【作者】 王霞；

【导师】 沈中元； 张志芳；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2006， 硕士

【摘要】 用随机克隆法,建立柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedrovirus,ApNPV)的质粒基因组文库:通过对插入片段进行克隆鉴定和序列分析,首次获得ApNPV的晚期表达因子3(later expression factor 3,lef-3)基因、蜕皮甾体尿苷二磷酸葡萄糖基转移酶基因(ecdysteroid UDP-glucosyltransferase,egt)、晚期表达因子1(lef-1)等基因,对其进行同源性比对分析。克隆得到了ApNPV PstI和XhoI两个片段,其中一个片断经序列分析表明:该片段含有完整的lef-3基因,与其它来源的lef-3基因具有很高的同源性。ApNPV lef-3基因的阅读框为1110个核苷酸,编码369个氨基酸,编码一种DNA单链结合蛋白(single-stranded DNA-binding protein,SSB)。在lef-3基因上游的互补序列有一编码127个氨基酸序列的不完全的阅读框。 第二个片段总长度为3388 bp,经序列分析表明:该片段包括5个完整读码框,与苜蓿银纹夜蛾多粒包埋核型多角体病毒(Autographa californica nucleopolyhedrovirus,AcNPV)ORF13/OpNPV ORF12同源性较高的基因、晚期表达因子1(later expression factor 1,lef-1)、缺失的Ecdysteroid UDP-Glucosyltransferase基因(egt)、da26、da16。其中egt基因编码蜕皮甾体尿苷二磷酸葡萄糖基转移酶;lef-1编码的是晚期表达因子1(LEF-1),分析得到LEF-1的氨基酸序列有3个高度保守同源区,具有引发酶的活性:da26基因编码结构蛋白BV/ODV-E26,这个蛋白在出芽型病毒(budded virus,BV)和包埋型病毒(occlusion-derived virus,ODV)中都存在。 本论文首次应用家蚕-杆状病毒表达系统表达嗜酸性α-淀粉酶,此酶基因长3906 bp,编码1301个氨基酸,蛋白质理论分子量140 KD。将克隆进大肠杆菌表达载体pET-22b(+)的amy基因克隆到转移载体pVL1393中,用常规方法获得病毒,感染家蚕后嗜酸性α-淀粉酶得到正确表达,检测表达产物具有淀粉酶活性。更多还原

【Abstract】 The gene encoding single-stranded DNA-binding protein(SSB) late expression factor 3 was cloned from Pst I to Xho I fragment of Antheraea pemyi nucleopolyhedrovirus (ApNPV) genome, which is fisrt found in ApNPV. Nucleotide sequence analysis of fragment revealed that lef-3 is a whole open reading frame of 1110 bp codes for 369 amino acids. Compared to the LEF-3 from other baculovirus, it was shown that the homologous on amino acid level was high. LEF-3 has identified as being essential for baculovirus DNA replication. LEF-3 proteins contain a conserved pattern of amino acids similar to this SSB motif in their amino-terminal regions, indicating that this region may be important for LEF-3 function. Upstream the lef-3 there is a sequence encoded 127 amino acids, but it is a part open reading frame. According to analysis of homologous this nucleotide sequence is similar to open reading frame 73 (ORF) of Orgyia pseudotsugata nucleopolyhedrovirus (OpNPV). At 3’end there is no homologous sequence compared to OpNPV and Choristoneura fumiferana nucleopolyhedrovirus (CfNPV), which indicates particularity of ApNPV.We also fisrt found the gene encoding protein of similar to Autographa californica nucleopolyhedrovirus (AcNPV) ORF 13 ORF 12, LEF-1, truncated Ecdysteroid UDP-glucosyltransferase (EGT), BV/ODV-E26, DA 16, which were also cloned from another Pst I to Xho I fragment of ApNPV genome. Compared to the five genes from other baculovirus, they were shown that the homologous on amino acid level was high. Among the five genes: lef-1 is believed to encode LEF-1. LEF-1 has identified as being essential for baculovirus DNA replication and contains three conserved pattern of amino acids, indicating that these region may be important for LEF-1 function and LEF-1 also has the activity of primase. Downstream the lef-1 there is a sequence encoded 79 amino acids named egt, which encodes EGT. Compared to egt from other baculovirus, it is deficient. da26 encodes BV/ODV-E26, which is the structure protein of budded virus, (BV) and occlusion-derived virus(ODV).The α-amylase from the Gram-positive Alicylobacillus acidocaldarius was one kind of thermoacidophilic enzyme. It was expressed by Bombyx mori NPV expression vector fisrt. The nucleotide sequence of the gene amy was 3 906bp, comprising one open reading frame encoding a polypeptide of 1 301 amino acids, the calculated molecular weight of the a-amylase AMY was about 140KD. AMY was expressed in silkworm larva by injection with the recombinant virus. The expressed products showed amylase activity.更多还原