摘要
目的 以鸡痘病毒为载体，表达ＨＩＶ－１ Ｇａｇ蛋白，进而研制ＡＩＤＳ疫苗。方法 以鸡痘病毒２８２Ｅ４株 非必需区ＴＫ基因为侧翼，插入ＶＶ突变型Ｐ７．５串联启动子和牛痘病毒Ａ包涵体晚期启动子与２０个串联的突变型 早期痘苗病毒Ｐ７．５启动子组成复合启动子及ＬａｃＺ报告基因构建重组表达质粒ｐＵＴＡＬ。在ＡＴ１－Ｐ７．５复合启动子 下游，插入编码中国流行株 ＨＩＶ－１ Ｇａｇ基因（Ｂ亚型），构建了重组表达质粒ｐＵＴＡＬ－Ｇａｇ。重组质粒Ｇａｇ与ＦＰＶ共 转染ＣＥＦ细胞，进行同源重组。通过ＢＵｄＲ加压筛选，Ｘ－ｇａｌ染色，获得重组鸡痘病毒ｖＵＴＡＬＧ，用于感染ＢＨＫ细胞 并免疫小鼠。结果 经Ｗｅｓｔｅｒｎ ｂｌｏｔ检测，重组病毒表达了Ｇａｇ蛋白相对分子质量分别为５５０００、４８０００、２４０００和 １７０００，为Ｇａｇ蛋白的Ｐ５５、Ｐ４８、Ｐ２４和Ｐ１７蛋白。重组病毒免疫小鼠可诱导产生特异性抗体。结论 所重组的病毒 成功地表达了目的蛋白并进行了正确加工。
Objective To express HIV-1 Gag protein in fowlpox virus( FPV) and develop AIDS vac-cine. Methods By insterting a combined promoter consisting of 20 tandem mutant early p7.5 promoters of vaccinia virus( VV) . a ATI promoter of cowpox virus and other 16 tandem mutant early p7.5 promoter of VV as well as report gene LacZ into the Ned site of TK gene of fowlpox virus strain 282E4, a FPV expression vector pUTAL was constructed. The recombinant expression plasmid pUTAL/Gag was constructed by inserting the Gag gene of HIV-1 subtype B into the downstream of ATI-p7. 5 combined promoter and transfected to CEF cells pre-infected with FPV strain 282E4,then a recombinant virus vUTALG was obtained by BudR pressure screen-ing and X-gal staining. The expressed product was detected by Western blot and observed under electron scope. The recombinant was used for infecting BHK cell and immunizing mice. Results Western blot showed that Gag protein was expressed in vUTALG and lysed to p55, p48, p24 and p17 proteins. Electron microscopy showed reverse transcription virus-like particles formed by Gag protein in CEF cells. Gag protein was also ex-pressed in mammal cell BHK infected with the recombinant virus vUTALG,and specific antibody response was induced in the mice immunized with the virus. Conclusin HIV-1 Gag protein is successfully expressed in fowlpox virus.
出处
《中国生物制品学杂志》
CAS
CSCD
2001年第3期132-135,共4页
Chinese Journal of Biologicals
基金
国家杰出青年基金会(39251197)资助
