目的研究重组毕赤酵母表达的乙型肝炎病毒preS2S蛋白的纯化方法及免疫效果。方法甲醇诱导含preS2S基因的重组毕赤酵母菌,采用蔗糖区带速率离心、Butyl-S-Sepharose 4FF疏水层析和DEAE-Sepharose Fast Flow离子交换层析等方法,对表达产...目的研究重组毕赤酵母表达的乙型肝炎病毒preS2S蛋白的纯化方法及免疫效果。方法甲醇诱导含preS2S基因的重组毕赤酵母菌,采用蔗糖区带速率离心、Butyl-S-Sepharose 4FF疏水层析和DEAE-Sepharose Fast Flow离子交换层析等方法,对表达产物进行纯化。纯化后蛋白经SDS-PAGE和Western blot方法鉴定,制备成疫苗,并进行效力试验。放射免疫法检测免疫小鼠血清抗体浓度,计算ED50和相对效力。ELISA法检测血清中的preS2S抗体。结果纯化后的preS2(epi)S蛋白相对分子质量约为27000,且能与特异性抗体结合。两批纯化蛋白制备的疫苗ED50分别为0.35和0.48μg/ml,参比苗为0.52μg/ml。2批疫苗的体外相对效力分别为1.5和1.1,均合格,血清抗体平均浓度为808和784mIU/ml,参比苗为312mIU/ml。2批疫苗免疫小鼠后均产生了preS2抗体。结论该纯化方法实用有效,纯化后的preS2(epi)S蛋白比S蛋白具有更好的免疫原性。展开更多
乙型肝炎病毒(Hepatitis B Virus,HBV)外膜蛋白含PreS1、PreS2和S 3种蛋白,位于病毒颗粒的外表面,参与病毒颗粒的吸附、组装与成熟,在保护性免疫应答反应中起重要作用,HBV感染肝细胞所合成的外膜蛋白对肝细胞的生长、代谢,甚至是恶性转...乙型肝炎病毒(Hepatitis B Virus,HBV)外膜蛋白含PreS1、PreS2和S 3种蛋白,位于病毒颗粒的外表面,参与病毒颗粒的吸附、组装与成熟,在保护性免疫应答反应中起重要作用,HBV感染肝细胞所合成的外膜蛋白对肝细胞的生长、代谢,甚至是恶性转化产生一系列重要的生物学调节效应,外膜蛋白的变异使得HBV对受染肝细胞的影响非常复杂且成多样化,在病毒性乙型肝炎慢性化及其相关疾病演变过程的分子病理机制扮演重要角色。展开更多
The DNA fragments coding for preS2(120 146) and preS1(21 47) amplified by PCR were fused to both 5′ and 3′ ends of S gene at the position of amino acid 223. The fusion gene was placed downstream of the promoter P7.5...The DNA fragments coding for preS2(120 146) and preS1(21 47) amplified by PCR were fused to both 5′ and 3′ ends of S gene at the position of amino acid 223. The fusion gene was placed downstream of the promoter P7.5 of the universal vaccinia viral vector pGJP 5 and the recombinant vaccinia virus vS2SS1 was then selected by \%in vivo\% homogeneous recombination. Fusion protein S2SS1 could be expressed in the mammalian cells infected with vS2SS1. The investigation of expression, secretion, antigenicity and particle assembly of the S2SS1 protein demonstrated that S2SS1 protein could be assembled into particles which presented preS1, preS2 and S antigenicity and be efficiently secreted from the cells. It also showed that the level of its expression and secretion approached to that of the S protein expressed by the recombinant vaccinia virus.展开更多
文摘目的研究重组毕赤酵母表达的乙型肝炎病毒preS2S蛋白的纯化方法及免疫效果。方法甲醇诱导含preS2S基因的重组毕赤酵母菌,采用蔗糖区带速率离心、Butyl-S-Sepharose 4FF疏水层析和DEAE-Sepharose Fast Flow离子交换层析等方法,对表达产物进行纯化。纯化后蛋白经SDS-PAGE和Western blot方法鉴定,制备成疫苗,并进行效力试验。放射免疫法检测免疫小鼠血清抗体浓度,计算ED50和相对效力。ELISA法检测血清中的preS2S抗体。结果纯化后的preS2(epi)S蛋白相对分子质量约为27000,且能与特异性抗体结合。两批纯化蛋白制备的疫苗ED50分别为0.35和0.48μg/ml,参比苗为0.52μg/ml。2批疫苗的体外相对效力分别为1.5和1.1,均合格,血清抗体平均浓度为808和784mIU/ml,参比苗为312mIU/ml。2批疫苗免疫小鼠后均产生了preS2抗体。结论该纯化方法实用有效,纯化后的preS2(epi)S蛋白比S蛋白具有更好的免疫原性。
文摘乙型肝炎病毒(Hepatitis B Virus,HBV)外膜蛋白含PreS1、PreS2和S 3种蛋白,位于病毒颗粒的外表面,参与病毒颗粒的吸附、组装与成熟,在保护性免疫应答反应中起重要作用,HBV感染肝细胞所合成的外膜蛋白对肝细胞的生长、代谢,甚至是恶性转化产生一系列重要的生物学调节效应,外膜蛋白的变异使得HBV对受染肝细胞的影响非常复杂且成多样化,在病毒性乙型肝炎慢性化及其相关疾病演变过程的分子病理机制扮演重要角色。
文摘The DNA fragments coding for preS2(120 146) and preS1(21 47) amplified by PCR were fused to both 5′ and 3′ ends of S gene at the position of amino acid 223. The fusion gene was placed downstream of the promoter P7.5 of the universal vaccinia viral vector pGJP 5 and the recombinant vaccinia virus vS2SS1 was then selected by \%in vivo\% homogeneous recombination. Fusion protein S2SS1 could be expressed in the mammalian cells infected with vS2SS1. The investigation of expression, secretion, antigenicity and particle assembly of the S2SS1 protein demonstrated that S2SS1 protein could be assembled into particles which presented preS1, preS2 and S antigenicity and be efficiently secreted from the cells. It also showed that the level of its expression and secretion approached to that of the S protein expressed by the recombinant vaccinia virus.
