the expression plasmid pEUTMT1, the pES2ST1 which contains heat-shock groESL promoter, rbcS-polyA terminator and kanamycin resistance gene elements was constructed. Then using EcoRI and SacI to digest chromosome DNA of Synechococcus sp.PCC7942 and plasmid pES2ST1, the production of random digested fragments of chromosome DNA were linked with pES2ST1 to construct new donor plasmids. The donor plasmids were transformed into Synechococcus sp. PCC7942 and the transformants were obtained by kanamycin screening. The result of PCR and southern blot showed that the S 2S gene had been transferred into host chromosome. Induced by heat shock, the northern-blot result showed that HBsAg gene had been transcripted. Detected by chemistry irradiance method, it showed that the expression amount of HBsAg S 2S fragment reach 0.78～0.64ng/ml, which is about 1.1×10 -6～1.5×10 -6 of total solution protein.更多还原