丙肝病毒融合抗原基因NS_3-C定点整合入衣藻叶绿体基因组的研究

NS_3-C Chimeric Antigen Gene of Hepatitis C Virus was Introduced Site- specifically into Chloroplast Genome of Chlamydomonas reinhardtii

用ＰＣＲ 方法从丙型肝炎病毒（ＨＣＶ） ｃＤＮＡ 文库中克隆了两段ＤＮＡ 片段，即ＨＣＶ 基因组非结构ＮＳ３区抗原基因（约０．７ ｋｂ）和核心抗原Ｃ区抗原基因（约０．６ ｋｂ）的ｃＤＮＡ 片段。在两段ｃＤＮＡ 间加入连接肽Ｓｅｒ－ Ｐｒｏ－ Ｇｌｙ－ Ｓｅｒ 的密码子序列，构建成融合抗原基因ＮＳ３－ Ｃ。将该融合基因与衣藻叶绿体基因ａｔｐＡ 的启动子和ｒｂｃＬ 基因的３′末端连接，得到丙肝病毒融合抗原基因ＮＳ３－ Ｃ表达盒，再将该表达盒与选择标记基因ａａｄＡ 表达盒和衣藻叶绿体基因组同源片段连接，构建成衣藻叶绿体转化载体ｐＳＳ６。基因枪法转化衣藻叶绿体，经壮观霉素筛选获得转化再生的单藻落，对转基因衣藻的ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交分析表明，融合抗原基因ＮＳ３－ Ｃ已整合到衣藻叶绿体基因组中。

Two DNA fragments encoding the nucleocapsid (C) region protein and the non-structural region 3 (NS 3 ) protein of hepatitis C virus(HCV) were amplified from cDNA library by using PCR method. The 5′ terminal of C cDNA fragment was linked up with the 3′ terminal of NS 3 cDNA fragment by a oligonucleotide linker Ser-Pro-Gly-Ser to form a chimeric gene NS 3 -C, which was placed under the control of the chloroplast atpA promoter and rbcL 3′ region of Chlamydomonas reinhardtii to construct the chimeric gene NS 3 -C cassette. Then the NS 3 -C cassette was linked with selectable gene aadA cassette and the chloroplast homologous fragments of Chlamydomonas reinhardtii to generate transformation vector pSS6. Chloroplasts of Chlamydomonas reinhardtii were transformed by particle bombardment. Plastid transformants were selected by their resistance to 100 mg/L of spectinomycin. PCR and Southern hybridization analysis showed that the chimeric gene NS 3 -C had been integrated into chloroplast genome of Chlamydomonas reinhardtii.