DENG Xiao-Zhao, DIAO Zhen-Yu, HE Liang, JIAO Ren-Liang and ZHANG Lin-Yuan. Cloning and sequencing of the Helicoverpa armigera nuclear polyhedrosis virus DNA polymerase gene[J]. Virologica Sinica, 1998, 13(1).
Citation: DENG Xiao-Zhao, DIAO Zhen-Yu, HE Liang, JIAO Ren-Liang, ZHANG Lin-Yuan. Cloning and sequencing of the Helicoverpa armigera nuclear polyhedrosis virus DNA polymerase gene .VIROLOGICA SINICA, 1998, 13(1) : 76.

用杆状病毒载体在家蚕细胞中表达HBeAg基因

  • 以PCR技术扩增含有PreC信号肽序列及完整的HBeAg基因的序列(即HBcAg基因5′端447bp),在5′端加上合适的酶切位点,克隆到家蚕核多角体病毒转移载体pBm030上,与野生型BmNPVDNA共转染家蚕BmN细胞,空斑纯化后得到多角体基因失活的重组病毒。ELISA法测定表明培养液上清中HBeAg效价达1∶32000,细胞内HBeAg效价为1∶2000,培养液及细胞内的HBcAg含量极低(<1∶160)。研究结果表明,BmN细胞能正确识别与切割HBeAg信号肽序列,所表达的HBeAg效价高,纯度好,明显优于大肠杆菌表达系统

Cloning and sequencing of the Helicoverpa armigera nuclear polyhedrosis virus DNA polymerase gene

  • A HBeAg gene fragment, which has some restriction endonucleased sites on both 5′ ends, including pre c signal peptide sequence and 5′ 447 bp of the HBcAg gene was amplified by PCR. The HBeAg gene fragment was cloned into the BmNPV transfer vector pBm030 and the chimeric vector pBmHBe was constructed. The BmN cells were co transfected with pBmHBe and Wt BmNPV DNA,therefore, the recombinant viruses were obtained by plaque purification. Analysis of the HBeAg antigenecity by ELISA showed that the highest titer in the cell cultural medium was up to a dilution of 1∶32000. Although HBeAg protein also presents in the BmN cells the titer was only 1:2000. The HBcAg protein was fewer than HBeAg (<1∶160) whatever in culture medium and in cells. the results showed that the BmN cells can recognize the HBeAg signal peptide sequence and cut it correctly for HBeAg. The BmN PV BmN cell system is considered to be much better than E.coli system for producing HBeAg protein.

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    Cloning and sequencing of the Helicoverpa armigera nuclear polyhedrosis virus DNA polymerase gene

    • 1. Nanjing Command Military Medical Institute,Nanjing 210002

    Abstract: A HBeAg gene fragment, which has some restriction endonucleased sites on both 5′ ends, including pre c signal peptide sequence and 5′ 447 bp of the HBcAg gene was amplified by PCR. The HBeAg gene fragment was cloned into the BmNPV transfer vector pBm030 and the chimeric vector pBmHBe was constructed. The BmN cells were co transfected with pBmHBe and Wt BmNPV DNA,therefore, the recombinant viruses were obtained by plaque purification. Analysis of the HBeAg antigenecity by ELISA showed that the highest titer in the cell cultural medium was up to a dilution of 1∶32000. Although HBeAg protein also presents in the BmN cells the titer was only 1:2000. The HBcAg protein was fewer than HBeAg (<1∶160) whatever in culture medium and in cells. the results showed that the BmN cells can recognize the HBeAg signal peptide sequence and cut it correctly for HBeAg. The BmN PV BmN cell system is considered to be much better than E.coli system for producing HBeAg protein.

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