中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2012年
2期
96-98
,共3页
揣侠%陈红%杨扬%王文%文波%王刚%黄保英%邓瑶%谭文杰
揣俠%陳紅%楊颺%王文%文波%王剛%黃保英%鄧瑤%譚文傑
췌협%진홍%양양%왕문%문파%왕강%황보영%산요%담문걸
肝炎病毒,乙型%基因组%表达的序列标记%高压水动力法
肝炎病毒,乙型%基因組%錶達的序列標記%高壓水動力法
간염병독,을형%기인조%표체적서렬표기%고압수동역법
Hepatitis B virus%Genome%Expressed sequence tags%Hydrodynamic injection
目的 构建四种不同结构的1.3倍全基因乙型肝炎病毒(HBV)转基因载体,观察HBV抗原在体内外表达水平的差异.方法 克隆1.3倍的HBV基因至慢病毒转基因质粒pCS-CG并取代其中的EGFP表达盒,构建四种结构的pCS-HBV1.3质粒,体外转染Huh 7细胞和尾静脉高压水动力法注射C57BL/6小鼠后,ELISA方法分别检测细胞上清和小鼠血清中HBsAg和HBeAg的表达.结果 成功构建了四种不同结构的pCS-HBV1.3质粒,四种质粒HBV S抗原与e抗原在体内外的表达趋势一致,即乙肝抗原表达水平在正向插入的转基因质粒中高于反向插入；HBV抗原表达水平还与载体序列中RRE调控元件和cPPT调控元件前的基因序列长度有关.结论 筛选获得了可在体内外高效表达HBV抗原的转基因载体可应用于乙肝病毒体内外感染模型的建立与应用.
目的 構建四種不同結構的1.3倍全基因乙型肝炎病毒(HBV)轉基因載體,觀察HBV抗原在體內外錶達水平的差異.方法 剋隆1.3倍的HBV基因至慢病毒轉基因質粒pCS-CG併取代其中的EGFP錶達盒,構建四種結構的pCS-HBV1.3質粒,體外轉染Huh 7細胞和尾靜脈高壓水動力法註射C57BL/6小鼠後,ELISA方法分彆檢測細胞上清和小鼠血清中HBsAg和HBeAg的錶達.結果 成功構建瞭四種不同結構的pCS-HBV1.3質粒,四種質粒HBV S抗原與e抗原在體內外的錶達趨勢一緻,即乙肝抗原錶達水平在正嚮插入的轉基因質粒中高于反嚮插入；HBV抗原錶達水平還與載體序列中RRE調控元件和cPPT調控元件前的基因序列長度有關.結論 篩選穫得瞭可在體內外高效錶達HBV抗原的轉基因載體可應用于乙肝病毒體內外感染模型的建立與應用.
목적 구건사충불동결구적1.3배전기인을형간염병독(HBV)전기인재체,관찰HBV항원재체내외표체수평적차이.방법 극륭1.3배적HBV기인지만병독전기인질립pCS-CG병취대기중적EGFP표체합,구건사충결구적pCS-HBV1.3질립,체외전염Huh 7세포화미정맥고압수동역법주사C57BL/6소서후,ELISA방법분별검측세포상청화소서혈청중HBsAg화HBeAg적표체.결과 성공구건료사충불동결구적pCS-HBV1.3질립,사충질립HBV S항원여e항원재체내외적표체추세일치,즉을간항원표체수평재정향삽입적전기인질립중고우반향삽입；HBV항원표체수평환여재체서렬중RRE조공원건화cPPT조공원건전적기인서렬장도유관.결론 사선획득료가재체내외고효표체HBV항원적전기인재체가응용우을간병독체내외감염모형적건립여응용.
Objective To character HBV antigen expression in vitro and in vivo transduced by different transgenic plasmids carrying infectious genome of hepatitis B virus ( HBV ). Methods We constructed four different lentiviral transfer plasmids( carrying 1.3 full-length genome of HBV,by replacing the EGFP express box in pCS-CG plasmid with HBV genome and with different structural element,named as pCS-HBV1.3 (pCS-HBV1.3 X,pCS-HBV1.3 P,pCS-HBV1.3 N and pCS-HBV1.3 K).We detected the expression of HBsAg and HBeAg by ELISA in different time after transfected Huh 7 cells or hydrodynamic injection into C57 BL/6 mice with transfer plasmids pCS-HBV,respectively.Results We detected significant expression of HBsAg over 5 days after transfected Huh 7 cells ( in vitro ) or hydrodynamic injection into C57BL/6 mice(in vivo) with transfer plasmids pCS-HBV1.3 X,pCS-HBV1.3 P and pCS-HBV1.3 K.The expression level and dynamics of HBsAg and HBeAg in the sera of mice is consistent with that of in the supematant of Huh-7 cell.Furthermore,the expression of HBV antigens were modulated by the direction and position of HBV insert,also by some lentivira] vector cis-elements ( cPPT and RRE).Conclusion The optimal lentiviral transfer plasmids (pCS-HBV1.3 X,pCS-HBV1.3 P and pCS-HBV1.3 K) could be further used for establishment and application of HBV transgenic cell or animal model.
