A spontaneous mutant that produces a single abnormally large cubic polyhedron per infected cell was isolated from a polyhedra-positive recombinant Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Both wild-type and mutant virus produce two forms of virus particles, budded virions and occluded virions. However, occluded virions are not found within the polyhedra of cells infected with mutant virus, as with the wild-type virus. These large cubic polyhedra do not have the typical lattice-like structure normally seen in wild-type polyhedra and are noninfectious. Spodoptera frugiperda 9 (SF9) cells which were infected with this virus had low infectivity to larvae. No significant alterations were found in the viral genome by restriction enzyme analysis, and no mutations were found in the 25K gene. A single point mutation resulting in an amino acid change of Gly25 to Asp was identified in the polyhedrin gene. A transfer vector containing the entire polyhedrin gene including the point mutation was constructed and used to cotransfect Sf9 cells with a polyhedron-negative recombinant virus. Large cubic polyhedra were once again observed, confirming that the Gly25 to Asp mutation is responsible for the formation of abnormal polyhedra.

载脂蛋白A-I（apoA-I）是高密度脂蛋白中最主要的蛋白，在胆固醇代谢及抗动脉粥样硬化等疾病的发生和控制中有重要作用。大量研究表明apoA-I在肝脏细胞表面有特异性受体，使apoA-I有可能成为治疗肝脏疾病的靶向药物的载体。进一步探索apoA-1的结构与功能，及其在新药开发中的应用，迫切需要用基因工程的方法高效表达生产apoA-I. 本文采用Bac-To-Bac杆状病毒表达系统构建了表达人。apoA-I的两个重组杆状病毒，一是表达去除了天然信号肽（pre）序列，但保留pro序列的proapoA-I；另一个用一种异源的信号肽-蛇磷脂酶A2抑制因子o亚基信号肽引导表达成熟apoA-I；不带有信号肽序列的proapoA-I蛋白表达后主要在细胞内，但在感染的晚期也有相当量的重组蛋白被分泌到细胞培养液中。用蛇磷脂酶A2抑制因子a亚基信号肽序列引导表达的成熟apoA-I蛋白在感染甲期就可以被分泌到培养液中。在此基础上，本文组合使用DEAE-sepharose阴离子交换柱层析和phenyl sepharose疏水层析分离纯化胞内表达的proapoA-I蚩门，取得了初步的浓缩和纯化效果。而对于分泌表达的成熟apoA-I，分别使用了制备性非变性聚丙烯酰胺凝胶电泳和疏水层析进行纯化。非变性聚丙烯酰胺凝胶电泳对于保持蛋白的生物活性是有利的，其纯化效果也相对较好。在使用phenyl sepharose疏水柱层析分离成熟apoA-I时，也尝试了多种洗脱的方法，结果发现用30%丙二醇和70%丙二醇的分步洗脱效果和效率最佳。比较了各种分离纯化的手段以及研究了在实验过程中出现的问题，本文作者推测apoAI蛋白自身的多态性以及与脂质不同程度的结合是造成分离纯化困难和得率较低的一个重要原因。设想将分泌表达在上清中的apoA-I在分离纯化之前先脱脂，再通过疏水柱层析，有可能优化纯化效果和效率，这有待于进一步试验。

乙型肝炎是一个世蚧范围的传染病，在我国为害尤其严重。乙型肝炎病毒（hepatitis B virus, HBV）作为引起人类急性、慢性肝炎的主要病原体之一，属嗜肝DNA病毒科。虽然其基因组仅有3.2kb，但基复制和致病机理过程复杂，突变众多，目前还没有根治的方法。因此，发展抗乙肝药物的研究显得尤为必要。乙肝病毒聚合酶（HBV polymerase）是病毒复制的关键酶，具有TP（蛋白引物）、间隔区（删除大部分后不影响功能）、RT（逆转录酶）、RnaseH等多个功能区域，是抗乙肝药物作用的理想位点。乙肝聚合酶很难在感染细胞中分离纯化，也一直很难在哺乳动物细胞中大量重组表达，严重阻碍了抗乙肝药物和乙肝疫苗的研究。本研究选取高复制型的临床分离株HBV-56，克隆拼接了全长的多聚酶基因，并将基插入穿梭质粒，通过重组穿梭质粒在细菌细胞内与带有腺病毒基因组的质粒发生同源重组，得到带有HBV多聚酶的重组腺病毒质粒。该质粒线性化后转染人胚肾细胞转化细胞系293，成功地得了重组病毒。用该重组病毒感染293细胞系，SDS-PAGE检测，发现了新的蛋白的合成，该蛋白质的分子量与文献报道的乙肝聚合酶的分子量一致，用PCR的方法，初步证明了所表达的乙肝聚合酶具有逆转录酶的活性，合成了对应于HBV聚合酶基因的DNA分子。这一结果将促进对于抗乙肝药物研究和对乙肝病毒复制机制的认识。

Midgut epithelial ceils were isolated from fifth-instar Pseudaletia unipuncta larvae by collagenase treatment of midgut tissue, and cultured in TNM-FH medium. Long-term continuous culture and maintenance of midgut cells were achieved with P. unipuncta armyworm intestinal cells. Several ceils lines were obtained from these P. unipuncta prima3 cultures, and they have been subcultured and maintained for over 24 too. The three major midgut cell types were present in the cultures, including stern (regenerative), columnal, and goblet cells. In vitro morphogenesis and differentiation of columnar and goblet cells from stem ceils were observed. There appeared to be a cycle of cell death of goblet and columnar cells followed by their replacement from stem cells evely 7-8 wk. After approximately six passages, the cell density in Tflasks appeared to be somewhat constant, reaching 103-104 cells per milliliter of medium. The columnar cells are round to rectangular in shape and possess a brush border, while the goblet cells have a classic flask-like shape with a central cavity. Peritrophic membrane-like secretions were observed in all the culture flasks. Infection of these ceils with multiply embedded nucleopolyhedrovirus was confirmed, and we conclude that these midgut cells can be used as an in vitro model system to study early events in baculovirus infection.

颗粒体病毒的增强蛋白（enhanctn）是一种能显著提高核型多角体病毒（NPV）对昆虫感染力的病毒蛋白。构建了一种不形成多角体但表达粉纹夜蛾颗粒体病毒增强蛋白的重组病毒A出BH TnEn，将它与野生型AcMNPV共同感染SF21细胞，经SDS-PAGE、免疫印迹分析、荧光免疫等方法检测证实，增强蛋白与多角体可在同一细胞中同时表达，而且发现所形成的病毒多角体带有增强蛋白。这表明，可以通过混合感染的方式生产带有增强蛋白的病毒多角体。