哈尔滨医科大学学报
哈爾濱醫科大學學報
합이빈의과대학학보
JOURNAL OF HARBIN MEDICAL UNIVERSITY
2014年
4期
263-267
,共5页
王天真%赵然%韩昌松%张玉华%叶盛前
王天真%趙然%韓昌鬆%張玉華%葉盛前
왕천진%조연%한창송%장옥화%협성전
肝癌%HBV%基因型%载体
肝癌%HBV%基因型%載體
간암%HBV%기인형%재체
liver cancer%HBV%genotype%vector
目的：构建A、B、C和D四种基因型HBV克隆载体，分析和比较它们在HepG2和Huh7细胞中的表达和复制。方法利用分子生物学技术将连接在pUC19上的A、B、C和D四种基因型HBV全序列连接到改建的pIRES2-EGFP真核表达载体中；脂质体转染法将构建的重组载体瞬时转染入HepG2和Huh7细胞中；利用载体所携带的绿色荧光蛋白在显微镜下观察转染效率，并利用ELISA和荧光定量PCR技术检测细胞培养上清中HBsAg、HBeAg和HBV DNA的表达水平。结果经鉴定，重组HBV真核表达载体构建成功，在HepG2和Huh7细胞中转染效率在50％～60％之间，四种基因型HBV在宿主细胞中均有稳定的复制和表达，但不同基因型在表达水平上存在差异。结论成功构建A、B、C和D四种基因型HBV真核表达载体，各基因型在HepG2和Huh7肝癌细胞系中均表现出较高的转染效率，为后续HBV及其基因型研究提供了实验载体。
目的：構建A、B、C和D四種基因型HBV剋隆載體，分析和比較它們在HepG2和Huh7細胞中的錶達和複製。方法利用分子生物學技術將連接在pUC19上的A、B、C和D四種基因型HBV全序列連接到改建的pIRES2-EGFP真覈錶達載體中；脂質體轉染法將構建的重組載體瞬時轉染入HepG2和Huh7細胞中；利用載體所攜帶的綠色熒光蛋白在顯微鏡下觀察轉染效率，併利用ELISA和熒光定量PCR技術檢測細胞培養上清中HBsAg、HBeAg和HBV DNA的錶達水平。結果經鑒定，重組HBV真覈錶達載體構建成功，在HepG2和Huh7細胞中轉染效率在50％～60％之間，四種基因型HBV在宿主細胞中均有穩定的複製和錶達，但不同基因型在錶達水平上存在差異。結論成功構建A、B、C和D四種基因型HBV真覈錶達載體，各基因型在HepG2和Huh7肝癌細胞繫中均錶現齣較高的轉染效率，為後續HBV及其基因型研究提供瞭實驗載體。
목적：구건A、B、C화D사충기인형HBV극륭재체，분석화비교타문재HepG2화Huh7세포중적표체화복제。방법이용분자생물학기술장련접재pUC19상적A、B、C화D사충기인형HBV전서렬련접도개건적pIRES2-EGFP진핵표체재체중；지질체전염법장구건적중조재체순시전염입HepG2화Huh7세포중；이용재체소휴대적록색형광단백재현미경하관찰전염효솔，병이용ELISA화형광정량PCR기술검측세포배양상청중HBsAg、HBeAg화HBV DNA적표체수평。결과경감정，중조HBV진핵표체재체구건성공，재HepG2화Huh7세포중전염효솔재50％～60％지간，사충기인형HBV재숙주세포중균유은정적복제화표체，단불동기인형재표체수평상존재차이。결론성공구건A、B、C화D사충기인형HBV진핵표체재체，각기인형재HepG2화Huh7간암세포계중균표현출교고적전염효솔，위후속HBV급기기인형연구제공료실험재체。
Objective To construct A , B, C and D four kinds of HBV genotypes cloning vec-tors, and to analyze and compare their expression and replication in HepG 2 and Huh7 cells. Methods Whole sequences of A , B, C and D genotype HBV in pUC 19 were connected with alternative pIRES2-EGFP eukaryotic vector using molecular biology techniques , respectively . The recombinant vectors were transiently transfected into HepG 2 and Huh7 cells by liposome transfection .In order to determine the transfection efficiency , green fluorescent protein was ob-served under the microscope .Meanwhile , ELISA and fluorescence quantitative PCR were used to detect the expression levels of HBsAg , HBeAg and HBV DNA in the supernatant .Results HBV recombinant eukaryotic expression vector was successfully constructed , and the transfect-ed efficiency was about 50%~60%in HepG2 and Huh7 cells.Four kinds of HBV genotypes replicated and expressed stablely in the host cells , but there was difference on the expression levels of different genotypes .Conclusion Eukaryotic expression vector with A , B, C and D four kinds of HBV genotypes are successfully constructed , and show high transfection efficiency in HepG2 and Huh7 liver cancer cells .The results provide experimental support for the subse-quent HBV and genotype research .
