1.Expression of report gene in insect cells by a new tranfer vector with baculovirus early promoter
Xiaozhao DENG ; Fanxiu ZHU ; Zhenyu DIAO ; Jian GAO ; Yipeng QI
Journal of Medical Postgraduates 2001;14(3):200-203,206
Objectives:Using IE 1 gene promoter of Autograph californica nuclear polyhedrosis virus(AcNPV),a transfer vector with an immediately early gene promoter was constructed. Methods:Transfer vector pAcPIneo which contains neomycin resistance gene(neo)coding sequence downstream of IE 1-promoter was constructed and cotransfected with the wild type of AcNPV DNA into Sf9 insect cells.Recombinant virus was selected by G418 resistance since the neo gene can be expressed in Sf9 cells. Results:Northern blot hybridzation with 32 P labeled neo gene fragement as probe showed that the neogene was integrated in plogene of AcNPV genome. Conclusions:Transfer vector with an immediately early gene promoter of baculovirus was constructed successfully,the neo gene was transcribed from the immediately early phase to the very late phase in infected cells.
2.Application of HBeAg produced in prokaryotic cells and eukaryotic cells
Liang HE ; Xiaozhao DENG ; Zhenyu DIAO ; Zongan ZHOU ; Jishan ZHENG ; Jian GAO
Journal of Medical Postgraduates 2001;14(3):204-206
Objectives: To express HBeAg in prokaryotic and eukaryotic cells and compare the two types of HBeAg in the anti-HBeAg testing. Methods: HBeAg was expressed both in E.coli cells and in silk worm cells, purified by Sephacryl S-200.HBeAg protein concentration and antigenic titer were determined respectively by ultraviolet-spectroscopy and EIA. Results: HBeAg produced by E.coli cells: Activation ratio was 10 000/mg, HBeAg/HBcAg = 50; The specificity in testing anti-HbeAg was 96%;HBeAg produced by silk worm cells: Activation ratio was 160 000/mg, HBeAg/HBcAg = 5 000, The specificity in testing anti-HbeAg was 100%. Conclusions: HBeAg produced by eukaryotic cells contained much lower proportion of HbcAg and higher activation ratio, which therefore bring about a possibility to improve the quality of the kit for testing Anti-HBe.
3.Distribution of hemorrhagic fever with renal syndrome virus in gamasid mites and chigger mites.
Yun ZHANG ; Jin ZHU ; Xiaozhao DENG ; Guanghua WU ; Jiaju ZHANG ; Yanping ZHOU
Chinese Journal of Preventive Medicine 2002;36(4):232-234
OBJECTIVETo study the distribution of hemorrhagic fever with renal syndrome virus (HFRSV) in mites.
METHODSIn situ reverse transcription-polymerase chain reaction (IS RT-PCR) was used for detecting the distribution of HFRSV in mites.
RESULTSHFRSV RNA was mainly located in ovary and mid-gut tissues of gamasid mites and chigger mites. The positive signal intensity in the third and fourth generations of gamasid mite was stronger than that in the first and second generations, and that in nymph of chigger mite more than larva.
CONCLUSIONBoth chigger mite and gamasid mite could play an important role in the transmission of HFRSV.
Animals ; Digestive System ; virology ; Female ; Hantaan virus ; genetics ; growth & development ; In Situ Hybridization ; methods ; Male ; Mites ; virology ; Nymph ; virology ; Ovary ; virology ; RNA, Viral ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods