HIV－1gag基因重组痘苗病毒的构建和高效表达

High Level Expression of Human immunodeficiency Virus Type 1 (HIV 1) Gag Gene in Vaccinia Viruses

Ｇａｇ蛋白是人Ⅰ型免疫缺陷病毒（ＨＩＶ－１）重要的结构蛋白，可诱导机体产生体液和细胞免疫应答。本试验将ＨＩＶ－１不同长度的长末端重复序列（ＬＴＲ）和全长的ｇａｇＯＲＦ置于痘病毒启动子控制下，经过同源重组、红细胞吸附试验筛选和间接免疫荧光试验鉴定，分离了６株重组痘苗病毒。免疫印迹（Ｗｅｓｔｅｒｎｂｌｏｔ）和免疫酶试验检测表明，６株病毒均能表达ｇａｇ基因，其中以ｖ１６ＬＧ△２的Ｇａｇ蛋白表达量最高，占细胞裂解物上清的５．１９％，相当于１４．７μｇ／１０６细胞，接近杆状病毒的表达产量。重组蛋白主要为ｐ５５ｇａｇ蛋白前体，但也有一部分被加工，生成成熟蛋白ｐ２４ｇａｇ、ｐ１７ｇａｇ及其中间蛋白。表达的重组蛋白可形成病毒样粒子（ＶＬＰ）。

By inserting the gag gene at the downstream of Cowpox A type inclusion body (ATI) promoter or a hybrid promoter composed of ATI and 16 tandemly repeated mutant early p7.5 promoter, recombinant vaccinina viruses containing region of the gag ORF and different truncated domains of long terminal repeats (LTR) of human immunodeficiency virus type 1 (HIV 1) were constructed. Indirect immunofluorescence assay (IFA), Western blot and immunoenzyme assay (IEA) showed that all the recombinant vaccinia viruses expressed the Gag protein. Among them, v16LG△2,containing TAR and the downstream sequence, produced the highest level of Gag protein. Densitometric analysis of the stained gels showed that 5.192% Gag protein was contained in the lysate of BHK21 cell infected with v16LG△2, i. e. 10 6 cells infected with the produced at least 14.7 μg Gag protein. This expression efficiency reached the level of foreign protein produced in recombinant baculovirus. Observation with electron microscope revealed that the recombinant Gag protein assembled into virus like particles and buded from cell plasma membrance. Recombinant vaccinia viruses containing gag gene were able to elicit specific antibody against HIV 1 in immunized mice. The present data may be useful to the development of AIDS vaccine.