| In the life cycle of baculovirus, DNA replication acts as the focus role. The homologous regions (hrs), which intersperse among the baculovirus genome, have the function of ori. of baculovirus DNA replication, and during the process of baculovirus genome transcription, they can also augment the expression of some early genes. Homologous region 3 (hr3) involved in this study was from BmNPV ZJ-8 strain, and it contains three 72bp conservative repeating units, i.e. three EcoRl little segments, each EcoRl little segment contains both a 30bp imperfect palindrome with EcoRl site in center and a 13bp stem-and-Ioop structure on both sides. It has been proved that hr3 has the function of ori. of baculovirus DNA replication, and specific silkworm cell factors bind it. In order to take more investigation on the enhancer function of BmNPV hr3 towards immediate early gene ie-1 promoter of different viruses, and more investigation on the correlation between hr3 and host cells, we cloned luciferase gene under the control of BmNPV or AcMNPV ie-1 promoter on hr3's upstream to construct the transient expression reporting plasmids. After transient expression in the BmN, Bm5, sf-21 cell lines and silkworm larvae of 5th instar, separately, we measured the activity of luciferase to study the enhancer activity of hr3 toward ie-1 promoter in different hosts.Analysis of the transient expression of the reporting plasmids with or without hr3 in BmN, Bm5, sf-21 cell lines and silkworm larvae of 5th instar showed that hr3 has obvious enhancer activity, 6980 times at the most, towards ie-1 promoter in cell lines and silkworm larva of 5th instar. Although luciferase gene's expression level in the three cell lines were different, enhancing times of hr3 to ie-1 promoter was almost the same. It suggested that the mechanism of hr3 in different lepidoptra section hosts be the same. Among baculovirus immediate early promoters, ie-1 promoter's activity is the strongest, though it is lower than the polyhedrin promoters. By using ie-1 promoter, we can make foreign genes expressed during the immediate early period when the viruses destroy the host cells less, so the expressed product be modified better. Hr3 is one of the strongest enhancer reported, so the compose of hr3 and ie-1 promoter can be used to construct stable insect cell expression vector and transgenic silkworms. As the first time, we used lipofectin to transfer foreign gene to silkworm lymphocyte to express transiently. This experiment proved the facility of transferring foreign gene to silkworm larva by using lipofectin. Meanwhile this experiment started a more facile, more economic and more authentic method to study BmNPV replication-transcription element through transient expression in vivo.We have known that hr3 has three EcoRl little segments. Based on this special structure characteristic, we constructed a series of transient expresson reporting plasmids with 108bp or 200bp or hr3 segments. This three segments have two halves of 30bp imperfect palindrome separated by the 13bp stem-and-loop conservative sequence, one EcoRl little segments, hr3 segment, respectively. In order to illustrate the correlation between hr3 structure and function, we investigated the enhancer function of hr3 sub-structure segments at different regions by the transient expression analysis in BmN cells. The results showed that 30bp imperfect palindrome takes the main enhancer function in hr3, the more the quantity of whole 30bp imperfect palindrome, the stronger the enhancer function.In the aspect of appliance study, in order to elevate the expression efficiency of BEVS for phytase gene, we constructed two different phytase transfer expression vectors pVL1393phyffland pBmOSOphy. By comparing the expression efficiency of pVL1393phy, pBm030phy with the former pAKSphy to screen phytase transfer expression vectors with the highest phytase expression efficiency, we found that pVL1393phy has the highest efficiency. |
PDF Full Text Request