猴免疫缺陷病毒SIV核心蛋白基因在大肠杆菌中表达的研究

Studies on the Expression of SIV Core P27 Gene inE . coli

应用多聚酶链反应（ＰＣＲ），直接从ＳＩＶ感染的猴艾滋病（ＳＡＩＤＳ）模型猴的外周血淋巴细胞总ＤＮＡ中扩增出７６７ｂｐ的ＳＩＶ核心蛋白Ｐ２７基因片段。扩增产物经ＥｃｏＲＩ及ＳａｌＩ双酶切后，克隆入相同酶切的表达质粒ｐＢＶ２２０中，获得含ＳＩＶ核心蛋白基因片段的重组质粒ｐＢＶＳＧ，并进行ＤＮＡ序列分析。用该重组质粒转化大肠杆菌ＤＨ５ａ经筛选、增殖及４２℃温度诱导，ＳＤＳ－ＰＡＧＥ表明外源基因表达蛋白含量占菌体总蛋白１４．５％，Ｗｅｓｔｅｒｎ－ｂｌｏｔ证实表达产物能被ＳＩＶＰ２７单克隆抗体及ＳＡＩＤＳ模型猴血清中特异性抗体识别。

IV core P27 gene was directly amplified from the monkey's PBMCinfected with SIVmac by method of PCR. The P27 gene was digestedwith EcoRI and SalI, and inserted into the EcoRI and Sall site of the exp-ression vector PBV220. The recombinant plasmid named PBVSG was vertifiedby DNA sequencing. When the expression plasmid PBVSG was transferred toE.coli DH5a and then induced at 40 , a product with the expected molec-ular weight ( 27KD ) was observed, which was approximately 14.5%of the total bacterial proteins. By Western blotting, the expressed proteinretained the reactivity with the SIV P27 monoclonal antibody and thespecific anti-SIV serum antibody in SAIDS model monkeys.