人β防御素2(HBD2)与人乳头瘤病毒HPV16E6羧基端编码基因的克隆及其在真核细胞中的表达  

Gene cloning and eukaryotic expression of human β defensin 2 and HPV16E6 C-terminal region.

作　　者：孙艳[1] 李明[1] 黄宁[1] 吴琦[1] 王伯瑶[1] 

机构地区：[1]四川大学华西医学中心感染免疫研究室,成都610041

出　　处：《西部医学》2003年第4期289-292,共4页Medical Journal of West China

基　　金：中华纽约医学会基金 (编号 :98681 ) ;国家自然科学基金 (编号 :3980 0 1 4 6)

摘　　要：目的　构建人β防御素与人乳头瘤病毒 HPV16 E6羧基端基因的真核表达质粒 ,并检测其在真核细胞中的表达情况 ,探讨其作为 DNA疫苗治疗宫颈癌的可行性。方法　利用分子克隆技术 ,将 HBD2基因片段与 HPV16 E6羧基端基因片段连接 ,插入真核表达质粒 pc DNA3.1,经测序鉴定后 ,用阳离子脂质体法转染真核细胞 COS7,RT- PCR及免疫组化法检测其表达。结果　重组质粒 pc DNA3.1/ HBD2 - HPV16 E6 C转染 COS7细胞 4 8小时后 ,RT- PCR扩增出插入的 HBD2 - HPV16 E6 C片段 ,免疫组化染色呈棕黄色阳性反应。结论　人 β防御素与人乳头瘤病毒 HPV16 E6羧基端融合基因能在真核细胞中有效表达 ,为今后进行整体动物 DNA免疫试验奠定了基础。Objective To construct eukaryotic expression plasmid pcDNA3.1/HBD2-HPV16E6C and detect the expression of the plassmid in eukaryocyte. Methods Using cloning technique,the cDNA fragments of HBD2 gene and HPV16E6C gene were constructed into eukaryotic expression plasmid pcDNA3.1.The inserted target genes in the plasmid were verified by nucleotide sequecing.COS7 cell line was transfected with the recombinant plasmid pcDNA3.1/HBD2-HPV16E6C using lipofectamine reagent.RT-PCR and immunohistochemistry were performed to identify the expression of fusion gene HBD2-HPV16E6C in the transfected cells. Results The sequence of DNA fragments amplified by PCR is identifical to that published in GeneBank. Digestion of plasmid with restriction enzyme liberated DNA fragments with expected size. Fusion gene HBD2-HPV16E6C was expressed in COS7 after transfection.Conclusion The recombinant plasmid pcDNA3.1/HBD2-HPV16E6C was successfully .This study provides a good basis for further researcher on HPV16E6C DNA vaccine.

关 键 词：人Β防御素2 人乳头瘤病毒 分子克隆 真核细胞 

分 类 号：R392.11[医药卫生—免疫学]