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使甲型肝炎病毒基因组cDNA能够在减毒伤寒杆菌中表达,从而通过口服此种减毒伤寒杆菌达到对甲肝免疫的作用。将甲肝病毒编码区cDNA插入高效表达质粒PBV221,转化大肠杆菌DH5α,筛选并鉴定阳性克隆。将阳性克隆用电转移法转化减毒的伤寒杆菌BRD509。用温度诱导法诱导目的基因在伤寒杆菌中表达。通过Western Blot和ELISA检测表明,表达蛋白可与人抗甲肝IgG发生特异性反应。用此伤寒杆菌口服或腹腔注射免疫小鼠,小鼠血清检测到抗甲型肝炎病毒的抗体。结论:甲肝病毒cDNA可以在减毒伤寒杆菌中表达,且表达的蛋白质在体内、外有免疫原性。
Hepatitis A virus genomic cDNA can be expressed in attenuated Salmonella typhimurium so as to achieve the effect of hepatitis A immunity by oral administration of such attenuated Salmonella typhi. The coding region of hepatitis A virus was inserted into the high expression plasmid PBV221 and transformed into E. coli DH5α. The positive clones were screened and identified. The positive clones were transformed into attenuated Salmonella typhimurium BRD509 by electrotransfer method. Induction of the target gene by temperature induction in Salmonella typhi. Western Blot and ELISA showed that the expressed protein reacted specifically with human anti-A IgG. With this Salmonella typhimurium oral or intraperitoneal injection of immunized mice, mouse serum anti-hepatitis A virus was detected. Conclusion: Hepatitis A virus cDNA can be expressed in attenuated Salmonella typhi, and the expressed protein is immunogenic both in vivo and in vitro.