中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
8期
1718-1720
,共3页
肝炎病毒,乙型%腺病毒载体%同源重组%抗原检测
肝炎病毒,乙型%腺病毒載體%同源重組%抗原檢測
간염병독,을형%선병독재체%동원중조%항원검측
Hepatitis B virus%Adenovirus vector%Homologous recombination%Antigen assay
目的 应用腺病毒AdEasy系统构建含1.3拷贝乙型肝炎病毒(HBV)基因组的腺病毒载体,并鉴定目的蛋白的表达.方法 采用基因重组技术将4.1 kb的1.3拷贝HBV基因序列克隆在腺病毒穿梭质粒上,通过在大肠杆菌内与骨架质粒的同源重组,构建重组腺病毒质粒,经293T细胞包装后产生重组腺病毒,感染肝癌细胞后检测乙型肝炎表面抗原(HBsAg)、乙型肝炎病毒e抗原(HBeAg)、HBV DNA和乙型肝炎病毒X蛋白(HBX)蛋白的表达,以转HBV基因组的HepG2.2.15肝癌细胞株为对照,同时检测上述指标含量.结果 双酶切下的1.3拷贝HBV序列成功克隆在腺病毒穿梭质粒上,与骨架质粒同源重组后的腺病毒质粒转染293T包装细胞后产生了重组腺病毒,感染HepG2细胞后能在细胞培养上清中检测HBsAg和HBeAg的含量分别为70.70、5.50 IU/ml,HBV DNA含量为1.2×107 copies/ml.同期检测的HepG2.2.15细胞内HBsAg和HBeAg的含量分别为3.51、44.85 IU/ml,HBV DNA含量为7.4×104 copies/ml.Western blot检测可见被感染的HepG2细胞有HBX蛋白的表达.结论 应用AdEasy系统成功构建含1.3拷贝HBV基因组的重组腺病毒载体,经包装细胞产生的病毒感染靶细胞后能检测到HBV抗原的表达.
目的 應用腺病毒AdEasy繫統構建含1.3拷貝乙型肝炎病毒(HBV)基因組的腺病毒載體,併鑒定目的蛋白的錶達.方法 採用基因重組技術將4.1 kb的1.3拷貝HBV基因序列剋隆在腺病毒穿梭質粒上,通過在大腸桿菌內與骨架質粒的同源重組,構建重組腺病毒質粒,經293T細胞包裝後產生重組腺病毒,感染肝癌細胞後檢測乙型肝炎錶麵抗原(HBsAg)、乙型肝炎病毒e抗原(HBeAg)、HBV DNA和乙型肝炎病毒X蛋白(HBX)蛋白的錶達,以轉HBV基因組的HepG2.2.15肝癌細胞株為對照,同時檢測上述指標含量.結果 雙酶切下的1.3拷貝HBV序列成功剋隆在腺病毒穿梭質粒上,與骨架質粒同源重組後的腺病毒質粒轉染293T包裝細胞後產生瞭重組腺病毒,感染HepG2細胞後能在細胞培養上清中檢測HBsAg和HBeAg的含量分彆為70.70、5.50 IU/ml,HBV DNA含量為1.2×107 copies/ml.同期檢測的HepG2.2.15細胞內HBsAg和HBeAg的含量分彆為3.51、44.85 IU/ml,HBV DNA含量為7.4×104 copies/ml.Western blot檢測可見被感染的HepG2細胞有HBX蛋白的錶達.結論 應用AdEasy繫統成功構建含1.3拷貝HBV基因組的重組腺病毒載體,經包裝細胞產生的病毒感染靶細胞後能檢測到HBV抗原的錶達.
목적 응용선병독AdEasy계통구건함1.3고패을형간염병독(HBV)기인조적선병독재체,병감정목적단백적표체.방법 채용기인중조기술장4.1 kb적1.3고패HBV기인서렬극륭재선병독천사질립상,통과재대장간균내여골가질립적동원중조,구건중조선병독질립,경293T세포포장후산생중조선병독,감염간암세포후검측을형간염표면항원(HBsAg)、을형간염병독e항원(HBeAg)、HBV DNA화을형간염병독X단백(HBX)단백적표체,이전HBV기인조적HepG2.2.15간암세포주위대조,동시검측상술지표함량.결과 쌍매절하적1.3고패HBV서렬성공극륭재선병독천사질립상,여골가질립동원중조후적선병독질립전염293T포장세포후산생료중조선병독,감염HepG2세포후능재세포배양상청중검측HBsAg화HBeAg적함량분별위70.70、5.50 IU/ml,HBV DNA함량위1.2×107 copies/ml.동기검측적HepG2.2.15세포내HBsAg화HBeAg적함량분별위3.51、44.85 IU/ml,HBV DNA함량위7.4×104 copies/ml.Western blot검측가견피감염적HepG2세포유HBX단백적표체.결론 응용AdEasy계통성공구건함1.3고패HBV기인조적중조선병독재체,경포장세포산생적병독감염파세포후능검측도HBV항원적표체.
Objective To construct recombinant adenovirus vector containing 1.3-fold overlength hepatitis B virus (HBV) genome and to identify the expression of the targeted proteins.Methods 1.3-fold overlength HBV genome of 4.1 kilobases was obtained by digesting the plasmid pT-HBV1.3 with restriction endonucleases and subcloned into shuttle vector pAdTrack-TOX.The resultant vector pAdTrack-HBV1.3 was linearized by endonuclease Pme Ⅰ and electroporated into competent E.coli BJ5183 containing the adenoviral backbone plasmid for homologous recombination.Then the confirmed recombinant adenovirus vector pAdEasy-HBV1.3 was constructed and transferred into 293T packaging cells.The recombinant adenovirus Ad-HBV1.3 was generated and amplified in 293T cells.HepG2 cells were cultured in DMEM plus 10％ fetal bovine serun (FBS) and infected with Ad-HBV1.3.Ninety-six h after infection,the supernatant was collected and assayed for HBsAg,HBeAg and HBV DNA content.The infected cells were washed 5 to 6 times with PBS and pellets collected for protein extraction.Hepatitis B virus x-protein (HBX) expression was detected by Western blotting.Correspondingly,HepG2.2.15 was used as positive control cell model as it stably expressed HBV antigens.Results pAdTrack-HBV1.3 was successfully constructed by ligating 1.3-fold HBV genome to pAdTrack-TOX,and the presence and orientation of HBV genome were confirmed by sequencing.Also,the recombinant adenovirus vector pAdEasy-HBV1.3 was successfully obtained by homologous recombination in BJ5183.Green fluorescent protein (GFP) expression was observed in 293T cells after transfection of pAdEasy-HBV1.3 and generation of recombinant adenovirus Ad-HBV1.3 was conformed.Contents of HBsAg,HBeAg and HBV DNA assayed in the supernatant of HepG2 cells at 4th day post-infection with..Ad-HBV1.3 were 70.70,5.50 IU/ml and 1.2 × 107 copies/ml,respectively.Also the contents of HBsAg,HBeAg and HBV DNA in the supernatant of cultured HepG2.2.15 cells were assayed as high as 3.51,44.85 IU/ml and 7.4 × 104 copies/ml respectively.HBV X protein expression was also observed in infected cells.Conclusion The recombinant adenovirus vector containing 1.3-fold HBV genome was constructed by AdEasy system and high level production of HBV antigens could be assayed in HepG2 cells infected with recombinant adenovirus Ad-HBV1.3.
