Nucleotide sequence of the p26 gene in Bombyx mori nuclear is virus and its recombinant protein secretion in infected cells.

完整的鯉魚性腺激素基因構築於苜蓿夜蛾核多角體病毒之置換載體中, 和 家蠶核多角體病毒基因體DNA共同轉移感染至家蠶細胞,確實能夠選到交換 成功之重組病毒。此重組病毒於感染細胞後第5天，合成鯉魚性腺激素的a 或b次單元，試驗結果顯示具備a 次單元訊號胜太的a或b次單元蛋白在細 胞中合成後，幾乎完全被分泌至胞外。另一方面，共同轉移感染的重複試 驗，由於重組病毒出現的機率過低，以致無法得到交換成功的重組病毒。 為促進利用家蠶核多角體病毒在細胞內合成B 型肝炎表面抗原的分泌，將 鯉魚性腺激素a 次單元的訊號胜太inframe 構築於B 型肝炎表面抗原pre- S2基因上游，並將此重組基因殖入利用p10 基因啟動子之家蠶核多角體病 毒置換載體pBmp10MZ。經過多次重複試驗共篩到158 株疑似交換成功，具 多角體、在X-gal 培養基中呈白色的重組病毒，然而利用PCR 反應檢查結 果顯示，這些病毒皆不含B型肝炎表面抗原基因，說明以含lacZ基因的病 毒與不含lacZ基因的置換載體交換挑選重組病毒的策略，有技術上的困難 。為檢查位於p10 基因上游的核甘酸序列與苜蓿夜蛾核多角體病毒相同區 域之相似程度，本試驗進行此段DNA 序列的選殖與定序，結果發現此段區 域與苜蓿夜蛾核多角體病毒位於p10 基因上游的p26基因在核甘酸序列 有95.0% 的相似性。且家蠶核多角體病毒p10 基因上游的這段區域，有一 可轉譯成240 個氨基酸之open reading frmae,與苜蓿夜蛾核多角體病毒 p26 基因的氨基酸序列有95.8% 之相似性，分析家蠶核多角體病毒p26 的 氨基酸序列，並沒有發現任何特定的motifs。The Bombyx mori nuclear polyhedrosis virus (BmNPV) genome and the Autographa californica nuclear polyhedrosis virus (AcMNPV) transfer vectors containing the complete carp gonadotropin (GTH) gene were simultaneously cotransfected into BmN cells. The insertion of carp GTH gene into th BmNPV genome was achieved. Monolayers of BmN cells were infected with recombinant virus. Five days post-infection, the carp GTH a or b-subunit was synthesized and entirely secreted from BmN cells into media. Subsequently, determination of the virus titer suffered from failure in forming single plaques. The cotransfection was repeated and no more recombinant virus could be obtained. In order to improve the secretion of hepatitis B surface antigen (HBsAg) that was synthesized in BmN cells infected with recombinant BmNPV, the carp GTH a-signal peptide was constructed inframe upstream of the HBsAg pre-S2 gene. The chimeric gene was inserted into the BmNPV transfer vector, pBmp10MZ, downstream of the p10 promoter. From 4 cotransfection experiments, 158 occlusion-positive and b-galactosidase- negative recombinant virus were obtained via plaque assay (9 times) or end-point dilution (9 times) experiments. Polymerase chain reactions (PCR) were conducted to examine the recombinant virus for the presence of the chimeric HBsAg. The results indicated that despite of their lack of b-galactosidase activity, no HBsAg gene was detectable in thses recombinant viruses. These results suggested that selection for the replacement of the lacZ gene carried on the parent virus could be technically difficult. The DNA fragment upstream of the BmNPV p10 gene was cloned and sequenced, so that it could be compared with the nucleotide sequence upstream of the AcMNPV p10 gene. An open reading frame (ORF) encoding a polypeptide of 240 amino acids was detected. It shares 95.8% amino acid sequence similarity with that of AcMNPV p26 protien.Analysis of the BmNPV p26 amino acid sequence did not reveal any structural motifs