乙型肝炎病毒表面抗原S基因在人5型重组腺病毒中的表达及其特性  

Expression of hepatitis B virus S genes in Ad5 vector

作　　者：蒋国桥[1] 石长信[1] 周伟[1] 洪涛[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《中华实验和临床病毒学杂志》1997年第4期323-324,共2页Chinese Journal of Experimental and Clinical Virology

基　　金：国家863高科技生物技术领域基金

摘　　要：从美国Ｗｙｅｔｈ公司引进ｐＡｄ５△Ｅ３载体，将乙型肝炎病毒表面抗原基因（ＨＢＶＳ），ＰｒｅＳ２＋Ｓ，腺病毒晚期表达盒＋ＨＢＶＳ基因插入Ｅ３区，与ＥｃｏＲⅠ消化的Ａｄ５ＤＮＡＡ片段共转染细胞获得重组腺病毒，相应命名为ｒＡｄ５Ｓ，ｒＡｄ５ＭＳ，ｒＡｄ５Ｓ２Ｓ。对所获得的重组病毒进行聚合酶链反应（ＰＣＲ）分析，证实重组病毒中含有目的基因。用ＥＬＩＳＡ分析了ｒＡｄ５ＭＳ的表达量，其病变细胞和上清液滴度均为１∶４，表明表达产物能分泌到细胞外，而另两株重组病毒ＥＬＩＳＡ为阴性。进一步放射免疫检测表明，三株重组病毒Ｓ抗原皆为阳性，结果说明Ｅ３区启动子能表达外源基因，所构建的腺病毒晚期表达盒能显著提高表达效率。实验还同时探讨了重组病毒表达特性和重组病毒遗传稳定性，并发现只要５型腺病毒ＤＮＡ酶切完全，不分离、纯化ＥｃｏＲⅠ消化的Ａ片段，共转染中也不会出现野毒株背景问题从美国Ｗｙｅｔｈ公司引进ｐＡｄ５△Ｅ３载体，将乙型肝炎病毒表面抗原基因（ＨＢＶＳ），ＰｒｅＳ２＋Ｓ，腺病毒晚期表达盒＋ＨＢＶＳ基因插入Ｅ３区，与ＥｃｏＲⅠ消化的Ａｄ５ＤＮＡＡ片段共转染细胞获得重组腺病毒，相应命名为ｒＡｄ５Ｓ，ｒＡｄ５ＭＳ，ｒＡｄ５Ｓ２Ｓ。对所获?The hepatitis B virus (HBV) S gene, PreS2+S genes and the late phase expression cassette (MTI)+HBV S genes were separately cloned into Ad5 vector downstream of E3 promoter (pAd5△E3 provided by Wyeth Co.). The above constructed plasmids and Ad5 DNA EcoR Ⅰ A fragment were cotransfected into 293 cells. The progeny adenoviruses named rAd5S, rAd5MS, rAd5S2S were harvested for analysis. The recombinants were isolated and analyzed by PCR, using two primers specific to the HBV S genes. The expressed products were detected by ELISA and RIA. The recombinant containing MIT+HBV S genes (rAd5MS) was identified to be ELISA positive, whereas the other two recombinants (rAd5S, rAd5S2S) were negative to ELISA, but positive to RIA. The results indicated that adenovirus E3 early promoter could express the inserted foreign genes, and MIT worked well in the E3 region of Ad5 and could increase the expression capacity of the recombinants. The conditions for foreign gene expression and genetic stability of the recombinant viruses were studied in detail. There was no wild Ad5 discovered during the cotransfection experiments. The present study provides some experiences for studying adenovirus recombination.

关 键 词：腺病毒 载体 乙型肝炎病毒 病毒抗原 基因重组 

分 类 号：R446.5[医药卫生—诊断学]